Obligate coupling of CFTR pore opening to tight nucleotide-binding domain dimerization
The cystic fibrosis transmembrane conductance regulator ( CFTR ) is a chloride ion channel which belongs to the superfamily of ATP binding cassette ( ABC ) proteins ( Riordan et al . , 1989 ) . Following phosphorylation of the cytosolic regulatory ( R ) domain by cAMP-dependent protein kinase ( PKA ) , CFTR channels are gated by cytosolic ATP ( Figure 1a ) . The ion pore within the TMDs opens upon formation of a tight head-to-tail NBD dimer stabilized by two molecules of ATP occluded at the dimer interface ( Vergani et al . , 2005 ) ; each nucleotide is sandwiched between the conserved Walker A and B sequences of one NBD and the signature sequence of the other ( Smith et al . , 2002; Chen et al . , 2003 ) . Pore closure accompanies NBD dimer disruption prompted by ATP hydrolysis at the composite interfacial site harbouring the Walker motifs of NBD2 ( site 2 ) ( Vergani et al . , 2005 ) : the other site ( site 1 ) is catalytically inactive retaining ATP bound throughout multiple gating cycles ( Aleksandrov et al . , 2002; Basso et al . , 2003 ) . As evidence of such strict coupling between NBD and transmembrane domain ( TMD ) movements , a hydrogen bond between the side chains of NBD2 Walker-A threonine ( T1246 ) and an arginine ( R555 ) adjacent to the NBD1 signature sequence was shown to form in open , but not in closed , channels ( Vergani et al . , 2005 ) . These two positions have co-evolved as a pair ( Vergani et al . , 2005 ) , and in crystal structures of nucleotide-bound ABC NBD dimers the analogous side chains also form a hydrogen bond , the arginine serving as hydrogen donor and the threonine/serine side chain as acceptor ( Smith et al . , 2002; Chen et al . , 2003 ) . Furthermore , by disturbing proper spacing between the donor and acceptor sites , both shortening of the R555 and lengthening of the T1246 side chains ( by single mutations R555K and T1246N , respectively ) prevent , whereas the double mutation R555K-T1246N restores , the formation of this stabilizing hydrogen bond ( Vergani et al . , 2005 ) . 10 . 7554/eLife . 18164 . 003Figure 1 . The P355A-K978C double mutation increases both spontaneous and ATP-dependent Po . ( a , c ) Inward macroscopic ( a ) WT and ( c ) P355A-K978C CFTR currents at −80 mV , and their dependence on ATP and PKA ( black bars ) . In ( a ) a 30 s segment of spontaneous current ( gray box ) is magnified in the inset . In ( c ) bath chloride ( green bars ) was repeatedly replaced by gluconate ( red bars ) to determine baseline current ( gray dashed line ) . ( b ) Two alternative mechanisms of spontaneous gating in cartoon representation . TMDs ( gray ) , TMD-NBD interface formed by intracellular loops ( light violet ) , NBD1 ( green ) , NBD2 ( blue ) , membrane ( yellow ) . ( d ) Spontaneous Po ( solid bars ) and Po in 2 mM ATP ( striped bars ) for WT and P355A-K978C ( 'background' , BG ) CFTR ( n = 5–21 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 18164 . 00310 . 7554/eLife . 18164 . 004Figure 1—figure supplement 1 . Replacement of bath chloride with gluconate abolishes CFTR currents at -80 mV . Normalized macroscopic current-voltage relationships of WT CFTR under chloride ( out ) /chloride ( in ) ( green symbols ) and chloride ( out ) /gluconate ( in ) ( red symbols ) ionic conditions ( n = 6–9 ) . For both ionic conditions macroscopic steady CFTR currents in 2 mM ATP , and background currents in the absence of ATP , were sampled at membrane voltages ranging from −80 to +80 mV , and ATP-dependent currents normalized to that measured at +80 mV . The Red solid line is a fit to the Goldman-Hodgkin-Katz equation , the green solid line is a linear interpolation between data points . Note mild inward rectification in symmetrical chloride ( Cai et al . , 2003 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 18164 . 00410 . 7554/eLife . 18164 . 005Figure 1—figure supplement 2 . High ATP-dependent Po due to background mutation facilitates counting channels . ( a–b ) , Open probabilities in 2 mM ATP , following PKA removal , for WT CFTR and for the four constructs that form the mutant cycle . Po was estimated from the last ~2-minutes of the 2 . 5-minute segments of recording in 2 mM ATP , bracketing the 5-minute ATP-free period during which spontaneous activity was sampled ( n = 6–17 ) . ( a ) Front bracket , ( b ) rear bracket ( see Figure 2a–d ) . DOI: http://dx . doi . org/10 . 7554/eLife . 18164 . 005 Wild-type ( WT ) CFTR channels open occasionally even in the absence of ATP ( Szollosi et al . , 2010 ) ( Figure 1a , inset ) , but it is unclear whether this reflects the occasional spontaneous formation of the NBD dimer ( Figure 1b , top ) , or occasional pore openings independent of NBD dimerization ( Figure 1b , bottom ) . The significance of this question goes beyond the scarcity of WT spontaneous openings . Indeed , the strictness of coupling between NBD and TMD movements is a strongly debated fundamental question , key for understanding the forces that drive the normal , ATP-dependent functional cycle of ABC proteins ( Vergani et al . , 2005; Csanády et al . , 2010; Kirk and Wang , 2011; Jih and Hwang , 2013; Okeyo et al . , 2013 ) . Moreover , ivacaftor , the only potentiator drug to be approved so far for clinical use in CF patients , acts by promoting such spontaneous , ATP-independent , openings of G551D CFTR , the third most common disease mutant ( Bompadre et al . , 2007; Jih et al . , 2013 ) . However , mechanistic understanding of spontaneous gating has been limited by its vanishingly small open probability ( Po ) , far too low to be reliably quantified ( Figure 1a ) . Recently , mutations in the third intracellular loop ( position 978 , between TM helices 8 and 9 ) ( Wang et al . , 2010 ) , and at the cytosolic end of pore-lining TM helix 6 ( position 355 ) ( Wei et al . , 2014 ) , were both found to strongly enhance spontaneous activity . Furthermore , these 'gain-of-function' effects proved additive in the double-mutant P355A–K978C , shifting spontaneous Po into a range where quantitative biophysical studies become tractable ( Wei et al . , 2014 ) . Here we exploit this double mutant as a background to address the mechanism of spontaneous openings .
Whereas WT CFTR channel currents decline rapidly to baseline upon ATP removal ( Figure 1a ) , in patches containing P355A-K978C CFTR channels ( Figure 1c ) a readily measurable fraction of the chloride current resisted even prolonged removal of bath ATP , but could be abolished by removal of permeating chloride ions ( red bars; see also Figure 1—figure supplement 1 ) . Moreover , for WT CFTR , ATP-dependent currents are very small prior to exposure to PKA , and decline by ~50% when PKA is withdrawn ( Figure 1a ) , signalling strong phosphorylation dependence of its Po ( Szollosi et al . , 2010 ) . In contrast , ATP-evoked P355A-K978C CFTR currents were large already before exposure to PKA , and were little further enhanced by the addition of – or reduced following removal of – the kinase ( Figure 1c ) , suggesting a large Po for unphosphorylated ( pre-PKA ) double-mutant channels in ATP , and close-to-maximal Po when the channels were either fully phosphorylated ( in PKA ) or partially dephosphorylated ( post-PKA ) . The robust spontaneous activity of P355A-K978C channels in the absence of ATP allowed reliable sampling of the characteristics of spontaneous gating in microscopic patches ( Figure 2a ) , in which the number of active channels could be confidently estimated from bracketing high-Po segments of recordings in ATP ( Figure 1—figure supplement 2 ) . Thus , for pre-phosphorylated channels the P355A-K978C background mutation increased spontaneous Po from 0 . 000053 ± 0 . 000021 ( n = 5 ) to 0 . 15 ± 0 . 03 ( n = 21 ) ( Figure 1d , WT vs . BG [background] ) , and Po in ATP from 0 . 11 ± 0 . 04 ( n = 6 ) to 0 . 80 ± 0 . 02 ( n = 14 ) ( Figure 1d , striped bars ) . 10 . 7554/eLife . 18164 . 006Figure 2 . Arg 555 and Thr 1246 become energetically coupled upon spontaneous pore opening . ( a–d ) , Microscopic currents at −80 mV , and their dependence on ATP and PKA ( black bars ) , for ( a ) the P355A-K978C background construct , and for this background construct carrying ( b ) the R555K or ( c ) T1246N single mutations or ( d ) the R555K-T1246N double mutation . ( e–g ) , Spontaneous open probabilities ( e , n = 19–21 ) , opening rates ( f , n = 15–19 ) , and closing rates ( g , n = 15–19 ) of the background construct ( black bars ) , and of constructs carrying additional R555K ( blue bars ) , T1246N ( red bars ) , or R555K-T1246N ( violet bars ) mutations . ( h–j ) , Thermodynamic mutant cycles showing mutation-induced changes ( h ) in the stability of the spontaneous open state relative to the closed state , and ( i–j ) in the height of the activation free energy barriers for spontaneous opening ( i ) and closure ( j ) . Each corner is represented by the side chains at positions 555 and 1246; k , Boltzmann's constant , T , absolute temperature . DOI: http://dx . doi . org/10 . 7554/eLife . 18164 . 006 This double-mutant CFTR background allowed us to study , using the thermodynamic double mutant cycle formalism ( Materials and methods ) , changes in energetic coupling associated with spontaneous gating between the same two NBD-interface side chains previously used ( Vergani et al . , 2005 ) to demonstrate NBD dimer formation and disruption in CFTR channels opening and closing in ATP . Mutating either the arginine at position 555 to a lysine , or the threonine at position 1246 to an asparagine , resulted in a large reduction in spontaneous Po ( Figure 2b–c 0 . 034 ± 0 . 007 ( n = 18 ) and 0 . 044 ± 0 . 009 ( n = 19 ) , respectively , Figure 2e , blue and red bar ) , that is , in the closed-open equilibrium constant in the absence of ATP . This reduction in the stability of the open ( O ) state relative to the closed ( C ) state ( ΔΔG ( O−C ) ∘ = +1 . 82 ± 0 . 39 kT and +1 . 54 ± 0 . 39 kT , respectively , in R555K and T1246N single mutants; Figure 2h , numbers on top-across , and left-downward , arrows of the mutant cycle ) is due to the loss in both single mutants of microscopic interactions , between the targeted side chain and the rest of the protein , that in WT channels combine to increase the stability of the open state . If the side chains of residues R555 and T1246 did not interact in WT CFTR , or if their interaction were to remain unaltered during spontaneous gating , then any impact on the spontaneous channel activity of mutating either single position should be independent of the nature of the side chain found at the other position: that is , mutational effects at the two positions should add energetically . Any non-additive behaviour would signal gating-associated dynamic changes in the interaction energy between the R555 and T1246 side chains . In stark contrast to the single mutants , the double mutant R555K-T1246N ( Figure 2d ) displayed a high spontaneous Po ( 0 . 11 ± 0 . 02 ( n = 20 ) ; Figure 2e , violet bar ) , comparable to that of the background construct . Thus , the effects on the spontaneous Po of either single mutation depended strongly on the nature of the residue at the other position: the spontaneous open-channel state was destabilized relative to the closed-channel state when the R555K mutation was introduced into a WT background ( blue vs . black bar in Figure 2e ) , but was stabilized when the same mutation was introduced into a T1246N background ( violet vs . red bar in Figure 2e ) . The difference between ΔΔG ( O−C ) ∘ along two parallel sides of the mutant cycle quantifies the change in interaction strength between the two target side chains that accompanies a spontaneous pore opening ( ΔΔGint ( O−C ) ; see Materials and methods ) , and revealed a significant ( p = 0 . 0004 ) change of –2 . 90 ± 0 . 49 kT ( Figure 2h , center ) in R555-T1246 interaction energy between ATP-free closed- and open-channel states . In principle , the negative sign of ΔΔGint ( O−C ) is compatible with the existence of either a stabilizing interaction in the spontaneously formed open-channel state , or a destabilizing interaction in the closed state . However , earlier work reported a lack of interaction between the R555 and T1246 side chains in closed channels , regardless of the presence of ATP in site 2 ( Vergani et al . , 2005 ) . Thus , the only plausible explanation is a stabilizing interaction , consistent with the formation of a hydrogen bond , in the spontaneous open state in WT channels that is lost in both single mutants , but is restored in the double mutant . The inevitable implication of these findings is that the NBD dimer interface must tighten within site 2 upon pore opening , whether or not bound ATP is present . Conformational transitions between stable open-channel ( burst ) and closed-channel ( interburst ) states of CFTR reflect passages across the transition ( T‡ ) state ( Figure 3a ) , a high free-energy transient conformation . The rates of spontaneous opening and closure of each molecular channel type , obtained as the inverse of the mean interburst ( τib ) and burst ( τb ) durations , respectively , report on transition-state stability: mutation-induced fractional changes in spontaneous opening and closing rates quantitate changes in transition-state free energy relative to the closed ( ΔΔG ( T−C ) ∘ ) and open ( ΔΔG ( T−O ) ∘ ) ground states , respectively . Specifically , the difference between such ΔΔG∘ values along two parallel sides of a mutant cycle quantify the changes in coupling strength between the two target side chains upon reaching the transition state during opening ( ΔΔGint ( T−C ) , Figure 2i ) or closure ( ΔΔGint ( T−O ) , Figure 2j ) . Kinetic analysis revealed that the reduced spontaneous open probabilities of both single mutants R555K and T1246N ( Figure 2e , blue and red bar ) were caused predominantly by a markedly reduced opening rate ( Figure 2f , blue and red bar ) , which was restored in the double mutant ( Figure 2f , violet bar ) . Consequently , a significant ( p = 0 . 0001 ) negative value of –2 . 30 ± 0 . 46 kT ( Figure 2i , center ) was obtained for the change in coupling free energy between the target side chains upon reaching the transition state from the closed state ( ΔΔGint ( T−C ) ) . In contrast , closing rates were little affected by the mutations ( Figure 2g ) , yielding a value of +0 . 62 ± 0 . 34 kT for ΔΔGint ( T−O ) , not significantly different from zero ( Figure 2j , center ) . These results imply that in WT channels the hydrogen bond between the R555 and T1246 side chains is already formed in the transition state for spontaneous opening , its strength being little further altered when the protein reaches the stable open state ( Figure 3b ) . Thus , the NBD dimer interface tightens within site 2 already in the transition state for unliganded opening ( Figure 3a , center ) , just as it does during the opening of ATP-bound channels ( Vergani et al . , 2005; Sorum et al . , 2015 ) . 10 . 7554/eLife . 18164 . 007Figure 3 . Mechanism of spontaneous pore openings , and energetic effects of bound ATP and gain-of-function mutations . ( a ) Cartoon representation of domain organization in closed ( C ) , transition ( T‡ ) , and open ( O ) states , during spontaneous gating . Color coding as in Figure 1b , blue and red circles identify target positions . ( b ) Changes in energetic coupling between the target positions associated with spontaneous gating in the background construct . ( c ) Free energy profiles of gating for WT ( orange line ) and P355A-K978C ( black line ) CFTR in the absence of ATP , and of hydrolysis-deficient K1250R CFTR in saturating ATP ( gray line ) ( Vergani et al . , 2005 ) . Black and gray downward arrows illustrate the energetic effects of the background double mutation and of the presence of ATP bound in both composite sites . DOI: http://dx . doi . org/10 . 7554/eLife . 18164 . 007
While ATP-driven CFTR pore openings are known to be coupled to the formation of a tight NBD dimer ( Vergani et al . , 2005 ) , the mechanism of spontaneous openings , observable in the absence of ATP , is unknown . Here we show that spontaneous openings are also linked to tightening of the NBD interface around composite site 2 , just as in the presence of bound ATP . Such strict coupling between NBD and pore movements – regardless of the absence or presence of ATP – strongly constrains mechanistic models of ATP-dependent channel gating , and understanding of the underlying driving forces , which are likely conserved among ABC proteins . It also helps explain modulatory effects of mutations , or of physiological or pharmacological gating regulators . For example , the G551D CF mutation converts ATP at site 2 into an inhibitory ligand , due to electrostatic repulsion between the γ-phosphate of ATP bound to the Walker motifs , and the introduced negative charge in the signature sequence ( Lin et al . , 2014 ) . Such an effect would not be expected if the opposing NBD interfaces did not approach each other in the open-channel state , but is readily explained by openings being strictly linked to NBD dimer formation even in the G551D mutant . Furthermore , although reported low-probability ATP-independent pore openings of a CFTR construct lacking NBD2 ( CFTR residues 1–1197; [Wang et al . , 2007] ) can clearly not be linked to NBD dimer formation , the present results argue that upon pore opening its remaining NBD-TMD coupling machinery undergoes movements similar to those that accompany NBD dimerization when neither 'limb' is truncated . Because of the tightened NBD interface , the ATP binding sites of open CFTR channels are likely inaccessible to the bulk solution even in the absence of bound ATP , just as in its presence ( Chaves and Gadsby , 2015 ) , suggesting that ATP binding/unbinding can take place only in the closed-channel state . Two immediate corollaries ensue . First , the ATP-free open-channel state is unlikely to be an integral component of the ATP-dependent gating cycle ( Szollosi et al . , 2010; but , cf . Jih et al . , 2013 ) . Second , despite suggested similarities ( Wang et al . , 2010; Kirk et al . , 2011; Okeyo et al . , 2013 ) , the principles that drive gating of CFTR channels are fundamentally different from those of ligand-gated channels , such as the nicotinic acetylcholine receptor . Ligand-gated channels obey cyclic equilibrium mechanisms in which ligand binding/unbinding can occur both in the closed- and the open-channel state , and the activating effect of the ligand is based on the thermodynamic principle of detailed balance which constrains the product of the equilibrium constants around a cycle to be unity: thus , opening ( 'isomerization' ) is driven by the concurrent conformational change of the ligand binding site which increases its affinity for the ligand ( Grosman and Auerbach , 2001 ) . But in CFTR the ligand binding site is occluded in open channels , so that an open-state 'binding affinity' cannot be defined ( the rates for both ATP binding and unbinding are zero ) . Thus , even when disruption of ATP hydrolysis forces CFTR channels to gate at equilibrium , that gating follows a reversible linear sequence ( cf . , Figure 3a ) , rather than a cycle subject to the principle of detailed balance . Strong correlation between effects on unliganded and liganded channel activity , and energetic additivity of such effects , has been reported for a range of diverse 'allosteric modulators' acting on various regions of the channel . For instance , both unliganded and liganded Po is increased in a physiological context by R-domain phosphorylation , presumably by altering R-domain interactions with the TMDs ( Wang et al . , 2010 ) , or in a therapeutic context by the CF drug ivacaftor , believed to target the TMDs ( Jih et al . , 2013 ) . Further , distinct mutations in intracellular loops increase ( Wang et al . , 2010; Wei et al . , 2014 ) or decrease ( Wang et al . , 2014 ) Po for both unliganded and liganded CFTR . The present results suggest a simple , unified explanation for all these findings: energetic stabilization or destabilization of the inherent open-channel-state structure ( Figure 3a , right ) , which we show here is similarly organized in the absence or presence of ATP . Comparison of gating parameters for WT CFTR and our background mutant reveals the mechanism by which gain-of-function mutations promote spontaneous activity ( Figure 3c , black vs . orange free energy profiles ) . The increase in spontaneous Po by the P355A-K978C double mutation ( Figure 1d , BG vs . WT ) amounts to a stabilization of the spontaneous open state by ~8 . 1 kT ( Figure 3c , right black arrow ) , and is due not only to an increased frequency of openings but also to a slower rate of closure , because spontaneous τb is substantially prolonged ( from 60 ± 9 ms ( n = 5 ) to 1566 ± 235 ms ( n = 19 ) ) , suggesting an increase in ΔΔG ( T−O ) ∘ of ~3 . 3 kT relative to WT . Together , these data imply that these gain-of-function mutations stabilize the transition state by far less ( only by ~4 . 8 kT; Figure 3c , left black arrow ) than the open state , as expected for perturbations that affect protein positions which have not yet reached their fully open-like conformations in the transition state . This is consistent with a similar finding for an NBD-TMD interface position during ATP-dependent opening ( Sorum et al . , 2015 ) . Thus , just as for ATP-bound channels , the pore is likely still closed in the high-energy evanescent transition state for spontaneous openings ( Figure 3a , center cartoon ) . Finally , what can we infer about the mechanism by which binding of the natural ligand ATP promotes CFTR channel opening ? The presence of bound ATP does not alter the global arrangement of conformations: rather ATP acts as a 'molecular glue' to stabilize the transition state and the open state . Because ATP-induced openings are terminated by ATP hydrolysis ( Csanády et al . , 2010 ) , energetic interpretation of ATP-binding induced gating effects cannot be based on the comparison of WT gating in the presence and absence of ATP ( Figure 1d , WT+ATP vs . WT ) . However , comparing gating parameters for spontaneous activity of WT CFTR and ATP-driven activity of hydrolysis-incompetent mutant CFTR channels offers some insight into the impact of ATP binding on gating energetics ( Figure 3c , gray vs . orange free energy profiles ) . Mutations that disrupt ATP hydrolysis in site 2 all prolong τb and reduce CFTR gating to an equilibrium , by forcing the channels to close back through the same high-energy transition state they had traversed upon opening ( cf . Figure 3a ) ( Vergani et al . , 2005; Csanády et al . , 2010; Sorum et al . , 2015 ) . For instance , comparison of Po and τb of the site 2 Walker-A mutant K1250R CFTR in saturating ATP ( Po ~ 0 . 6 , τb = 9323 ms [Vergani et al . , 2005] ) with those of spontaneously gating WT CFTR suggests that the presence of bound ATP stabilizes the open-channel state by ~10 . 3 kT ( Figure 3c , right gray arrow ) and increases ΔΔG ( T−O ) ∘ by ~5 . 1 kT , respectively , which implies that bound ATP stabilizes the transition state by only ~5 . 2 kT ( Figure 3c , left gray arrow ) . Thus , intriguingly , although the NBD interface is already tightened within site 2 in the ATP-bound transition state ( Vergani et al . , 2005; Sorum et al . , 2015 ) , the 'molecular glue' effect of bound ATP is further strengthened when the channel reaches the stable open state . The implication is that some rearrangements must still occur within the dimer interface between the transition state and the open state . One possibility is that , in the transition state , site 1 has not yet reached its full open-channel-like conformation . Perhaps the site 2 'glue' bonds fully already in the transition state , whereas bonding of the site 1 'glue' is completed only in the open-channel state . Further work will be needed to address the precise timing of site 1 motions during channel opening , and their contributions to CFTR gating energetics .
Mutations introduced ( Stratagene , QuikChange ) into pGEMHE-CFTR were confirmed by automated sequencing; cDNA was transcribed in vitro ( Ambion , mMessage T7 ) and cRNA stored at −80°C . Oocytes were isolated from anaesthetized adult female Xenopus laevis frogs following Institutional Animal Care Committee guidelines , injected with 0 . 1–10 ng cRNA in a fixed 50 nl volume , and stored at 18°C as described ( Szollosi et al . , 2010 ) . Current recordings were obtained 1–3 days after injection . The patch pipette solution contained ( in mM ) : 136 NMDG-Cl , 2 MgCl2 , 5 HEPES , pH = 7 . 4 with NMDG . Bath solution contained ( in mM ) : 134 NMDG-Cl , 2 MgCl2 , 5 HEPES , 0 . 5 EGTA , pH = 7 . 1 with NMDG , and was freshly supplemented with 3 mM DTT to prevent thiol oxidation of the cysteine side chain engineered into position 978 . MgATP ( 2 mM ) was added from a 400 mM aqueous stock solution ( pH = 7 . 1 with NMDG ) . The catalytic subunit of PKA ( 300 nM , Sigma ) was applied for ~1 min to activate CFTR channels . Experiments were done at 25°C . Currents were recorded at a membrane potential of −80 mV , digitized at 10 kHz , and recorded to disk after on-line Gaussian filtering at 2 kHz . For macroscopic recordings of P355A-K978C CFTR , baseline current was estimated by brief replacements of bath chloride ions with gluconate ( Figure 1c , red bars ) ; at −80 mV no CFTR currents are observable under such ionic conditions ( Figure 1—figure supplement 1 ) . For microscopic recordings channels were pre-phosphorylated by PKA , and gating in the absence of ATP was observed for 5 min , bracketed by 2 . 5 min exposures to ATP . The number of active channels was estimated based on the high-Po bracketing segments ( cf . , Figure 1—figure supplement 2 ) . To allow for loss of ATP even from the higher affinity site 1 , which retains ATP for tens of seconds ( Tsai et al . , 2010 ) , spontaneous gating parameters were extracted from the last ~4 min stretches of the 5 min ATP-free segments; precise starting points for the quasi-steady segment to be analyzed were chosen in each patch based on visual inspection . Segments of current recording were digitally filtered at 50 Hz , and idealized by half-amplitude threshold crossing . Open probabilities in the absence ( Figure 2e ) and presence ( Figure 1—figure supplement 2 ) of ATP were calculated from the events lists as the time-average of the fraction of open channels . Spontaneous channel opening and closing rates were calculated as described ( Vergani et al . , 2005 ) , from ATP-free segments containing no more than 7 superimposed channel openings . A closed-open-blocked ( C-O-B ) kinetic scheme , which separates brief ( ~10 ms ) flickery closures ( to state B ) from long ( >1 s ) interburst closures ( to state C ) , was fitted by maximum likelihood to the set of dwell-time histograms for all conductance levels , to obtain microscopic transition rates rCO , rOC , rOB , and rBO , while accounting for a fixed dead time of 6 ms ( Csanády , 2000 ) . Mean burst ( τb ) and interburst ( τib ) durations were calculated as τb= ( 1/rOC ) ( 1+rOB/rBO ) and τib=1/rCO , and channel opening and closing rates defined as 1/τib and 1/τb , respectively . Changes in interaction strength between pairs of residues associated with various steps of the gating cycle were estimated using mutant cycle analysis , as described previously ( Vergani et al . , 2005 ) . For a conformational transition from state A to state B of the protein , energetic coupling ( ΔΔGint ) between positions 1 and 2 is defined as the difference between mutation-induced changes in the stability of state B relative to state A along parallel sides of the mutant cycle , i . e . , ( 1 ) ΔΔGint=ΔΔGB−A0 ( X′Y→X′Y′ ) −ΔΔGB−A0 ( XY→XY′ ) =ΔΔGB−A0 ( XY′→X′Y′ ) −ΔΔGB−A0 ( XY→X′Y ) Here the superscripts refer to the pairs of residues found at target positions 1 and 2 of the four studied constructs ( e . g . , XY: residue X at position 1 and residue Y at position 2; XY': residue X at position 1 and residue Y' at position 2; etc . ) , and arrows represent substitutions in a particular background ( e . g . , XY→XY': mutation of position 2 from Y to Y' , with X at position 1 ) . If states A and B are stable ( 'ground' ) states of the protein , then the energetic stability of state B relative to state A can be calculated from the measured equilibrium constant ( Keq ) as ΔGB−A0=−kTlnKeq ( k is Boltzmann's constant , T is absolute temperature ) , and hence the two terms in Equation 1 are obtained as follows:ΔΔGB−A0 ( X′Y→X′Y′ ) =ΔGB−A0 ( X′Y′ ) −ΔGB−A0 ( X′Y ) =−kTln ( Keq ( X′Y′ ) /Keq ( X′Y ) ) , andΔΔGB−A0 ( XY→XY′ ) =ΔGB−A0 ( XY′ ) −ΔGB−A0 ( XY ) =−kTln ( Keq ( XY′ ) /Keq ( XY ) ) . In this study , we used the equilibrium constant between the spontaneous closed- and open-channel state , Keq=Po/ ( 1−Po ) ( Figure 2h ) . Analogously , mutation-induced changes in activation free energy barriers , calculated from fractional changes in transition rates , can also be substituted into Equation 1 . For instance , for the transition A→T‡→B ( T‡ is the transition state ) the two terms for Equation 1 are obtained as follows:ΔΔGT−A0 ( X′Y→X′Y′ ) =−kTln ( rAB ( X′Y′ ) /rAB ( X′Y ) ) , andΔΔGT−A0 ( XY→XY′ ) =−kTln ( rAB ( XY′ ) /rAB ( XY ) ) , where rAB is the transition rate from state A to state B . In this study , we calculated mutation-induced changes in activation free energy barriers for channel opening ( ΔΔGT−C0 ) and closing ( ΔΔGT−O0 ) from the fractional changes in opening and closing rates , respectively ( Figure 2i–j ) . All ΔΔGs are given as mean ± SEM . Estimates of SEM for ΔΔGo values were obtained as follows . All ΔΔG values are functions of the form ΔΔG=kTln ( u/v ) of two independent random variables u and v which represent a specific gating parameter ( Keq , rCO , or rOC ) of two different channel constructs . Assuming that u and v are normally distributed with mean m ( mu , mv ) and variance σ2 ( σu2 , σv2 ) , the variance of ln ( u/v ) is given byvar ( ln ( u/v ) ) =∫−∞∞∫−∞∞ln2 ( u/v ) ⋅12π⋅e− ( u−mu ) 22σu2⋅e− ( v−mv ) 22σv2dudv− ( ∫−∞∞∫−∞∞ln ( u/v ) ⋅12π⋅e− ( u−mu ) 22σu2⋅e− ( v−mv ) 22σv2dudv ) 2 The above integrals cannot be performed analytically , but the functions ln ( u/v ) and ln2 ( u/v ) can be expanded into power series around ( mu , mv ) . Taking the terms only up to second order , the integrals can be performed , giving the approximationvar ( ln ( u/v ) ) ≈σu2mu2+σv2mv2+12⋅σu2mu2⋅σv2mv2−14⋅ ( σu4mu4+σv4mv4 ) The power series approximations hold as long as σu≪mu , σv≪mv; thus , for σu=0 . 2mu , σv=0 . 2mv this estimate of the variance for ln ( u/v ) is still quite accurate , and was used to obtain var ( ΔΔGB-A0 ) values . Since the numbers of observations for each pair of constructs ( e . g . , nXY and nXY' ) was similar , standard errors were calculated using the mean values for n . E . g . , SEM ( ΔΔGB−A0 ( XY→XY′ ) ) =var ( ΔΔGB−A0 ( XY→XY′ ) ) /n¯ , where n¯= ( nXY+nXY' ) /2 . Variances for energetic coupling were obtained as var ( ΔΔGint ) =var ( ΔΔGB−A0 ( X′Y→X′Y′ ) ) +var ( ΔΔGB−A0 ( XY→XY′ ) ) . Finally , since the numbers of observations for each corner of a cycle ( nXY , nXY' , nX'Y , nX'Y' ) were similar , standard errors for energetic coupling were calculated as SEM ( ΔΔGint ) =var ( ΔΔGint ) /n¯ , where n¯= ( nXY+nXY'+nX'Y+nX'Y' ) /4 . The values of ΔΔGint obtained from the above two types of mutant cycle are conventionally interpreted to reflect the change in the strength of the interaction ( in WT ) between the two target residues X and Y upon transiting from stable ground state A either to stable ground state B , or to transition state T‡ , respectively . This interpretation assumes that the native interaction between residues X and Y is destroyed in both single mutants and the double mutant ( valid , e . g . , for side chain truncations ) ( Horovitz , 1996 ) . However , if an interaction is restored in the double mutant X'Y' , then the measured ΔΔGint reports the sum of the changes in interaction strengths for residue pairs X-Y ( in WT ) and X'-Y' ( in the double mutant ) . Choosing such mutations ( e . g . , ( Vergani et al . , 2005 ) ; present study ) provides a convenient signal amplification which facilitates separating the signal from noise . All data are presented as mean ± S . E . M , estimated from current recordings obtained from n ( ≥5 ) patches as specified in each figure legend . All data were included in the analysis . Statistical significance was quantified using Student's t test , and differences are reported as significant for p<0 . 05 .
In CFTR , the chloride channel mutated in cystic fibrosis ( CF ) patients , ATP-binding-induced dimerization of two cytosolic nucleotide binding domains ( NBDs ) opens the pore , and dimer disruption following ATP hydrolysis closes it . Spontaneous openings without ATP are rare in wild-type CFTR , but in certain CF mutants constitute the only gating mechanism , stimulated by ivacaftor , a clinically approved CFTR potentiator . The molecular motions underlying spontaneous gating are unclear . Here we correlate energetic coupling between residues across the dimer interface with spontaneous pore opening/closure in single CFTR channels . We show that spontaneous openings are also strictly coupled to NBD dimerization , which may therefore occur even without ATP . Coordinated NBD/pore movements are therefore intrinsic to CFTR: ATP alters the stability , but not the fundamental structural architecture , of open- and closed-pore conformations . This explains correlated effects of phosphorylation , mutations , and drugs on ATP-driven and spontaneous activity , providing insights for understanding CF mutation and drug mechanisms .
A protein pore called the CFTR channel allows chloride ions to move through the membrane of the cells that line the airways and some other parts of the human body . Mutations in the genes that encode CFTR may reduce the number of pores at the cell surface or stop them from working properly . When this happens , these cells cannot transport enough chloride , which causes the disease cystic fibrosis . CFTR contains two regions that lie inside the cell known as nucleotide binding domains ( NBDs ) . These domains bind to the chemical energy molecule ATP , and when ATP does bind , two NBDs associate to form a dimer and the pore in CFTR opens . The CFTR channel can occasionally open in a spontaneous way that does not require ATP . However , it was not clear whether NBDs also formed dimers when CFTR opened in this way . This is because spontaneous opening could reflect NBDs occasionally forming a dimer without ATP binding or it could occur when the pore occasionally opens without the NBDs forming a dimer . To explore whether opening of the pore always requires NBD dimerization , Mihályi et al . studied the behaviour of single human CFTR channels produced in frog eggs . Normal channels and mutant ones ( which show differences in spontaneous opening ) were used , and the change in the way NBDs interacted when the channels spontaneously opened or closed was investigated . Mihályi et al . found that the NBD dimer forms when the pore spontaneously opens , demonstrating that this step happens both with and without ATP . The result demonstrates that NBD dimer formation and pore movement are strictly coupled and that this is an inbuilt property of the CFTR protein . When ATP binds , this only changes how stable the open-pore and closed-pore structures of CFTR are but does not alter the fundamental architecture of the channel . These new findings will be of interest to researchers studying a large group of transport proteins related to CFTR called ABC proteins . Furthermore , a drug called ivacaftor stimulates spontaneous opening of CFTR , and has recently been approved for clinical use to treat people with mutations in CFTR . As such , the new findings will be also useful to help researchers understand how ivacaftor stimulates the CFTR pore to open .