An Additional Ca
Transmembrane protein 16A (TMEM16A) belongs to the TMEM16 family whose members include both Ca 2+ -activated chloride channels (CaCCs)  and dual functional Ca 2+ -activated non-selective ion channels and phospholipid scramblases . The CaCC TMEM16A plays an important role in regulating smooth muscle contraction, gut motility, fluid secretion, nociception, and anxiety . Abnormal expression of TMEM16A is intimately associated with numerous types of cancers, such as gastrointestinal stromal tumors; head and neck squamous carcinomas; and breast, pancreatic, and prostate cancer, thus highlighting its potential as a biomarker and promising anticancer therapeutic target . As a polymodal sensor, the gating landscape of TMEM16A is shaped by the synergistic action of Ca 2+ binding and membrane depolarization, both of which promote channel opening . TMEM16A’s channel activity is also positively regulated by the phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) . Extensive structural and functional studies have advanced our mechanistic understanding of TMEM16A’s Cl − permeation, Ca 2+ -dependent gating, and its regulation by lipids. Assembled as a homodimer, each TMEM16A subunit contains 10 membrane-spanning segments with its own independent pore . Transmembrane helix (TM)3–TM8 form the pore-gate domain that is essential for ion permeation, Ca 2+ sensing, and PIP 2 regulation, whereas TMs 1, 2, 9, and 10 form the supporting domain that establishes the dimer interface . The hourglass-shaped permeation pathway is composed of residues from TM3 to TM7, many of which have been implicated in controlling anion selectivity . Binding of intracellular Ca 2+ to five highly conserved acidic residues and an asparagine from TM6–TM8  not only induces a prominent conformational rearrangement of TM6 to help open the steric gate but also relieves a second, electrostatic gate imposed by the Ca 2+ -binding acidic residues, subsequently allowing chloride conduction . Intriguingly, recent structural studies of TMEM16F and TMEM16K scramblases have revealed yet another Ca 2+ binding site in TM2 and TM10 of the supporting domain . We refer to this additional Ca 2+ site as the “third Ca 2+ site” to distinguish it from the primary “orthosteric sites” in TM6–TM8. Despite not being resolved initially, it was later suggested that the Ca 2+ -bound TMEM16A structure  also harbors an electron density at the equivalent site, presumably deriving from a bound Ca 2+ ion . This assumption is supported by the fact that the Ca 2+ -coordinating residues at the third site are highly conserved among TMEM16 proteins . More importantly, the distant location of this Ca 2+ site from the pore-gate domain  also suggests that TMEM16 proteins may undergo a long-range allosteric modulation, a regulatory mechanism common in many ion channels and membrane receptor proteins that harbor multiple topologically distinct, yet tightly coupled, sensors . Nevertheless, lack of functional evidence raises questions on the existence and functional relevance of this Ca 2+ site in regulating TMEM16 proteins. Interestingly, a missense mutation of a third Ca 2+ -coordinating residue, D615N, in TMEM16K was identified in a spinocerebellar ataxia type 10 (SCAR10) patient with unknown patho-physiology . Therefore, understanding the functional role of this third Ca 2+ binding site not only informs an allosteric gating mechanism of TMEM16 proteins but also provides potential insight into the molecular patho-physiology of TMEM16K variant in SCAR10. To define the functional existence and the molecular basis for channel-gating modulation by the third Ca 2+ binding site, we studied the TMEM16A CaCC using electrophysiology, mutagenesis, and Cd 2+ -mediated metal bridging. Here, we show that the third site in TM2 and TM10 binds intracellular Ca 2+ with high affinity. Notably, mutating the Ca 2+ -binding residues at this site not only significantly reduces its Ca 2+ sensing but also directly affects the channel’s activation gate. Using Cd 2+ -mediated metal bridging of cysteines engineered to the third Ca 2+ site, we further demonstrate that the conformational states of TM2 and TM10 can strongly influence TMEM16A’s channel opening in a manner that is independent of the orthosteric Ca 2+ sites. Our functional analyses thus unveil an unknown regulatory mechanism in which channel activation is facilitated by Ca 2+ binding to the TM2–TM10 site. We anticipate that our present findings could stimulate future mechanistic investigations on the allosteric regulation and the development of allosteric modulators for TMEM16 ion channels and phospholipid scramblases. 

Mutation of the Putative Third Ca 2+ Site Residues Alters TMEM16A’s Ca 2+ Sensing Given the structural and sequence conservation between mammalian TMEM16 proteins at the putative third Ca 2+ binding site ( and – ), we examined whether the equivalent site in TMEM16A contributes to channel gating by measuring the Ca 2+ -dependent activation of the alanine mutations of E425 and K428 from TM2 and of D879 and D884 from TM10 . Paradoxically, the K428A and D884A mutations markedly reduce, while E425A and D879A enhance, the apparent Ca 2+ sensitivity of TMEM16A ( and ; ). Similar phenotypes were also observed in the charge-reversing mutations of these residues . Although they do not tell us whether the third Ca 2+ site residues indeed bind Ca 2+ , these results offer clear evidence to support that this putative Ca 2+ site in TM2 and TM10 significantly impacts the Ca 2+ -dependent activation of TMEM16A. TMEM16A’s D884 residue is absolutely conserved among mammalian TMEM16 proteins and corresponds to TMEM16K D615, whose D615N variant was identified in a patient with autosomal recessive SCAR10 ( , , and ) . To gain some insight into the mutational effect of TMEM16K D615N, we measured the Ca 2+ sensitivity of the equivalent TMEM16A D884N mutant . Similar to D884A, the D884N mutation also significantly reduces TMEM16A Ca 2+ sensitivity, albeit to a lesser extent ( and ; ). Although this observation implies a possibility that D615N also impairs TMEM16K’s function, the previous study by found that this mutant displayed no obvious defects in its lipid scrambling under both Ca 2+ -free and saturating Ca 2+ conditions using reconstituted liposomes. We speculated that the mutational effect of TMEM16K D615N may require a more rigorous assessment of its Ca 2+ dose-dependent lipid scrambling, especially considering the relatively lower sensitivity/resolution of lipid scrambling assays compared to that of electrophysiology as well as the less disruptive Asp-to-Asn substitution. Taken together, our single point mutation characterizations of TMEM16A reveal that disrupting the putative third Ca 2+ binding site in TM2 and TM10 alters channel activation. Disrupting the Third Ca 2+ Site Residues Alters Channel Activation independent of Ca 2+ Considering the pronounced effects from the alanine and charge-reversing mutations of the third Ca 2+ site on channel activation, we speculated that such perturbations could either affect the orthosteric-site-mediated, Ca 2+ -dependent gating or modulate the channel’s activation gate independent of Ca 2+ . To test whether disrupting the third Ca 2+ site residues would remotely impact TMEM16A’s activation gate in the pore-gate domain independent of Ca 2+ binding, we utilized the gain-of-function mutant Q645A , which allows us to measure TMEM16A’s channel activation in the complete absence of Ca 2+ . To accurately quantify their voltage-dependent activation, we normalized the measured conductance in the absence of Ca 2+ to the maximal conductance (G max ), which was obtained by exposing the excised membrane patches to saturating 100 μM Ca 2+ . Interestingly, alanine mutations of the putative third Ca 2+ site residues in the Q645A background markedly shift the conductance-voltage (G-V) curves in the absence of Ca 2+ . Whereas E425A and D879A enhance channel activation and shift the G-V curve toward more a negative voltage range, K428A and D884A shift it toward more positive voltages. Furthermore, the charge-reversing mutants E425K and D879K also promote, whereas K428E and D884K reduce, TMEM16A’s channel activation in the absence of Ca 2+ . The G/G max -V curves of these mutant channels did not go beyond 0.5 , which prevents us from extrapolating the half-maximal activation voltage (V 1/2 ) values for further comparisons. Nonetheless, the mutational effects of the third Ca 2+ site residues on TMEM16A’s Ca 2+ -independent activation strikingly mirror their respective effects on the channel’s Ca 2+ -dependent activation seen in the wild-type (WT) background . Because the orthosteric Ca 2+ sites in TM6–TM8 account for the measured Ca 2+ -dependent currents in the WT background , the observed alterations of TMEM16A’s Ca 2+ sensitivity from the third site mutations should arise from their Ca 2+ -independent allosteric effects on channel gating. Therefore, the paradoxical effects of the third Ca 2+ site mutants on WT TMEM16A’s Ca 2+ sensitivity  likely reflect a net result of the Ca 2+ -dependent activation by the orthosteric Ca 2+ sites and the Ca 2+ -independent allosteric effects from perturbation of the third Ca 2+ site on the channel’s activation gate. The Putative Third Ca 2+ Site in TM2 and TM10 Binds Ca 2+ Because neutralizing the orthosteric Ca 2+ binding residues severely diminishes Ca 2+ -dependent activation in TMEM16A , it is thus intriguing that neutralizing E425A and D879A mutants at the third Ca 2+ site enhance, whereas K428A and D884A decrease, the apparent Ca 2+ sensitivity of TMEM16A . However, the fact that these mutants are sufficient to sensitize or attenuate channel activation in the absence of Ca 2+ binding is suggestive of their potential pleiotropic effects on not only the channel’s activation gate  but also Ca 2+ binding to this third site. We reasoned that the mutational effects on channel activation from disrupting the third Ca 2+ site residues could be confounded by the intact orthosteric Ca 2+ sites. Thus, to specifically investigate the Ca 2+ binding properties of the third site, we utilized the E701K and D734R mutations, two charge-reversing mutations of the Ca 2+ -coordinating E701 and D734 on TM7 and TM8, respectively , previously shown to abolish Ca 2+ binding to the orthosteric Ca 2+ sites . We also introduced the Q645A mutation on TM6 to allow the channel to adopt a partially released steric gate that can be opened via membrane depolarization . Remarkably, the resulting triple mutant Q645A/E701K/D734R (henceforth referred to as QA/EK/DR) channels display robust Ca 2+ sensitivity, as Ca 2+ application strongly potentiates the basal current mediated by Q645A . Because the E701K and D734R mutations abolish Ca 2+ binding to the orthosteric sites, we attributed the observed Ca 2+ sensing capability of QA/EK/DR to the third Ca 2+ site in TM2–TM10 due to the following lines of evidence. First, by analyzing the Ca 2+ -sensitive currents, which were obtained by subtracting the basal current from each Ca 2+ -elicited steady-state current , we found that the third Ca 2+ site has an estimated Ca 2+ half-maximal activation concentration (EC 50 ) of ~320 nM ± 10 nM , which is well within physiological range and is thus unlikely to be mediated by the already impaired orthosteric sites. To rule out any potential confounding effects from Ca 2+ -dependent rundown on our Ca 2+ EC 50 analysis, we performed rundown-corrected measurements of the QA/EK/DR mutant and obtained a comparable Ca 2+ EC 50 value of ~360 nM ± 20 nM . Second, consistent with its location closer to the cytosolic side, the Ca 2+ sensitivity of the third site shows no apparent voltage dependence , which is distinct from the highly voltage-dependent Ca 2+ sensitivity of the orthosteric Ca 2+ binding sites, owing to their location deep into the membrane field . Finally, we designed single alanine mutations of the third Ca 2+ site—E425A, K428A, D879A and D884A—in the context of the QA/EK/DR background, examined their Ca 2+ sensitivity, and found that they substantially reduce the Ca 2+ -dependent currents of the QA/EK/DR channels . The reduced Ca 2+ -dependent currents are not due to decreased channel expression, as these quadruple mutants display robust channel activation in the absence of Ca 2+ ( – , , and ). Notably, alanine mutations of the three acidic residues, E425, D879, and D884, most dramatically attenuate the third site’s Ca 2+ sensing, whereas that of the basic K428 residue causes a less pronounced reduction, consistent with the potential structural role of K428 in stabilizing the third Ca 2+ site . Our observation that E425A and D884A strongly reduce the Ca 2+ sensitivity of the third site is particularly fascinating in light of the recent structural studies showing that the equivalent E395-D859 and E259-D615 residues in TMEM16F and TMEM16K, respectively, directly coordinate the bound third Ca 2+ ion via their side-chain carboxylates . Taken together, our characterizations of the third Ca 2+ site on the QA/EK/DR background demonstrate that Ca 2+ binds to this site with high affinity to promote channel opening. Charged Residues at the Third Ca 2+ Site Play Differential Roles in Ca 2+ Binding To further explore the functional roles of the third Ca 2+ site residues, we carried out Ca 2+ -dependent current measurements of all six combinations of double mutants at the third Ca 2+ site on the QA/EK/DR background. Provided the strongly reduced Ca 2+ sensitivity by single alanine mutations of E425, D879, and D884 , we reasoned that double-mutant variants comprising two acidic residues at the third site should abolish its Ca 2+ binding. In agreement with this hypothesis, we found that the acidic double mutants E425A/D879A, E425A/D884A, and D879A/D884A completely lose their Ca 2+ sensing capability ( , , and ). The loss of Ca 2+ sensitivity by the basic/acidic E425A/K428A double mutant further highlights the importance of E425 at the third Ca 2+ site . It is worth noting that the lack of Ca 2+ sensing by these mutants is not due to impaired membrane trafficking, as evidenced by their robust membrane expression  and voltage-dependent channel activation in the absence of Ca 2+ ( – , , and ). By contrast, the basic/acidic double mutants K428A/D879A and K428A/D884A exhibit an apparent Ca 2+ sensitivity comparable to that of D879A and D884A, respectively . The observation that neutralizing two acidic residues completely extinguishes its Ca 2+ sensing further strengthens our conclusion that the third site binds Ca 2+ to facilitate channel opening. The ability of K428A/D879A and K428A/D884A double mutants to retain some Ca 2+ sensitivity  allowed us to conduct double-mutant cycle analysis  to study the potential coupling between these charged residues ( , , and ). Interestingly, our analyses reveal that K428 is energetically coupled to D879 and to D884 with a significant energy of interaction (ΔΔG int ) of −1.07 ± 0.03 kcal · mol −1 and −0.98 ± 0.02 kcal · mol −1 , respectively ( and ). This suggests that, although K428 is unlikely to directly coordinate Ca 2+ , its energetically favorable interactions with D879 and D884 may serve an important structural role in stabilizing these first-shell acidic residues for Ca 2+ binding. Taken together, in excellent agreement with previous structural studies , our functional analyses not only validate that Ca 2+ , indeed, binds to the third site in TM2 and TM10 but also unveil the distinct roles of its charged residues in mediating Ca 2+ binding and regulating channel activation. TM2-TM10 Interaction Can Strongly Influence TMEM16A’s Channel Gating The multifaceted role of the third site in modulating TMEM16A’s Ca 2+ -dependent and Ca 2+ -independent channel activation prompted us to speculate that the conformation of TM2 and TM10 at this region may be key to allosterically controlling channel gating. To test this hypothesis, we performed double-cysteine substitutions to the third Ca 2+ site and sought to assess whether the transition metal ion Cd 2+ could form a metal bridge between TM2 and TM10 and how such restricting their conformation would impact channel activation. The readily reversible formation of Cd 2+ metal bridge permits temporal probing of the effects from crosslinking TM2 and TM10 on channel activity . By screening different TM2–TM10 interhelical pairs of cysteines, we found that application of Cd 2+ (0.5, 1, or 10 μM) largely abolishes channel activity of the double mutant E425C/D879C, but not WT or the single-cysteine mutants E425C and D879C, even under saturating 100 μM Ca 2+ . The partial inhibition seen in WT channels is likely caused by Cd 2+ competing with Ca 2+ for binding to the orthosteric Ca 2+ sites, as this inhibition is strongly attenuated by elevating intracellular Ca 2+ and is rapidly reversed upon Cd 2+ removal ( and ; ). In stark contrast, Cd 2+ -mediated inhibition of E425C/D879C is much longer lasting, as evidenced by the slow current recovery rate , which is indicative of a high-affinity Cd 2+ -E425C/D879C metal bridge. In fact, we found that Cd 2+ also potently inhibits the double mutant E425H/D879C ( , and ), in accordance with the ability of Cd 2+ to bridge a histidine and a cysteine . Cd 2+ elicits no obvious effects on other TM2–TM10 interhelical double-cysteine mutants, including E425C/D884C and K428C/D879C , presumably owing to a lack of optimal distance and/or orientation for Cd 2+ metal bridging. The K428C/D884C double mutant shows Ca 2+ -elicited currents that are too small for analysis . We also examined whether the inhibitory effect from chemically restraining TM2 and TM10 is dependent upon Ca 2+ binding to orthosteric sites. By again exploiting the QA/EK/DR background construct to interrogate the effects of Cd 2+ -mediated bridging in the absence of Ca 2+ , we found that 10 μM Cd 2+ markedly inhibits the voltage-dependent channel activity of E425C/D879C but not the Cd 2+ -insensitive E428C/D884C mutant . Taken together, these results reveal that altering the conformation of TM2–TM10 at the third Ca 2+ site by bridging the substituted cysteines E425C and D879C leads to profound channel inhibition in a manner that is independent of Ca 2+ binding to the orthosteric sites. 

2+ Site Residues Alters TMEM16A’s Ca 2+ Sensing Given the structural and sequence conservation between mammalian TMEM16 proteins at the putative third Ca 2+ binding site ( and – ), we examined whether the equivalent site in TMEM16A contributes to channel gating by measuring the Ca 2+ -dependent activation of the alanine mutations of E425 and K428 from TM2 and of D879 and D884 from TM10 . Paradoxically, the K428A and D884A mutations markedly reduce, while E425A and D879A enhance, the apparent Ca 2+ sensitivity of TMEM16A ( and ; ). Similar phenotypes were also observed in the charge-reversing mutations of these residues . Although they do not tell us whether the third Ca 2+ site residues indeed bind Ca 2+ , these results offer clear evidence to support that this putative Ca 2+ site in TM2 and TM10 significantly impacts the Ca 2+ -dependent activation of TMEM16A. TMEM16A’s D884 residue is absolutely conserved among mammalian TMEM16 proteins and corresponds to TMEM16K D615, whose D615N variant was identified in a patient with autosomal recessive SCAR10 ( , , and ) . To gain some insight into the mutational effect of TMEM16K D615N, we measured the Ca 2+ sensitivity of the equivalent TMEM16A D884N mutant . Similar to D884A, the D884N mutation also significantly reduces TMEM16A Ca 2+ sensitivity, albeit to a lesser extent ( and ; ). Although this observation implies a possibility that D615N also impairs TMEM16K’s function, the previous study by found that this mutant displayed no obvious defects in its lipid scrambling under both Ca 2+ -free and saturating Ca 2+ conditions using reconstituted liposomes. We speculated that the mutational effect of TMEM16K D615N may require a more rigorous assessment of its Ca 2+ dose-dependent lipid scrambling, especially considering the relatively lower sensitivity/resolution of lipid scrambling assays compared to that of electrophysiology as well as the less disruptive Asp-to-Asn substitution. Taken together, our single point mutation characterizations of TMEM16A reveal that disrupting the putative third Ca 2+ binding site in TM2 and TM10 alters channel activation. 

2+ Site Residues Alters Channel Activation independent of Ca 2+ Considering the pronounced effects from the alanine and charge-reversing mutations of the third Ca 2+ site on channel activation, we speculated that such perturbations could either affect the orthosteric-site-mediated, Ca 2+ -dependent gating or modulate the channel’s activation gate independent of Ca 2+ . To test whether disrupting the third Ca 2+ site residues would remotely impact TMEM16A’s activation gate in the pore-gate domain independent of Ca 2+ binding, we utilized the gain-of-function mutant Q645A , which allows us to measure TMEM16A’s channel activation in the complete absence of Ca 2+ . To accurately quantify their voltage-dependent activation, we normalized the measured conductance in the absence of Ca 2+ to the maximal conductance (G max ), which was obtained by exposing the excised membrane patches to saturating 100 μM Ca 2+ . Interestingly, alanine mutations of the putative third Ca 2+ site residues in the Q645A background markedly shift the conductance-voltage (G-V) curves in the absence of Ca 2+ . Whereas E425A and D879A enhance channel activation and shift the G-V curve toward more a negative voltage range, K428A and D884A shift it toward more positive voltages. Furthermore, the charge-reversing mutants E425K and D879K also promote, whereas K428E and D884K reduce, TMEM16A’s channel activation in the absence of Ca 2+ . The G/G max -V curves of these mutant channels did not go beyond 0.5 , which prevents us from extrapolating the half-maximal activation voltage (V 1/2 ) values for further comparisons. Nonetheless, the mutational effects of the third Ca 2+ site residues on TMEM16A’s Ca 2+ -independent activation strikingly mirror their respective effects on the channel’s Ca 2+ -dependent activation seen in the wild-type (WT) background . Because the orthosteric Ca 2+ sites in TM6–TM8 account for the measured Ca 2+ -dependent currents in the WT background , the observed alterations of TMEM16A’s Ca 2+ sensitivity from the third site mutations should arise from their Ca 2+ -independent allosteric effects on channel gating. Therefore, the paradoxical effects of the third Ca 2+ site mutants on WT TMEM16A’s Ca 2+ sensitivity  likely reflect a net result of the Ca 2+ -dependent activation by the orthosteric Ca 2+ sites and the Ca 2+ -independent allosteric effects from perturbation of the third Ca 2+ site on the channel’s activation gate. 

2+ Site in TM2 and TM10 Binds Ca 2+ Because neutralizing the orthosteric Ca 2+ binding residues severely diminishes Ca 2+ -dependent activation in TMEM16A , it is thus intriguing that neutralizing E425A and D879A mutants at the third Ca 2+ site enhance, whereas K428A and D884A decrease, the apparent Ca 2+ sensitivity of TMEM16A . However, the fact that these mutants are sufficient to sensitize or attenuate channel activation in the absence of Ca 2+ binding is suggestive of their potential pleiotropic effects on not only the channel’s activation gate  but also Ca 2+ binding to this third site. We reasoned that the mutational effects on channel activation from disrupting the third Ca 2+ site residues could be confounded by the intact orthosteric Ca 2+ sites. Thus, to specifically investigate the Ca 2+ binding properties of the third site, we utilized the E701K and D734R mutations, two charge-reversing mutations of the Ca 2+ -coordinating E701 and D734 on TM7 and TM8, respectively , previously shown to abolish Ca 2+ binding to the orthosteric Ca 2+ sites . We also introduced the Q645A mutation on TM6 to allow the channel to adopt a partially released steric gate that can be opened via membrane depolarization . Remarkably, the resulting triple mutant Q645A/E701K/D734R (henceforth referred to as QA/EK/DR) channels display robust Ca 2+ sensitivity, as Ca 2+ application strongly potentiates the basal current mediated by Q645A . Because the E701K and D734R mutations abolish Ca 2+ binding to the orthosteric sites, we attributed the observed Ca 2+ sensing capability of QA/EK/DR to the third Ca 2+ site in TM2–TM10 due to the following lines of evidence. First, by analyzing the Ca 2+ -sensitive currents, which were obtained by subtracting the basal current from each Ca 2+ -elicited steady-state current , we found that the third Ca 2+ site has an estimated Ca 2+ half-maximal activation concentration (EC 50 ) of ~320 nM ± 10 nM , which is well within physiological range and is thus unlikely to be mediated by the already impaired orthosteric sites. To rule out any potential confounding effects from Ca 2+ -dependent rundown on our Ca 2+ EC 50 analysis, we performed rundown-corrected measurements of the QA/EK/DR mutant and obtained a comparable Ca 2+ EC 50 value of ~360 nM ± 20 nM . Second, consistent with its location closer to the cytosolic side, the Ca 2+ sensitivity of the third site shows no apparent voltage dependence , which is distinct from the highly voltage-dependent Ca 2+ sensitivity of the orthosteric Ca 2+ binding sites, owing to their location deep into the membrane field . Finally, we designed single alanine mutations of the third Ca 2+ site—E425A, K428A, D879A and D884A—in the context of the QA/EK/DR background, examined their Ca 2+ sensitivity, and found that they substantially reduce the Ca 2+ -dependent currents of the QA/EK/DR channels . The reduced Ca 2+ -dependent currents are not due to decreased channel expression, as these quadruple mutants display robust channel activation in the absence of Ca 2+ ( – , , and ). Notably, alanine mutations of the three acidic residues, E425, D879, and D884, most dramatically attenuate the third site’s Ca 2+ sensing, whereas that of the basic K428 residue causes a less pronounced reduction, consistent with the potential structural role of K428 in stabilizing the third Ca 2+ site . Our observation that E425A and D884A strongly reduce the Ca 2+ sensitivity of the third site is particularly fascinating in light of the recent structural studies showing that the equivalent E395-D859 and E259-D615 residues in TMEM16F and TMEM16K, respectively, directly coordinate the bound third Ca 2+ ion via their side-chain carboxylates . Taken together, our characterizations of the third Ca 2+ site on the QA/EK/DR background demonstrate that Ca 2+ binds to this site with high affinity to promote channel opening. 

2+ Site Play Differential Roles in Ca 2+ Binding To further explore the functional roles of the third Ca 2+ site residues, we carried out Ca 2+ -dependent current measurements of all six combinations of double mutants at the third Ca 2+ site on the QA/EK/DR background. Provided the strongly reduced Ca 2+ sensitivity by single alanine mutations of E425, D879, and D884 , we reasoned that double-mutant variants comprising two acidic residues at the third site should abolish its Ca 2+ binding. In agreement with this hypothesis, we found that the acidic double mutants E425A/D879A, E425A/D884A, and D879A/D884A completely lose their Ca 2+ sensing capability ( , , and ). The loss of Ca 2+ sensitivity by the basic/acidic E425A/K428A double mutant further highlights the importance of E425 at the third Ca 2+ site . It is worth noting that the lack of Ca 2+ sensing by these mutants is not due to impaired membrane trafficking, as evidenced by their robust membrane expression  and voltage-dependent channel activation in the absence of Ca 2+ ( – , , and ). By contrast, the basic/acidic double mutants K428A/D879A and K428A/D884A exhibit an apparent Ca 2+ sensitivity comparable to that of D879A and D884A, respectively . The observation that neutralizing two acidic residues completely extinguishes its Ca 2+ sensing further strengthens our conclusion that the third site binds Ca 2+ to facilitate channel opening. The ability of K428A/D879A and K428A/D884A double mutants to retain some Ca 2+ sensitivity  allowed us to conduct double-mutant cycle analysis  to study the potential coupling between these charged residues ( , , and ). Interestingly, our analyses reveal that K428 is energetically coupled to D879 and to D884 with a significant energy of interaction (ΔΔG int ) of −1.07 ± 0.03 kcal · mol −1 and −0.98 ± 0.02 kcal · mol −1 , respectively ( and ). This suggests that, although K428 is unlikely to directly coordinate Ca 2+ , its energetically favorable interactions with D879 and D884 may serve an important structural role in stabilizing these first-shell acidic residues for Ca 2+ binding. Taken together, in excellent agreement with previous structural studies , our functional analyses not only validate that Ca 2+ , indeed, binds to the third site in TM2 and TM10 but also unveil the distinct roles of its charged residues in mediating Ca 2+ binding and regulating channel activation. 

The multifaceted role of the third site in modulating TMEM16A’s Ca 2+ -dependent and Ca 2+ -independent channel activation prompted us to speculate that the conformation of TM2 and TM10 at this region may be key to allosterically controlling channel gating. To test this hypothesis, we performed double-cysteine substitutions to the third Ca 2+ site and sought to assess whether the transition metal ion Cd 2+ could form a metal bridge between TM2 and TM10 and how such restricting their conformation would impact channel activation. The readily reversible formation of Cd 2+ metal bridge permits temporal probing of the effects from crosslinking TM2 and TM10 on channel activity . By screening different TM2–TM10 interhelical pairs of cysteines, we found that application of Cd 2+ (0.5, 1, or 10 μM) largely abolishes channel activity of the double mutant E425C/D879C, but not WT or the single-cysteine mutants E425C and D879C, even under saturating 100 μM Ca 2+ . The partial inhibition seen in WT channels is likely caused by Cd 2+ competing with Ca 2+ for binding to the orthosteric Ca 2+ sites, as this inhibition is strongly attenuated by elevating intracellular Ca 2+ and is rapidly reversed upon Cd 2+ removal ( and ; ). In stark contrast, Cd 2+ -mediated inhibition of E425C/D879C is much longer lasting, as evidenced by the slow current recovery rate , which is indicative of a high-affinity Cd 2+ -E425C/D879C metal bridge. In fact, we found that Cd 2+ also potently inhibits the double mutant E425H/D879C ( , and ), in accordance with the ability of Cd 2+ to bridge a histidine and a cysteine . Cd 2+ elicits no obvious effects on other TM2–TM10 interhelical double-cysteine mutants, including E425C/D884C and K428C/D879C , presumably owing to a lack of optimal distance and/or orientation for Cd 2+ metal bridging. The K428C/D884C double mutant shows Ca 2+ -elicited currents that are too small for analysis . We also examined whether the inhibitory effect from chemically restraining TM2 and TM10 is dependent upon Ca 2+ binding to orthosteric sites. By again exploiting the QA/EK/DR background construct to interrogate the effects of Cd 2+ -mediated bridging in the absence of Ca 2+ , we found that 10 μM Cd 2+ markedly inhibits the voltage-dependent channel activity of E425C/D879C but not the Cd 2+ -insensitive E428C/D884C mutant . Taken together, these results reveal that altering the conformation of TM2–TM10 at the third Ca 2+ site by bridging the substituted cysteines E425C and D879C leads to profound channel inhibition in a manner that is independent of Ca 2+ binding to the orthosteric sites. 

Here, we present functional evidence to establish that, in addition to the two orthosteric sites in the pore-gate domain, TMEM16A also possesses an additional Ca 2+ binding site comprising E425 and K428 in TM2 and D879 and D884 in TM10 . Our data not only consolidate recent structural findings of a third Ca 2+ binding site in TMEM16F and TMEM16K scramblases  but also functionally validate that the unassigned electron density observed in TM2–TM10 of the liganded TMEM16A structure, indeed, represents the third bound Ca 2+ ion . Allosteric Modulation of TMEM16A’s Gating via an Additional High-Affinity Ca 2+ Site In TMEM16 proteins, the pore-gate domain accommodates both the two orthosteric Ca 2+ sites and the activation gate, thereby allowing Ca 2+ binding to efficiently open the activation gate ( and ). This structural arrangement differs from that of the Ca 2+ -activated large conductance potassium BK channels, whose two Ca 2+ sensors are distantly located within disparate parts of the cytosolic gating ring and independently control activation via long-range allosteric coupling mechanisms . Therefore, the presence of an additional Ca 2+ site that is distant from the pore-gate domain raises the question of whether TMEM16 proteins may also utilize a long-range allosteric mechanism to regulate ion/lipid permeation gating. To address this question and to understand the relationship between the third Ca 2+ site and the orthosteric Ca 2+ sites, we used three different TMEM16A backgrounds: WT, Q645A, and QA/EK/DR. First, our Ca 2+ -dependent measurements on the WT background revealed the involvement of the third Ca 2+ site residues in channel activation . Second, the Q645A background allowed us to demonstrate that mutation of the third site residues alters channel activation independent of Ca 2+ binding to the orthosteric sites, which is in support of a long-range allosteric coupling between this additional Ca 2+ site and the activation gate. Finally, because simultaneous Ca 2+ binding to the orthosteric sites hinders assessment of the third site’s Ca 2+ sensitivity, we generated the QA/EK/DR construct and used it to reliably measure the Ca 2+ sensitivity of the third site. This approach allowed us to reveal that the third Ca 2+ site possesses a high affinity for Ca 2+ with an apparent dissociation constant (K D ) of ~320 nM, which is well suited for sensing physiological Ca 2+ concentrations . Our comprehensive mutagenesis experiments, in conjunction with double-mutant cycle analyses, further support that the three acidic residues E425, D879, and D884 directly participate in Ca 2+ binding, whereas K428 serves a stabilizing structural role ( and ). Interestingly, in addition to impairing Ca 2+ binding, disruption of the third Ca 2+ site also differentially alters channel gating in a Ca 2+ -independent manner , consistent with a pleiotropic role of these residues in both mediating Ca 2+ binding and regulating the channel’s gate. Conformationally Restricting TM2–TM10 at the Third Site Inhibits Channel Activation Our observation that chemically bridging E425C and D879C inhibits TMEM16A activation is both striking and counterintuitive, considering that Ca 2+ binding to the third site promotes channel opening . However, this is unsurprising, as we believe that Cd 2+ -mediated metal bridging likely elicits distinct effects from Ca 2+ binding to an intact third site based on the following reasons. First, in the intact site, the Ca 2+ ion is coordinated by the acidic residues E425, D879, and D884 ( and ) , while in the double cysteine-substituted mutant, Cd 2+ should be sandwiched between E425C and D879C. Second, given the shorter side chain of cysteine compared with those of aspartate and glutamate, the conformation resulting from Cd 2+ -E425C/D879C complex formation should, therefore, be distinct from that induced by Ca 2+ binding to the intact site. Finally, Cd 2+ coordination with E425C and D879C is more stable than Ca 2+ within the third-site acidic residues, as signified by the extremely slow off-rate of Cd 2+ that is characteristic of a cadmium-cysteine metal bridge . Although precisely how such stabilization of TM2 and TM10 prohibits channel activation requires additional studies, our data further underscore the importance of this site in channel gating. Potential Role of TM10’ in Allosteric Modulation by the Third Ca 2+ Site Our functional evidence suggests that Ca 2+ binding to the third site can lead to long-range allosteric effects on TMEM16A activation. However, it is still unclear how Ca 2+ binding energy at the third site is propagated to the pore-gate domain. We speculate that TM10’, a short α helix following TM10, may play an important role in mediating this allosteric coupling based on the following observations . First, it was shown that replacing or truncating the C-terminal region following TM10’ after the third Ca 2+ site at L904  or K895  markedly altered the Ca 2+ sensitivity of TMEM16A. Second, using chemical crosslinking experiments, suggested that TM10’ may regulate channel gating via its inter-subunit coupling to the TM2–TM3 loop, which was previously implicated in TMEM16A’s voltage-dependent channel activation . Finally, the recent structural study of TMEM16K suggested that TM10’ forms an inter-subunit contact with the TM2–TM3 loop (mostly β9–β10) and undergoes a pronounced swinging movement during Ca 2+ -dependent activation; this enables rearrangement of the TM2–TM3 loop and, subsequently, the N-terminal cytosolic domain (NCD), all of which culminate in the opening of TMEM16K’s lipid pathway . On the basis of these structural and functional observations, we suggest that Ca 2+ binding to the third site in TM2–TM10 likely induces a conformational change of TM10’, perhaps altering its inter-subunit interaction with the TM2–TM3 loop and, subsequently, the NCD to modulate channel opening . The lack of conformational changes at the third site upon Ca 2+ binding in the TMEM16A and TMEM16F structures likely reflects their desensitized/rundown states after Ca 2+ -dependent activation . Future studies are required to fully elaborate the molecular basis of this long-range allosteric coupling pathway between the third Ca 2+ site and the activation gate . In summary, our work builds upon previous structural studies and provides the physical basis underlying a long-range regulatory gating mechanism by which Ca 2+ binding to the TM2–TM10 site enhances channel opening. Our present findings also suggest that structurally conserved TMEM16 ion channels and scramblases may exploit a similar mode of allosteric regulation by Ca 2+ . We believe that the additional Ca 2+ site endows TMEM16 proteins with enhanced regulatory mechanisms, increasing their complexity necessary for diverse Ca 2+ signaling functions in various tissues. Mechanistic insight into this Ca 2+ binding site not only facilitates our understanding of the patho-physiology of TMEM16 mutations but may also assist future therapeutic design of allosteric modulators for the treatment of TMEM16-related diseases. 

2+ Site In TMEM16 proteins, the pore-gate domain accommodates both the two orthosteric Ca 2+ sites and the activation gate, thereby allowing Ca 2+ binding to efficiently open the activation gate ( and ). This structural arrangement differs from that of the Ca 2+ -activated large conductance potassium BK channels, whose two Ca 2+ sensors are distantly located within disparate parts of the cytosolic gating ring and independently control activation via long-range allosteric coupling mechanisms . Therefore, the presence of an additional Ca 2+ site that is distant from the pore-gate domain raises the question of whether TMEM16 proteins may also utilize a long-range allosteric mechanism to regulate ion/lipid permeation gating. To address this question and to understand the relationship between the third Ca 2+ site and the orthosteric Ca 2+ sites, we used three different TMEM16A backgrounds: WT, Q645A, and QA/EK/DR. First, our Ca 2+ -dependent measurements on the WT background revealed the involvement of the third Ca 2+ site residues in channel activation . Second, the Q645A background allowed us to demonstrate that mutation of the third site residues alters channel activation independent of Ca 2+ binding to the orthosteric sites, which is in support of a long-range allosteric coupling between this additional Ca 2+ site and the activation gate. Finally, because simultaneous Ca 2+ binding to the orthosteric sites hinders assessment of the third site’s Ca 2+ sensitivity, we generated the QA/EK/DR construct and used it to reliably measure the Ca 2+ sensitivity of the third site. This approach allowed us to reveal that the third Ca 2+ site possesses a high affinity for Ca 2+ with an apparent dissociation constant (K D ) of ~320 nM, which is well suited for sensing physiological Ca 2+ concentrations . Our comprehensive mutagenesis experiments, in conjunction with double-mutant cycle analyses, further support that the three acidic residues E425, D879, and D884 directly participate in Ca 2+ binding, whereas K428 serves a stabilizing structural role ( and ). Interestingly, in addition to impairing Ca 2+ binding, disruption of the third Ca 2+ site also differentially alters channel gating in a Ca 2+ -independent manner , consistent with a pleiotropic role of these residues in both mediating Ca 2+ binding and regulating the channel’s gate. 

Our observation that chemically bridging E425C and D879C inhibits TMEM16A activation is both striking and counterintuitive, considering that Ca 2+ binding to the third site promotes channel opening . However, this is unsurprising, as we believe that Cd 2+ -mediated metal bridging likely elicits distinct effects from Ca 2+ binding to an intact third site based on the following reasons. First, in the intact site, the Ca 2+ ion is coordinated by the acidic residues E425, D879, and D884 ( and ) , while in the double cysteine-substituted mutant, Cd 2+ should be sandwiched between E425C and D879C. Second, given the shorter side chain of cysteine compared with those of aspartate and glutamate, the conformation resulting from Cd 2+ -E425C/D879C complex formation should, therefore, be distinct from that induced by Ca 2+ binding to the intact site. Finally, Cd 2+ coordination with E425C and D879C is more stable than Ca 2+ within the third-site acidic residues, as signified by the extremely slow off-rate of Cd 2+ that is characteristic of a cadmium-cysteine metal bridge . Although precisely how such stabilization of TM2 and TM10 prohibits channel activation requires additional studies, our data further underscore the importance of this site in channel gating. 

2+ Site Our functional evidence suggests that Ca 2+ binding to the third site can lead to long-range allosteric effects on TMEM16A activation. However, it is still unclear how Ca 2+ binding energy at the third site is propagated to the pore-gate domain. We speculate that TM10’, a short α helix following TM10, may play an important role in mediating this allosteric coupling based on the following observations . First, it was shown that replacing or truncating the C-terminal region following TM10’ after the third Ca 2+ site at L904  or K895  markedly altered the Ca 2+ sensitivity of TMEM16A. Second, using chemical crosslinking experiments, suggested that TM10’ may regulate channel gating via its inter-subunit coupling to the TM2–TM3 loop, which was previously implicated in TMEM16A’s voltage-dependent channel activation . Finally, the recent structural study of TMEM16K suggested that TM10’ forms an inter-subunit contact with the TM2–TM3 loop (mostly β9–β10) and undergoes a pronounced swinging movement during Ca 2+ -dependent activation; this enables rearrangement of the TM2–TM3 loop and, subsequently, the N-terminal cytosolic domain (NCD), all of which culminate in the opening of TMEM16K’s lipid pathway . On the basis of these structural and functional observations, we suggest that Ca 2+ binding to the third site in TM2–TM10 likely induces a conformational change of TM10’, perhaps altering its inter-subunit interaction with the TM2–TM3 loop and, subsequently, the NCD to modulate channel opening . The lack of conformational changes at the third site upon Ca 2+ binding in the TMEM16A and TMEM16F structures likely reflects their desensitized/rundown states after Ca 2+ -dependent activation . Future studies are required to fully elaborate the molecular basis of this long-range allosteric coupling pathway between the third Ca 2+ site and the activation gate . In summary, our work builds upon previous structural studies and provides the physical basis underlying a long-range regulatory gating mechanism by which Ca 2+ binding to the TM2–TM10 site enhances channel opening. Our present findings also suggest that structurally conserved TMEM16 ion channels and scramblases may exploit a similar mode of allosteric regulation by Ca 2+ . We believe that the additional Ca 2+ site endows TMEM16 proteins with enhanced regulatory mechanisms, increasing their complexity necessary for diverse Ca 2+ signaling functions in various tissues. Mechanistic insight into this Ca 2+ binding site not only facilitates our understanding of the patho-physiology of TMEM16 mutations but may also assist future therapeutic design of allosteric modulators for the treatment of TMEM16-related diseases. 

RESOURCE AVAILABILITY Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Huanghe Yang ( huanghe.yang@duke.edu ). Materials Availability This study did not generate new unique reagents. Data and Code Availability This study did not generate unique datasets/code. EXPERIMENTAL MODEL AND SUBJECT DETAILS Cell lines Adherent human embryonic kidney 293T (HEK293T) cells, which were authenticated and tested negative for mycoplasma, were obtained from Duke Cell Culture Facility (ATCC CRL-11268). HEK293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, Sigma), 1% penicillin/streptomycin (GIBCO) in an incubator supplied with 5% CO 2 and kept at 37°C. HEK293T cells were passaged every 2–3 days and were used within 15 passages. METHOD DETAILS Cloning and Cell Culture Mouse TMEM16A (mTMEM16A, NCBI accession code NP_001229278) tagged with a C-terminal EGFP (in an N1 vector) was used for all experiments. This mTMEM16A(‘a’) isoform lacks the EAVK segment (‘c’) in the first intracellular loop. Individual point mutations of TMEM16A were generated using QuikChange site-directed mutagenesis kit (Agilent), and multiple mutations were generated sequentially. All PCR primers were purchased from IDT DNA Technologies (see the complete list of primers used for mutagenesis). Substituted amino acid sequences were validated by Sanger sequencing (Genewiz). Glass coverslips were pre-coated with poly-L-lysine (Sigma) and laminin (Sigma) for at least 1–2 h in a 24-well plate (Eppendorf) and were seeded with freshly dissociated HEK293T cells. HEK293T cells were transiently transfected in the following day using X-tremeGENE 9 DNA transfection reagent (Sigma). Each well was typically transfected with 200 to 250 ng of total DNA plasmid to achieve sufficient expression for current recordings. Electrophysiology Electrophysiology recordings were performed at room temperature within 24 to 36 h after transfection. Glass electrodes were made by pulling borosilicate capillaries (Sutter Instruments) and fire-polished with a microforge (Narishige). Electrodes typically had a resistance of 2–3 MΩ when filled with recording solutions. Excised inside-out membrane patches were obtained by rapidly pulling gigaohm (GΩ) cell-attached seals from HEK293T cells expressing fluorescently tagged TMEM16A channels. External (pipette) solution contains 140 mM NaCl, 10 mM HEPES, 2 mM MgCl 2 , and pH 7.3 (adjusted with NaOH). Bath solution contains 140 mM NaCl, 10 mM HEPES, 5 mM EGTA, and pH 7.4 (adjusted with NaOH). Intracellular (perfusion) solutions with less than 100 μM Ca 2+ contain were made by adding CaCl 2 to a solution containing 140 mM NaCl, 10 mM HEPES, 5 mM EGTA, and the amount of CaCl 2 added was calculated using WEBMAXC ( https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/ ) to achieve desired free Ca 2+ . The free Ca 2+ concentrations were further corrected using the ratiometric Ca 2+ dye Fura-2 (ATT Bioquest) by fitting their absorbances against a standard Ca 2+ curve generated from a Ca 2+ calibration kit (Biotum). For solution with 100 μM or 500 μM Ca 2+ , CaCl 2 was directly added to a solution containing 140 mM NaCl, 10 mM HEPES, pH 7.3. Application of intracellular solutions to excised inside-out patches was carried out using a pressurized perfusion apparatus (ALA-VM8, ALA Scientific Instruments) in which the glass electrodes containing the excised patches were placed directly in front of the perfusion outlet. For measurements of the Ca 2+ -dependent dose-response behavior of TMEM16A WT and mutant channels with intact (WT or Q645A backgrounds) or disrupted (QA/EK/DR background) orthosteric Ca 2+ sites, excised inside-out patches were held at +60 mV and TMEM16A-mediated outward currents (Cl − influx) were elicited by sequential application of EGTA-buffered internal solutions containing increasing Ca 2+ concentrations. Once the Ca 2+ -elicited currents reached a steady state, a Ca 2+ -free solution (140 mM NaCl, 10 mM HEPES, 5 mM EGTA, pH 7.3) was immediately applied to allow the channels to return the closed state (WT background channels) or the basal activity level (QA/EK/DR background channels). To ensure that current rundown does not impact our Ca 2+ -dependent measurements and quantifications, we also performed Ca 2+ -dependent recordings of the QA/EK/DR mutant using a rundown-corrected protocol akin to one reported previously . In this rundown-corrected protocol, after application of each test Ca 2+ concentration (0.255, 0.387, 0.94, 2.26, 6 and 100 μM), saturating 500 μM Ca 2+ was applied to obtain the reference peak current, which was used for normalizing the test Ca 2+ current responses. For current-voltage ( I-V ) recordings of the Q645A background construct-containing TMEM16A mutant channels in the absence of Ca 2+ , the excised membrane patches were held at 60 mV, and test voltage steps from 120 to +140 mV were applied to elicit channel opening in the absence of 0 Ca 2+ . The same patch was immediately exposed to 100 μM Ca 2+ and subjected to an I-V protocol in which the membrane was held at 0 mV and stepped to +140 mV and 60 mV. The steady-state currents measured during test voltage steps were used for subsequent analysis. For cadmium (Cd 2+ ) metal bridging experiments, the desired Cd 2+ concentrations were directly added to a solution containing 140 mM NaCl, 10 mM HEPES, 100 μM Ca 2+ , pH 7.4 using 1000× Cd 2+ stock solutions. 1000× Cd 2+ stock solutions (0.5, 1, 10 mM) were made from 10× serial dilutions from a 100 mM Cd 2+ stock solution using ultrapure water (Invitrogen). Excised inside-out patches were held at +60 mV using a gap-free protocol and TMEM16A channels were activated with the control 100 μM or 500 μM Ca 2+ solution for 2–3 s to obtain the reference peak current before being switched to a Cd 2+ -containing solution to assess current inhibition. Application of Cd 2+ -containing solution usually took 4–5 s until the appearance of the steady-state currents after which the control 100 μM or 500 μM Ca 2+ solution was re-applied (for ~30 s) to allow current recovery from Cd 2+ -mediated inhibition. For experiments assessing the effects of Cd 2+ metal bridge formation in the absence of Ca 2+ , E425C/D879C(QA/EK/DR) and E425C/D879C(QA/EK/DR) channels were first activated in the absence of Ca 2+ and immediately exposed to solution containing 10 μM Cd 2+ (140 mM NaCl, 10 HEPES, pH 7.4) using an I-V protocol. The membrane was held at 60 mV and the channels were activated via voltage steps from 120 to +140 mV. The E425C/D884C(QA/EK/DR) was used as a negative control for its lack of Cd 2+ sensitivity as seen in the WT background (see , , and ). All electrophysiology recordings were low-pass filtered at 5 kHz (Axopatch 200B) and digitally sampled at 10 kHz (Axon Digidata 1550A) and digitized by Clampex 10 (Molecular Devices). Confocal microscopy HEK293T cells were co-transfected with EGFP-tagged mouse TMEM16A mutant channels (mTMEM16A-EGFP) and OFPSpark®-tagged human phospholipid scramblase 1 (hPLSCR1-OFPSpark®, Sino Biological) at a 2:1 DNA ratio. hPLSCR1 is a single-pass transmembrane protein that favors plasma membrane expression . The fluorescently tagged hPLSCR1 was used as a plasma membrane marker in this study. Cells were imaged approximately 24–30 h after transfection. Fluorescent images were acquired using a Zeiss 780 inverted confocal microscope at 60x magnification. QUANTIFICATION AND STATISTICAL ANALYSIS Electrophysiological data analysis was done in Clampfit, Microsoft Excel and MATLAB (MathWorks). Fluorescence image analysis was done in ImageJ ( https://imagej.nih.gov/ij/ ). Ca 2+ -dependent channel activation For WT and mutant channels with intact orthosteric Ca 2+ sites, the Ca 2+ -elicited steady-state currents were normalized to the peak current (see ). For TMEM16A mutant channels with impaired orthosteric Ca 2+ sites (QA/EK/DR containing constructs), the basal current right after each Ca 2+ application was subtracted from the steady-state Ca 2+ -elicited current to avoid the issue with changing basal current, and these Ca 2+ -sensitive current components (see , , and ) were then normalized to the peak Ca 2+ -sensitive current (see , , and ). For rundown-corrected Ca 2+ -dependent measurements of the QA/EK/DR mutant , the Ca 2+ -sensitive current of each test Ca 2+ concentration was normalized to its respective reference peak current elicited by 500 μM Ca 2+ , and these reference peak-normalized values of the test Ca 2+ concentrations were used for constructing the dose-response curves. In all cases, the sigmoidal Ca 2+ dose-response curves were similarly constructed by fitting the normalized Ca 2+ -elicited currents using a non-linear regression curve fit with the equation: (Equation 1) 1 I max = 1 1 + E C 50 Ca 2 + H where I/I max is the normalized Ca 2+ -elicited current, [Ca 2+ ] is the applied free Ca 2+ concentration, H is the Hill coefficient, and EC 50 is the estimated concentration of Ca 2+ at which I/I max is 0.5. The EC 50 values from individual recordings are extracted for statistical analyses. Cd 2+ metal bridging experiments The extent of Cd 2+ -mediated inhibition of TMEM16A in the presence of 100 μM Ca 2+ was determined as: (Equation 2) Current ratio  = I C d 2 + I peak × 100 where I peak and I Cd2+ denote steady-state currents measured in the presence of 100 μM or 500 μM Ca 2+ before and during Cd 2+ application, respectively. To assess the extent of current recovery following Cd 2+ treatment, we determined the apparent off-rate of Cd 2+ . As in the case of Cd 2+ -insensitive channels such as WT or the single cysteine mutants E425C, D879C, the rapid current recovery of inhibition following Cd 2+ washout mostly represents the rate of displacement of Cd 2+ from the orthosteric Ca 2+ binding sites ( and ; ). On the other hand, in the Cd 2+ -sensitive double mutant E425C/D879C, the tight association between Cd 2+ and the substituted cysteines E425C and D879C mostly accounts for the much slower recovery rate ( and ; ). As a result, we used the time constant (τ) of current recovery after application of 1 μM Cd 2+ to quantitatively assess the effects of Cd 2+ crosslinking by fitting the recovering current with a standard exponential fit with the equation: (Equation 3) I ( t ) = I 0 + A ( 1 − e − t τ ) where I (t) represents the current at time point t, I 0 is the initial current amplitude, t is the time constant. Current-voltage (I-V) recordings For I-V measurements of Q645A mutant channels, we used the steady state currents obtained during the depolarization steps for analysis due to the rapid deactivation at hyperpolarization and the intrinsically low open probability of the Q645A mutant in the absence of Ca 2+ binding to the orthosteric sites . The calculated conductance from each test voltage step measured in the absence of Ca 2+ (first recording) was normalized to the peak conductance measured at +140 mV in the presence of 100 μM Ca 2+ (second recording). The resulting normalized G/G max -V curves were fit with a Boltzmann equation: (Equation 4) G G max = 1 1 + e − z F ( V m − V 1 / 2 ) R T where G/G max is the normalized channel conductance, z is the number of equivalent gating charges, V m is the applied membrane voltage, V 1/2 is the estimated voltage at which G/G max is 0.5, F is the Faraday’s constant, R is the gas constant, and T is the absolute temperature. Because of the low open probability of the Q645A-containing mutants in 0 Ca 2+ , their G/G max -V curves did not go beyond 0.5 . We therefore did not extract the V 1/2 values for further comparisons. Double mutant cycle analysis To assess the potential energetic coupling between the charged residues at the third Ca 2+ site, we extracted the Ca 2+ EC 50 values from our Ca 2+ -dependent dose-response measurements of single and double mutations of the third Ca 2+ site in the QA/EK/DR background. Because we were able to reliably measure the Ca 2+ dose-response curves of K428A/D879A(QA/EK/DR) and K428A/D884A(QA/EK/DR), the K428-D879 and K428-D884 residue pairs were further analyzed using double mutant cycle analysis by calculating the free energy change associated with a perturbation . For each pair of residues, namely X and Y, the free energy change associated with their perturbations via single or double mutations was calculated using the following equations: (Equation 5) Δ G X Y > X ′ Y = R T * I n ( E C 50 ( X ′ Y ) ) E C 50 ( X Y ) ) ( or Δ G ( 1 ) ) (Equation 6) Δ G X Y ′ > X ′ Y ′ = R T * I n ( E C 50 ( X ′ Y ′ ) E C 50 ( X Y ′ ) ) ( or Δ G ( 4 ) ) (Equation 7) Δ G X Y > X Y ′ = R T * I n ( E C 50 ( X Y ′ ) E C 50 ( X Y ) ) ( or Δ G ( 2 ) ) (Equation 8) Δ G X ′ Y > X ′ Y ′ = R T * I n ( E C 50 ( X ′ Y ′ ) E C 50 ( X ′ Y ) ) ( or Δ G ( 3 ) ) where XY denotes intact residues on the background; X’Y, XY’, X’Y’ denote mutations at X, Y, and both, respectively; EC 50 is the estimated half-maximal activation concentration of Ca 2+ . Note that the background refers the QA/EK/DR construct on which single and double mutations of the third site residue pairs are made. In principle, ΔG (1) and ΔG (4) are related and represent the free energy change as a result of mutation of X (or X’) on the background channel (ΔG (1) ) or on the Y’ background (ΔG (4) ). Similarly, ΔG (2) and ΔG (3) represent the free change associated with Y’ mutations on background and X’ background, respectively. If X and Y residues are energetically coupled, the free energy change associated with the double mutations–X’ mutation on Y’ background (ΔG (4) ) or Y’ mutation on X’ background (ΔG (3) )–would differ from that associated with the single mutations X’ and Y’, or ΔG (1) and ΔG (2) , respectively. Please also refer to – for the schematic diagrams of our double mutant cycle analyses. To obtain the standard error for the interaction energy (ΔΔG int ), the individually calculated free energy change (ΔG (s) ) values were used as: (Equation 9) Δ Δ G i n t = Δ G ( 4 ) − Δ G ( 1 ) = Δ G ( 3 ) − Δ G ( 2 ) The calculated ΔΔG int values were subsequently confirmed using the equation: (Equation 10) Δ Δ G i n t = R T * I n ( E C 50 ( X ′ Y ′ ) ⋅ E C 50 ( b g ) E C 50 ( X ′ Y ) ⋅ E C 50 ( X Y ′ ) ) Structural and sequence analysis PDB coordinate files were obtained from the Protein Data Bank ( https://www.rcsb.org/ ). Protein structure figures were prepared in Pymol (Schrödinger, Inc.). Protein sequence alignment was done in UniProt ( https://www.uniprot.org/align/ ). Statistical analysis Statistical analyses were performed in Prism software (GraphPad). Two-tailed Student’s t test was used for comparisons between two groups (paired or unpaired), and one-way ANOVA was used for comparisons between multiple data groups. Comparisons with p values < 0.05 are deemed to be statistically significant. Data in summary bar graphs are represented as mean ± standard deviation (SD) or mean ± standard error of the mean (s.e.m.), and each data point represents an independent recording. *, **, *** and **** represent statistical significance corresponding to p value < 0.05, < 0.01, < 0.001, < 0.0001, respectively. 

Lead Contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Huanghe Yang ( huanghe.yang@duke.edu ). Materials Availability This study did not generate new unique reagents. Data and Code Availability This study did not generate unique datasets/code. 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Huanghe Yang ( huanghe.yang@duke.edu ). 

This study did not generate new unique reagents. 

This study did not generate unique datasets/code. 

Cell lines Adherent human embryonic kidney 293T (HEK293T) cells, which were authenticated and tested negative for mycoplasma, were obtained from Duke Cell Culture Facility (ATCC CRL-11268). HEK293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, Sigma), 1% penicillin/streptomycin (GIBCO) in an incubator supplied with 5% CO 2 and kept at 37°C. HEK293T cells were passaged every 2–3 days and were used within 15 passages. 

Adherent human embryonic kidney 293T (HEK293T) cells, which were authenticated and tested negative for mycoplasma, were obtained from Duke Cell Culture Facility (ATCC CRL-11268). HEK293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, Sigma), 1% penicillin/streptomycin (GIBCO) in an incubator supplied with 5% CO 2 and kept at 37°C. HEK293T cells were passaged every 2–3 days and were used within 15 passages. 

Cloning and Cell Culture Mouse TMEM16A (mTMEM16A, NCBI accession code NP_001229278) tagged with a C-terminal EGFP (in an N1 vector) was used for all experiments. This mTMEM16A(‘a’) isoform lacks the EAVK segment (‘c’) in the first intracellular loop. Individual point mutations of TMEM16A were generated using QuikChange site-directed mutagenesis kit (Agilent), and multiple mutations were generated sequentially. All PCR primers were purchased from IDT DNA Technologies (see the complete list of primers used for mutagenesis). Substituted amino acid sequences were validated by Sanger sequencing (Genewiz). Glass coverslips were pre-coated with poly-L-lysine (Sigma) and laminin (Sigma) for at least 1–2 h in a 24-well plate (Eppendorf) and were seeded with freshly dissociated HEK293T cells. HEK293T cells were transiently transfected in the following day using X-tremeGENE 9 DNA transfection reagent (Sigma). Each well was typically transfected with 200 to 250 ng of total DNA plasmid to achieve sufficient expression for current recordings. Electrophysiology Electrophysiology recordings were performed at room temperature within 24 to 36 h after transfection. Glass electrodes were made by pulling borosilicate capillaries (Sutter Instruments) and fire-polished with a microforge (Narishige). Electrodes typically had a resistance of 2–3 MΩ when filled with recording solutions. Excised inside-out membrane patches were obtained by rapidly pulling gigaohm (GΩ) cell-attached seals from HEK293T cells expressing fluorescently tagged TMEM16A channels. External (pipette) solution contains 140 mM NaCl, 10 mM HEPES, 2 mM MgCl 2 , and pH 7.3 (adjusted with NaOH). Bath solution contains 140 mM NaCl, 10 mM HEPES, 5 mM EGTA, and pH 7.4 (adjusted with NaOH). Intracellular (perfusion) solutions with less than 100 μM Ca 2+ contain were made by adding CaCl 2 to a solution containing 140 mM NaCl, 10 mM HEPES, 5 mM EGTA, and the amount of CaCl 2 added was calculated using WEBMAXC ( https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/ ) to achieve desired free Ca 2+ . The free Ca 2+ concentrations were further corrected using the ratiometric Ca 2+ dye Fura-2 (ATT Bioquest) by fitting their absorbances against a standard Ca 2+ curve generated from a Ca 2+ calibration kit (Biotum). For solution with 100 μM or 500 μM Ca 2+ , CaCl 2 was directly added to a solution containing 140 mM NaCl, 10 mM HEPES, pH 7.3. Application of intracellular solutions to excised inside-out patches was carried out using a pressurized perfusion apparatus (ALA-VM8, ALA Scientific Instruments) in which the glass electrodes containing the excised patches were placed directly in front of the perfusion outlet. For measurements of the Ca 2+ -dependent dose-response behavior of TMEM16A WT and mutant channels with intact (WT or Q645A backgrounds) or disrupted (QA/EK/DR background) orthosteric Ca 2+ sites, excised inside-out patches were held at +60 mV and TMEM16A-mediated outward currents (Cl − influx) were elicited by sequential application of EGTA-buffered internal solutions containing increasing Ca 2+ concentrations. Once the Ca 2+ -elicited currents reached a steady state, a Ca 2+ -free solution (140 mM NaCl, 10 mM HEPES, 5 mM EGTA, pH 7.3) was immediately applied to allow the channels to return the closed state (WT background channels) or the basal activity level (QA/EK/DR background channels). To ensure that current rundown does not impact our Ca 2+ -dependent measurements and quantifications, we also performed Ca 2+ -dependent recordings of the QA/EK/DR mutant using a rundown-corrected protocol akin to one reported previously . In this rundown-corrected protocol, after application of each test Ca 2+ concentration (0.255, 0.387, 0.94, 2.26, 6 and 100 μM), saturating 500 μM Ca 2+ was applied to obtain the reference peak current, which was used for normalizing the test Ca 2+ current responses. For current-voltage ( I-V ) recordings of the Q645A background construct-containing TMEM16A mutant channels in the absence of Ca 2+ , the excised membrane patches were held at 60 mV, and test voltage steps from 120 to +140 mV were applied to elicit channel opening in the absence of 0 Ca 2+ . The same patch was immediately exposed to 100 μM Ca 2+ and subjected to an I-V protocol in which the membrane was held at 0 mV and stepped to +140 mV and 60 mV. The steady-state currents measured during test voltage steps were used for subsequent analysis. For cadmium (Cd 2+ ) metal bridging experiments, the desired Cd 2+ concentrations were directly added to a solution containing 140 mM NaCl, 10 mM HEPES, 100 μM Ca 2+ , pH 7.4 using 1000× Cd 2+ stock solutions. 1000× Cd 2+ stock solutions (0.5, 1, 10 mM) were made from 10× serial dilutions from a 100 mM Cd 2+ stock solution using ultrapure water (Invitrogen). Excised inside-out patches were held at +60 mV using a gap-free protocol and TMEM16A channels were activated with the control 100 μM or 500 μM Ca 2+ solution for 2–3 s to obtain the reference peak current before being switched to a Cd 2+ -containing solution to assess current inhibition. Application of Cd 2+ -containing solution usually took 4–5 s until the appearance of the steady-state currents after which the control 100 μM or 500 μM Ca 2+ solution was re-applied (for ~30 s) to allow current recovery from Cd 2+ -mediated inhibition. For experiments assessing the effects of Cd 2+ metal bridge formation in the absence of Ca 2+ , E425C/D879C(QA/EK/DR) and E425C/D879C(QA/EK/DR) channels were first activated in the absence of Ca 2+ and immediately exposed to solution containing 10 μM Cd 2+ (140 mM NaCl, 10 HEPES, pH 7.4) using an I-V protocol. The membrane was held at 60 mV and the channels were activated via voltage steps from 120 to +140 mV. The E425C/D884C(QA/EK/DR) was used as a negative control for its lack of Cd 2+ sensitivity as seen in the WT background (see , , and ). All electrophysiology recordings were low-pass filtered at 5 kHz (Axopatch 200B) and digitally sampled at 10 kHz (Axon Digidata 1550A) and digitized by Clampex 10 (Molecular Devices). Confocal microscopy HEK293T cells were co-transfected with EGFP-tagged mouse TMEM16A mutant channels (mTMEM16A-EGFP) and OFPSpark®-tagged human phospholipid scramblase 1 (hPLSCR1-OFPSpark®, Sino Biological) at a 2:1 DNA ratio. hPLSCR1 is a single-pass transmembrane protein that favors plasma membrane expression . The fluorescently tagged hPLSCR1 was used as a plasma membrane marker in this study. Cells were imaged approximately 24–30 h after transfection. Fluorescent images were acquired using a Zeiss 780 inverted confocal microscope at 60x magnification. 

Mouse TMEM16A (mTMEM16A, NCBI accession code NP_001229278) tagged with a C-terminal EGFP (in an N1 vector) was used for all experiments. This mTMEM16A(‘a’) isoform lacks the EAVK segment (‘c’) in the first intracellular loop. Individual point mutations of TMEM16A were generated using QuikChange site-directed mutagenesis kit (Agilent), and multiple mutations were generated sequentially. All PCR primers were purchased from IDT DNA Technologies (see the complete list of primers used for mutagenesis). Substituted amino acid sequences were validated by Sanger sequencing (Genewiz). Glass coverslips were pre-coated with poly-L-lysine (Sigma) and laminin (Sigma) for at least 1–2 h in a 24-well plate (Eppendorf) and were seeded with freshly dissociated HEK293T cells. HEK293T cells were transiently transfected in the following day using X-tremeGENE 9 DNA transfection reagent (Sigma). Each well was typically transfected with 200 to 250 ng of total DNA plasmid to achieve sufficient expression for current recordings. 

Electrophysiology recordings were performed at room temperature within 24 to 36 h after transfection. Glass electrodes were made by pulling borosilicate capillaries (Sutter Instruments) and fire-polished with a microforge (Narishige). Electrodes typically had a resistance of 2–3 MΩ when filled with recording solutions. Excised inside-out membrane patches were obtained by rapidly pulling gigaohm (GΩ) cell-attached seals from HEK293T cells expressing fluorescently tagged TMEM16A channels. External (pipette) solution contains 140 mM NaCl, 10 mM HEPES, 2 mM MgCl 2 , and pH 7.3 (adjusted with NaOH). Bath solution contains 140 mM NaCl, 10 mM HEPES, 5 mM EGTA, and pH 7.4 (adjusted with NaOH). Intracellular (perfusion) solutions with less than 100 μM Ca 2+ contain were made by adding CaCl 2 to a solution containing 140 mM NaCl, 10 mM HEPES, 5 mM EGTA, and the amount of CaCl 2 added was calculated using WEBMAXC ( https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/ ) to achieve desired free Ca 2+ . The free Ca 2+ concentrations were further corrected using the ratiometric Ca 2+ dye Fura-2 (ATT Bioquest) by fitting their absorbances against a standard Ca 2+ curve generated from a Ca 2+ calibration kit (Biotum). For solution with 100 μM or 500 μM Ca 2+ , CaCl 2 was directly added to a solution containing 140 mM NaCl, 10 mM HEPES, pH 7.3. Application of intracellular solutions to excised inside-out patches was carried out using a pressurized perfusion apparatus (ALA-VM8, ALA Scientific Instruments) in which the glass electrodes containing the excised patches were placed directly in front of the perfusion outlet. For measurements of the Ca 2+ -dependent dose-response behavior of TMEM16A WT and mutant channels with intact (WT or Q645A backgrounds) or disrupted (QA/EK/DR background) orthosteric Ca 2+ sites, excised inside-out patches were held at +60 mV and TMEM16A-mediated outward currents (Cl − influx) were elicited by sequential application of EGTA-buffered internal solutions containing increasing Ca 2+ concentrations. Once the Ca 2+ -elicited currents reached a steady state, a Ca 2+ -free solution (140 mM NaCl, 10 mM HEPES, 5 mM EGTA, pH 7.3) was immediately applied to allow the channels to return the closed state (WT background channels) or the basal activity level (QA/EK/DR background channels). To ensure that current rundown does not impact our Ca 2+ -dependent measurements and quantifications, we also performed Ca 2+ -dependent recordings of the QA/EK/DR mutant using a rundown-corrected protocol akin to one reported previously . In this rundown-corrected protocol, after application of each test Ca 2+ concentration (0.255, 0.387, 0.94, 2.26, 6 and 100 μM), saturating 500 μM Ca 2+ was applied to obtain the reference peak current, which was used for normalizing the test Ca 2+ current responses. For current-voltage ( I-V ) recordings of the Q645A background construct-containing TMEM16A mutant channels in the absence of Ca 2+ , the excised membrane patches were held at 60 mV, and test voltage steps from 120 to +140 mV were applied to elicit channel opening in the absence of 0 Ca 2+ . The same patch was immediately exposed to 100 μM Ca 2+ and subjected to an I-V protocol in which the membrane was held at 0 mV and stepped to +140 mV and 60 mV. The steady-state currents measured during test voltage steps were used for subsequent analysis. For cadmium (Cd 2+ ) metal bridging experiments, the desired Cd 2+ concentrations were directly added to a solution containing 140 mM NaCl, 10 mM HEPES, 100 μM Ca 2+ , pH 7.4 using 1000× Cd 2+ stock solutions. 1000× Cd 2+ stock solutions (0.5, 1, 10 mM) were made from 10× serial dilutions from a 100 mM Cd 2+ stock solution using ultrapure water (Invitrogen). Excised inside-out patches were held at +60 mV using a gap-free protocol and TMEM16A channels were activated with the control 100 μM or 500 μM Ca 2+ solution for 2–3 s to obtain the reference peak current before being switched to a Cd 2+ -containing solution to assess current inhibition. Application of Cd 2+ -containing solution usually took 4–5 s until the appearance of the steady-state currents after which the control 100 μM or 500 μM Ca 2+ solution was re-applied (for ~30 s) to allow current recovery from Cd 2+ -mediated inhibition. For experiments assessing the effects of Cd 2+ metal bridge formation in the absence of Ca 2+ , E425C/D879C(QA/EK/DR) and E425C/D879C(QA/EK/DR) channels were first activated in the absence of Ca 2+ and immediately exposed to solution containing 10 μM Cd 2+ (140 mM NaCl, 10 HEPES, pH 7.4) using an I-V protocol. The membrane was held at 60 mV and the channels were activated via voltage steps from 120 to +140 mV. The E425C/D884C(QA/EK/DR) was used as a negative control for its lack of Cd 2+ sensitivity as seen in the WT background (see , , and ). All electrophysiology recordings were low-pass filtered at 5 kHz (Axopatch 200B) and digitally sampled at 10 kHz (Axon Digidata 1550A) and digitized by Clampex 10 (Molecular Devices). 

HEK293T cells were co-transfected with EGFP-tagged mouse TMEM16A mutant channels (mTMEM16A-EGFP) and OFPSpark®-tagged human phospholipid scramblase 1 (hPLSCR1-OFPSpark®, Sino Biological) at a 2:1 DNA ratio. hPLSCR1 is a single-pass transmembrane protein that favors plasma membrane expression . The fluorescently tagged hPLSCR1 was used as a plasma membrane marker in this study. Cells were imaged approximately 24–30 h after transfection. Fluorescent images were acquired using a Zeiss 780 inverted confocal microscope at 60x magnification. 

Electrophysiological data analysis was done in Clampfit, Microsoft Excel and MATLAB (MathWorks). Fluorescence image analysis was done in ImageJ ( https://imagej.nih.gov/ij/ ). Ca 2+ -dependent channel activation For WT and mutant channels with intact orthosteric Ca 2+ sites, the Ca 2+ -elicited steady-state currents were normalized to the peak current (see ). For TMEM16A mutant channels with impaired orthosteric Ca 2+ sites (QA/EK/DR containing constructs), the basal current right after each Ca 2+ application was subtracted from the steady-state Ca 2+ -elicited current to avoid the issue with changing basal current, and these Ca 2+ -sensitive current components (see , , and ) were then normalized to the peak Ca 2+ -sensitive current (see , , and ). For rundown-corrected Ca 2+ -dependent measurements of the QA/EK/DR mutant , the Ca 2+ -sensitive current of each test Ca 2+ concentration was normalized to its respective reference peak current elicited by 500 μM Ca 2+ , and these reference peak-normalized values of the test Ca 2+ concentrations were used for constructing the dose-response curves. In all cases, the sigmoidal Ca 2+ dose-response curves were similarly constructed by fitting the normalized Ca 2+ -elicited currents using a non-linear regression curve fit with the equation: (Equation 1) 1 I max = 1 1 + E C 50 Ca 2 + H where I/I max is the normalized Ca 2+ -elicited current, [Ca 2+ ] is the applied free Ca 2+ concentration, H is the Hill coefficient, and EC 50 is the estimated concentration of Ca 2+ at which I/I max is 0.5. The EC 50 values from individual recordings are extracted for statistical analyses. Cd 2+ metal bridging experiments The extent of Cd 2+ -mediated inhibition of TMEM16A in the presence of 100 μM Ca 2+ was determined as: (Equation 2) Current ratio  = I C d 2 + I peak × 100 where I peak and I Cd2+ denote steady-state currents measured in the presence of 100 μM or 500 μM Ca 2+ before and during Cd 2+ application, respectively. To assess the extent of current recovery following Cd 2+ treatment, we determined the apparent off-rate of Cd 2+ . As in the case of Cd 2+ -insensitive channels such as WT or the single cysteine mutants E425C, D879C, the rapid current recovery of inhibition following Cd 2+ washout mostly represents the rate of displacement of Cd 2+ from the orthosteric Ca 2+ binding sites ( and ; ). On the other hand, in the Cd 2+ -sensitive double mutant E425C/D879C, the tight association between Cd 2+ and the substituted cysteines E425C and D879C mostly accounts for the much slower recovery rate ( and ; ). As a result, we used the time constant (τ) of current recovery after application of 1 μM Cd 2+ to quantitatively assess the effects of Cd 2+ crosslinking by fitting the recovering current with a standard exponential fit with the equation: (Equation 3) I ( t ) = I 0 + A ( 1 − e − t τ ) where I (t) represents the current at time point t, I 0 is the initial current amplitude, t is the time constant. Current-voltage (I-V) recordings For I-V measurements of Q645A mutant channels, we used the steady state currents obtained during the depolarization steps for analysis due to the rapid deactivation at hyperpolarization and the intrinsically low open probability of the Q645A mutant in the absence of Ca 2+ binding to the orthosteric sites . The calculated conductance from each test voltage step measured in the absence of Ca 2+ (first recording) was normalized to the peak conductance measured at +140 mV in the presence of 100 μM Ca 2+ (second recording). The resulting normalized G/G max -V curves were fit with a Boltzmann equation: (Equation 4) G G max = 1 1 + e − z F ( V m − V 1 / 2 ) R T where G/G max is the normalized channel conductance, z is the number of equivalent gating charges, V m is the applied membrane voltage, V 1/2 is the estimated voltage at which G/G max is 0.5, F is the Faraday’s constant, R is the gas constant, and T is the absolute temperature. Because of the low open probability of the Q645A-containing mutants in 0 Ca 2+ , their G/G max -V curves did not go beyond 0.5 . We therefore did not extract the V 1/2 values for further comparisons. Double mutant cycle analysis To assess the potential energetic coupling between the charged residues at the third Ca 2+ site, we extracted the Ca 2+ EC 50 values from our Ca 2+ -dependent dose-response measurements of single and double mutations of the third Ca 2+ site in the QA/EK/DR background. Because we were able to reliably measure the Ca 2+ dose-response curves of K428A/D879A(QA/EK/DR) and K428A/D884A(QA/EK/DR), the K428-D879 and K428-D884 residue pairs were further analyzed using double mutant cycle analysis by calculating the free energy change associated with a perturbation . For each pair of residues, namely X and Y, the free energy change associated with their perturbations via single or double mutations was calculated using the following equations: (Equation 5) Δ G X Y > X ′ Y = R T * I n ( E C 50 ( X ′ Y ) ) E C 50 ( X Y ) ) ( or Δ G ( 1 ) ) (Equation 6) Δ G X Y ′ > X ′ Y ′ = R T * I n ( E C 50 ( X ′ Y ′ ) E C 50 ( X Y ′ ) ) ( or Δ G ( 4 ) ) (Equation 7) Δ G X Y > X Y ′ = R T * I n ( E C 50 ( X Y ′ ) E C 50 ( X Y ) ) ( or Δ G ( 2 ) ) (Equation 8) Δ G X ′ Y > X ′ Y ′ = R T * I n ( E C 50 ( X ′ Y ′ ) E C 50 ( X ′ Y ) ) ( or Δ G ( 3 ) ) where XY denotes intact residues on the background; X’Y, XY’, X’Y’ denote mutations at X, Y, and both, respectively; EC 50 is the estimated half-maximal activation concentration of Ca 2+ . Note that the background refers the QA/EK/DR construct on which single and double mutations of the third site residue pairs are made. In principle, ΔG (1) and ΔG (4) are related and represent the free energy change as a result of mutation of X (or X’) on the background channel (ΔG (1) ) or on the Y’ background (ΔG (4) ). Similarly, ΔG (2) and ΔG (3) represent the free change associated with Y’ mutations on background and X’ background, respectively. If X and Y residues are energetically coupled, the free energy change associated with the double mutations–X’ mutation on Y’ background (ΔG (4) ) or Y’ mutation on X’ background (ΔG (3) )–would differ from that associated with the single mutations X’ and Y’, or ΔG (1) and ΔG (2) , respectively. Please also refer to – for the schematic diagrams of our double mutant cycle analyses. To obtain the standard error for the interaction energy (ΔΔG int ), the individually calculated free energy change (ΔG (s) ) values were used as: (Equation 9) Δ Δ G i n t = Δ G ( 4 ) − Δ G ( 1 ) = Δ G ( 3 ) − Δ G ( 2 ) The calculated ΔΔG int values were subsequently confirmed using the equation: (Equation 10) Δ Δ G i n t = R T * I n ( E C 50 ( X ′ Y ′ ) ⋅ E C 50 ( b g ) E C 50 ( X ′ Y ) ⋅ E C 50 ( X Y ′ ) ) Structural and sequence analysis PDB coordinate files were obtained from the Protein Data Bank ( https://www.rcsb.org/ ). Protein structure figures were prepared in Pymol (Schrödinger, Inc.). Protein sequence alignment was done in UniProt ( https://www.uniprot.org/align/ ). Statistical analysis Statistical analyses were performed in Prism software (GraphPad). Two-tailed Student’s t test was used for comparisons between two groups (paired or unpaired), and one-way ANOVA was used for comparisons between multiple data groups. Comparisons with p values < 0.05 are deemed to be statistically significant. Data in summary bar graphs are represented as mean ± standard deviation (SD) or mean ± standard error of the mean (s.e.m.), and each data point represents an independent recording. *, **, *** and **** represent statistical significance corresponding to p value < 0.05, < 0.01, < 0.001, < 0.0001, respectively. 

2+ -dependent channel activation For WT and mutant channels with intact orthosteric Ca 2+ sites, the Ca 2+ -elicited steady-state currents were normalized to the peak current (see ). For TMEM16A mutant channels with impaired orthosteric Ca 2+ sites (QA/EK/DR containing constructs), the basal current right after each Ca 2+ application was subtracted from the steady-state Ca 2+ -elicited current to avoid the issue with changing basal current, and these Ca 2+ -sensitive current components (see , , and ) were then normalized to the peak Ca 2+ -sensitive current (see , , and ). For rundown-corrected Ca 2+ -dependent measurements of the QA/EK/DR mutant , the Ca 2+ -sensitive current of each test Ca 2+ concentration was normalized to its respective reference peak current elicited by 500 μM Ca 2+ , and these reference peak-normalized values of the test Ca 2+ concentrations were used for constructing the dose-response curves. In all cases, the sigmoidal Ca 2+ dose-response curves were similarly constructed by fitting the normalized Ca 2+ -elicited currents using a non-linear regression curve fit with the equation: (Equation 1) 1 I max = 1 1 + E C 50 Ca 2 + H where I/I max is the normalized Ca 2+ -elicited current, [Ca 2+ ] is the applied free Ca 2+ concentration, H is the Hill coefficient, and EC 50 is the estimated concentration of Ca 2+ at which I/I max is 0.5. The EC 50 values from individual recordings are extracted for statistical analyses. 

2+ metal bridging experiments The extent of Cd 2+ -mediated inhibition of TMEM16A in the presence of 100 μM Ca 2+ was determined as: (Equation 2) Current ratio  = I C d 2 + I peak × 100 where I peak and I Cd2+ denote steady-state currents measured in the presence of 100 μM or 500 μM Ca 2+ before and during Cd 2+ application, respectively. To assess the extent of current recovery following Cd 2+ treatment, we determined the apparent off-rate of Cd 2+ . As in the case of Cd 2+ -insensitive channels such as WT or the single cysteine mutants E425C, D879C, the rapid current recovery of inhibition following Cd 2+ washout mostly represents the rate of displacement of Cd 2+ from the orthosteric Ca 2+ binding sites ( and ; ). On the other hand, in the Cd 2+ -sensitive double mutant E425C/D879C, the tight association between Cd 2+ and the substituted cysteines E425C and D879C mostly accounts for the much slower recovery rate ( and ; ). As a result, we used the time constant (τ) of current recovery after application of 1 μM Cd 2+ to quantitatively assess the effects of Cd 2+ crosslinking by fitting the recovering current with a standard exponential fit with the equation: (Equation 3) I ( t ) = I 0 + A ( 1 − e − t τ ) where I (t) represents the current at time point t, I 0 is the initial current amplitude, t is the time constant. 

For I-V measurements of Q645A mutant channels, we used the steady state currents obtained during the depolarization steps for analysis due to the rapid deactivation at hyperpolarization and the intrinsically low open probability of the Q645A mutant in the absence of Ca 2+ binding to the orthosteric sites . The calculated conductance from each test voltage step measured in the absence of Ca 2+ (first recording) was normalized to the peak conductance measured at +140 mV in the presence of 100 μM Ca 2+ (second recording). The resulting normalized G/G max -V curves were fit with a Boltzmann equation: (Equation 4) G G max = 1 1 + e − z F ( V m − V 1 / 2 ) R T where G/G max is the normalized channel conductance, z is the number of equivalent gating charges, V m is the applied membrane voltage, V 1/2 is the estimated voltage at which G/G max is 0.5, F is the Faraday’s constant, R is the gas constant, and T is the absolute temperature. Because of the low open probability of the Q645A-containing mutants in 0 Ca 2+ , their G/G max -V curves did not go beyond 0.5 . We therefore did not extract the V 1/2 values for further comparisons. 

To assess the potential energetic coupling between the charged residues at the third Ca 2+ site, we extracted the Ca 2+ EC 50 values from our Ca 2+ -dependent dose-response measurements of single and double mutations of the third Ca 2+ site in the QA/EK/DR background. Because we were able to reliably measure the Ca 2+ dose-response curves of K428A/D879A(QA/EK/DR) and K428A/D884A(QA/EK/DR), the K428-D879 and K428-D884 residue pairs were further analyzed using double mutant cycle analysis by calculating the free energy change associated with a perturbation . For each pair of residues, namely X and Y, the free energy change associated with their perturbations via single or double mutations was calculated using the following equations: (Equation 5) Δ G X Y > X ′ Y = R T * I n ( E C 50 ( X ′ Y ) ) E C 50 ( X Y ) ) ( or Δ G ( 1 ) ) (Equation 6) Δ G X Y ′ > X ′ Y ′ = R T * I n ( E C 50 ( X ′ Y ′ ) E C 50 ( X Y ′ ) ) ( or Δ G ( 4 ) ) (Equation 7) Δ G X Y > X Y ′ = R T * I n ( E C 50 ( X Y ′ ) E C 50 ( X Y ) ) ( or Δ G ( 2 ) ) (Equation 8) Δ G X ′ Y > X ′ Y ′ = R T * I n ( E C 50 ( X ′ Y ′ ) E C 50 ( X ′ Y ) ) ( or Δ G ( 3 ) ) where XY denotes intact residues on the background; X’Y, XY’, X’Y’ denote mutations at X, Y, and both, respectively; EC 50 is the estimated half-maximal activation concentration of Ca 2+ . Note that the background refers the QA/EK/DR construct on which single and double mutations of the third site residue pairs are made. In principle, ΔG (1) and ΔG (4) are related and represent the free energy change as a result of mutation of X (or X’) on the background channel (ΔG (1) ) or on the Y’ background (ΔG (4) ). Similarly, ΔG (2) and ΔG (3) represent the free change associated with Y’ mutations on background and X’ background, respectively. If X and Y residues are energetically coupled, the free energy change associated with the double mutations–X’ mutation on Y’ background (ΔG (4) ) or Y’ mutation on X’ background (ΔG (3) )–would differ from that associated with the single mutations X’ and Y’, or ΔG (1) and ΔG (2) , respectively. Please also refer to – for the schematic diagrams of our double mutant cycle analyses. To obtain the standard error for the interaction energy (ΔΔG int ), the individually calculated free energy change (ΔG (s) ) values were used as: (Equation 9) Δ Δ G i n t = Δ G ( 4 ) − Δ G ( 1 ) = Δ G ( 3 ) − Δ G ( 2 ) The calculated ΔΔG int values were subsequently confirmed using the equation: (Equation 10) Δ Δ G i n t = R T * I n ( E C 50 ( X ′ Y ′ ) ⋅ E C 50 ( b g ) E C 50 ( X ′ Y ) ⋅ E C 50 ( X Y ′ ) ) 

PDB coordinate files were obtained from the Protein Data Bank ( https://www.rcsb.org/ ). Protein structure figures were prepared in Pymol (Schrödinger, Inc.). Protein sequence alignment was done in UniProt ( https://www.uniprot.org/align/ ). 

Statistical analyses were performed in Prism software (GraphPad). Two-tailed Student’s t test was used for comparisons between two groups (paired or unpaired), and one-way ANOVA was used for comparisons between multiple data groups. Comparisons with p values < 0.05 are deemed to be statistically significant. Data in summary bar graphs are represented as mean ± standard deviation (SD) or mean ± standard error of the mean (s.e.m.), and each data point represents an independent recording. *, **, *** and **** represent statistical significance corresponding to p value < 0.05, < 0.01, < 0.001, < 0.0001, respectively. 

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