Electrical Phenotypes of Calcium Transport Mutant Strains of a Filamentous Fungus, Neurospora crassa

ABSTRACT We characterized the electrical phenotypes of mutants with mutations in genes encoding calcium transporters—a mechanosensitive channel homolog (MscS), a Ca2+/H+ exchange protein (cax), and Ca2+-ATPases (nca-1, nca-2, nca-3)—as well as those of double mutants (the nca-2 cax, nca-2 nca-3, and nca-3 cax mutants). The electrical characterization used dual impalements to obtain cable-corrected current-voltage measurements. Only two types of mutants (the MscS mutant; the nca-2 mutant and nca-2-containing double mutants) exhibited lower resting potentials. For the nca-2 mutant, on the basis of unchanged conductance and cyanide-induced depolarization of the potential, the cause is attenuated H+-ATPase activity. The growth of the nca-2 mutant-containing strains was inhibited by elevated extracellular Ca2+ levels, indicative of lesions in Ca2+ homeostasis. However, the net Ca2+ effluxes of the nca-2 mutant, measured noninvasively with a self-referencing Ca2+-selective microelectrode, were similar to those of the wild type. All of the mutants exhibited osmosensitivity similar to that of the wild type (the turgor of the nca-2 mutant was also similar to that of the wild type), suggesting that Ca2+ signaling does not play a role in osmoregulation. The hyphal tip morphology and tip-localized mitochondria of the nca-2 mutant were similar to those of the wild type, even when the external [Ca2+] was elevated. Thus, although Ca2+ homeostasis is perturbed in the nca-2 mutant (B. J. Bowman et al., Eukaryot. Cell 10:654–661, 2011), the phenotype does not extend to tip growth or to osmoregulation but is revealed by lower H+-ATPase activity.

N eurospora crassa has a relatively concise genome in which duplicate genes are few (11,12). The ready availability of knockout mutants for many of the known genes and open reading frames (ORFs) (9) simplifies the analysis of phenotypes associated with the absence of specific genes. This is of especial utility for studies of transport physiology, including explorations of the role of Ca 2ϩ transport in hyphal morphogenesis. The central role of calcium in the life of the cell is well known (2,7). In fungi, cytoplasmic Ca 2ϩ is maintained at a very low level of 0.1 to 0.2 M (29,36). Ca 2ϩ elevation induces signal transduction by Ca 2ϩ -dependent kinases, Ca 2ϩ /calmodulin, and calcineurin (45). Mutants of Ca 2ϩ signaling components affect morphogenesis (15,33), and hyphal tip growth requires a cytoplasmic tip-high Ca 2ϩ gradient (39).
Calcium signaling in N. crassa is considered unique in that the genome sequence does not reveal the presence of gene families common in other clades-inositol-1,4,5-trisphosphate and ryanodine receptors and ADP ribosyl cyclase-that play central roles in calcium signaling via release from internal stores (4,11). Overall, there are approximately 50 Ca 2ϩ signaling proteins, 20 Ca 2ϩ transport proteins, and about 20 Ca 2ϩ /calmodulin-regulated proteins (45) in N. crassa. A larger number of calcium transporters have been reported in Aspergillus species (46 channels, 97 cotransporters, and 105 ATPases) (1). Bowman et al. (5,6) undertook an extensive analysis of four of the major calcium transporters in N. crassa: cax, nca-1, nca-2, and nca-3. Briefly, cax encodes a calcium transporter located on the vacuolar membrane. nca-1, nca-2, and nca-3 encode Ca 2ϩ -ATPases located on the endoplasmic reticulum (nca-1) and both the vacuolar and plasma membranes (nca-2 and nca-3) (5). Among these four calcium transporters, only the nca-2 mutant exhibits elevated intracellular calcium levels and growth inhibition by elevated extracellular calcium levels (6). Some of these transporters may affect transport at the plasma membrane, so we undertook an electrophysiological character-ization of Ca 2ϩ transporter mutants in N. crassa (the cax, nca-1, nca-2, and nca-3 mutants, as well as nca-2-containing double mutants). Besides the Ca 2ϩ transport mutants, we also examined the mechanosensitive channel MscS (14). The MscS gene (NCU09595) was identified on the basis of a BLAST search as one of two MscS homologs (the other is NCU04207, no knockout of which is available). It contains a conserved region of the MscS superfamily (pfam00924), a Ca 2ϩ -binding EF-hand motif, and 7 predicted transmembrane segments.
N. crassa has been used extensively for electrophysiological characterizations since the first microelectrode measurements of its potential (40). Much of the potential is generated by the activity of a plasma membrane H ϩ -ATPase (16). A relation between the electrical properties of the plasma membrane and defects in Ca 2ϩ transport could arise from two sources. One is the contribution to the potential by electrogenic Ca 2ϩ transport at the plasma membrane. The other is an indirect phenotype of Ca 2ϩ regulation of ion-transporting systems at the plasma membrane, including the H ϩ -ATPase. Our objective was to determine what effects, if any, Ca 2ϩ transport mutants had on electrical properties. We present evidence that two mutants-the MscS and nca-2 mutants-do affect electrical transport at the plasma membrane.

Strains.
The genotypes of the strains used in the present study are shown in Table 1 (35) and about 130 M in YPD (3). In YPD plus 1 mM BAPTA, the free Ca 2ϩ concentration is about 100 nM (25). A plug of agar containing mycelium from the strains was placed in the center of the agar dishes and was grown in the dark at 28°C. Growth measurements were taken every 1 to 2 h, and the growth rates were calculated as colony diameter increases (in centimeters per hour). The same growth protocol was used to examine the effects of Zn 2ϩ (1, 5, or 10 mM ZnCl 2 ), Li ϩ (10, 20, or 40 mM LiCl), and Mg 2ϩ (25,200, or 400 mM MgCl 2 ) (all in YPD) on the nca-2 mutant.
For assessment of the effect of elevated cytoplasmic Ca 2ϩ concentrations on growth, an agar plug of mycelia was placed at the edge of a YPD plate onto which the Ca 2ϩ ionophore A23187 was spotted (10 l from a 9.5 mM stock in ethanol) (25). Growth was recorded using time lapse photography under constant light at room temperature (22 to 24°C). The time lapse data were reviewed to determine the effect on growth as the colony edge of the strains grew through the circular zone containing the ionophore.
To assess osmosensitivity, strains were plated onto YPD plates containing NaCl (0.1 to 1.2 M). A plug of agar containing mycelium from the strains was placed in the center of the agar dishes and was grown in darkness at 28°C. Growth measurements were taken every 1 to 2 h, and the growth rates were calculated as colony diameter increases (in centimeters per hour).
Since the growth of the nca-2 mutant is sensitive to elevated extracellular Ca 2ϩ concentrations (unlike that of the other mutants examined in this study [see Results]), the effects of Ca 2ϩ antagonists and agonists on the growth of the nca-2 mutant were examined. Small (30- (20). Growth at the colony edge was viewed with a 10ϫ objective on a Zeiss Axioskop 2 microscope (Carl Zeiss Canada, Ontario, Canada) and was imaged using 1-min time lapse photography with an Orca C4742-95 camera (Hamamatsu Photonics KK, Japan) using OpenLab software (version 3.1.7; Improvision, Coventry, United Kingdom). The fungus was treated with inhibitors by perfusion with 2 ml of OM containing 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8) (200 M), thapsigargin (100 M), cyclopiazonic acid (100 M), or 2-aminoethoxydiphenyl borate (2-APB) (50 M) (37). Growth rates of individual hyphae (usually 4 to 5 per treatment) were measured for 10 min before and after inhibitor treatment using ImageJ (34).
All growth experiments were run in triplicate.

Preparation of cultures for electrical experiments.
Conidia from stock cultures were transferred to a sheet of cellophane dialysis tubing (2.5 by 6 cm) (molecular weight cutoff, 12,000 to 14,000) laid on top of a VM agar plate. Inoculation was carried out along one side of the plate. The cultures were incubated overnight in the dark at 28°C. A section of the cellophane (1 by 3 cm) that included the growing edge of the colony was cut, transferred to the inside of a 30-mm culture dish lid, and fixed in the center of the dish by using thin strips of masking tape. The dish was then flooded with 3 ml of buffer solution (BS) containing (in millimolar concentrations) KCl (10), CaCl 2 (1), MgCl 2 (1), sucrose (133), and morpholineethanesulfonic acid (MES) (10); the pH was adjusted to 5.8 using 5 N KOH. After the dish was flooded, the colony edge was viewed to ensure that the hyphae had recommenced growth prior to electrical measurements. The measurements of wild-type and mutant strains were normally interspersed during experimental runs.
Electrical measurements. Single-barrel micropipettes were fabricated from borosilicate glass capillaries (outside diameter [o.d.], 1.0 mm; inside diameter [i.d.], 0.58 mm, with filament; Friedrich and Dimmock Inc., Millville, NJ) using a model P-30 micropipette puller (Sutter Instrument Co., Novato, CA). Both micropipettes were filled with a 3 M KCl solution and were inserted into microelectrode holders containing a Ag/AgCl electrode. The microelectrodes were connected to IE-251A electrometers (input impedance, 10 11 ⍀; Warner Instruments). The microelectrodes had tip resistances of about 20 to 30 M⍀. The circuit was completed with a salt bridge containing 3 M KCl in 2% (wt/vol) agar and a Ag/AgCl electrode. Large hyphae (15 to 20 m in diameter) with visible cytoplasmic flow (21) were selected for electrophysiological measurements. Dual impalements were conducted with a separation distance of 10 to 20 m (Fig. 1). After the second impalement, a voltage clamp protocol was performed using an operational amplifier configured for voltage clamp that was controlled by a data acquisition board (Scientific Solutions, Solon, OH) (22). The clamp protocol was a bipolar stepwise staircase of alternating positive and negative voltage clamps and resting potential with a clamp duration of 200 ms (the clamp duration ensured that clamping currents were at steady state). During the last 10 ms of the voltage clamp, both the clamping current and the clamped voltage (to ensure clamping fidelity) were sampled. For current-voltage measurements, sample sizes ranged from 10 to 15 separate preparations.
To obtain the current density (in milliamperes per square meter), the cable properties (32) of the hypha must be taken into account (Fig. 1) . At increasing distances from the site of current injection, the voltage change is attenuated exponentially, as expressed by the equation where V x is the voltage at distance x, V 0 is the voltage at the current injection site, x is the distance, and is the length constant. For the cylindrical geometry of the hypha, the current density (I m ) can be calculated as I/(2d), where I is the clamping current, d is the diameter of the impaled hypha, and is the length constant of the hyphae. Based on direct measurements of voltage attenuation along hyphae under the same preparation and measurement conditions (22), a length constant of 407 m was used to calculate the current density. Clamping currents at the extreme negative and positive voltages were occasionally clipped (so that the voltage could not be clamped with fidelity). The clipped clamping currents were discarded prior to estimating conductances (in millisiemens per square meter) from the slopes of linear regression fits of the currentvoltage relations. One concern that arises in quantifying current densities is the effect of multiple impalements on membrane resistance. That is, impalements may cause shunt resistances at the sites of impalement that attenuate voltage measurements because of significant voltage dividing. When dual impalements are performed, there can be a noticeable decrease in the measured potential. The effect was quantified as the ratio of the final potential (after the second impalement) to the initial potential (after the first impalement). The average ratio was 0.90 Ϯ 0.03 (n ϭ 26). The effect of the impalements will differ depending on the care taken during the impalement and the degree of robustness of recovery by the impaled hyphae, but the effect is fairly small, so no attempt was made to correct for shunt resistance. It does, however, lead to higher clamping currents and thus to an overestimate of current density by approximately 10%. Turgor measurements. Cultures for turgor measurements (23, 24) were prepared similarly to those for electrical measurements. Sections of mycelium grown overnight on cellophane dialysis tubing were cut, transferred to the lid of a 30-mm culture dish, and flooded with 3 ml of BS. Micropipettes were prepared with a large aperture tip and were filled with a silicone oil of low viscosity (polydimethylsiloxane, 1.5 centistokes; Dow Corning, Midland, WI). The micropipette was connected to a holder that, in turn, was connected to a micrometer-driven piston with thick-wall Teflon tubing. A pressure transducer (XT-190-300G; Kulite Semiconductor, Leonia, NJ) was mounted on the holder. Upon impalement of the hypha with the micropipette, the silicon oil/cell sap meniscus moved back from the tip. The pressure required to move the meniscus back to the micropipette tip was used as a measure of turgor. Hyphal mass flow (21) normally continued during the measurements, indicative of hyphal health. About 6 to 8 measurements (over 6 min) were made on a single hypha by drawing the meniscus back and then returning it to the micropipette tip (to ensure that the tip had not become plugged). Measurements for the wild type and the nca-2 mutant were normally interspersed during the experimental runs.
Ca 2؉ flux measurements. Cultures for Ca 2ϩ flux measurements were prepared similarly to those for electrical and turgor measurements. However, the mycelium was grown between two sheets of cellophane dialysis tubing overlaying YPD agar plates. This ensured that fluxes were being measured from a flat planar source. YPD contains about 0.13 mM Ca 2ϩ (3); thus, the mycelia would be Ca 2ϩ replete prior to the flux measurements so as to enhance our ability to measure the net efflux of Ca 2ϩ directly. Sections of mycelium grown overnight were cut, gently lifted with a small spatula, transferred to the lid of a 30-mm culture dish, taped down with masking tape, and flooded with 3 ml of OM. OM contains lower levels of Ca 2ϩ than YPD (increasing the signal-to-noise ratio for flux measurements) and has been used for previous Ca 2ϩ flux measurements (20).
The technique for measuring net ion fluxes (the scanning ion-selective electrode technique [SIET]) was recently described by Nguyen and Donini (31). The external steady-state diffusive gradient created by solute transport across the plasma membrane is measured by sampling the solute concentrations at two positions, one near and one far from the cell, with the ion-selective microelectrode. The flux can be calculated from the difference in concentration (10,17,22,42).
The micropipettes were pulled from borosilicate tubes (catalog no. TW150-4; World Precision Instruments, Sarasota, FL) to form a tip aperture of 5 to 8 m. The micropipettes were silanized by exposure to N,Ndimethyltrimethylsilylamine vapors after heating to 300°C. After cooling, the micropipette was filled with 100 mM CaCl 2 , and the tip was dipped in a Ca 2ϩ -selective cocktail (Ca 2ϩ ionophore I cocktail A; catalog no. 21048; Sigma-Aldrich) to form a column of 150 to 300 m. The microelectrode was mounted on the headstage of an ion/polarographic amplifier (IPA-2; Applicable Electronics, Forestdale, MA) using a AgCl/Ag half-cell. The reference electrode was 500 mM KCl in 2% agar connected to ground with a AgCl/Ag half-cell. Calibrations were performed with CaCl 2 with [Ca 2ϩ ] of 0.01, 0.1, and 1.0 mM. Slopes were 29.6 Ϯ 0.9 mV per decade difference in [Ca 2ϩ ] (n ϭ 4).
The ion-selective electrode was positioned as close as possible to the (D) after second impalement. Arrows in the sequence from A through D show the movement of a spherical particle through the hypha due to mass flow (21). Bar, 10 m. (Bottom) The equivalent electrical circuit of the hyphal segment. Due to the long cylindrical shape of the cell, the cell membrane resistances are in series. Thus, clamping currents are increasingly attenuated with increasing distance from the site of impalements as current passes through the plasma membrane into the external, grounded medium (22). Corrections for the cable properties of the hypha are described in Materials and Methods. mycelium and 50 m away to measure the diffusive gradient of Ca 2ϩ caused by ion flux at the surface of the mycelium. The differences in ion concentration were converted into a flux by assuming steady-state conditions and a planar source. Fluxes were corrected for background by measuring regions of the cellophane dialysis tubing into which the mycelium had not yet grown. To correct the area so as to account for spaces between hyphae, a threshold was applied to digital images of the mycelium in order to distinguish interhyphal space from the hyphae, and the hyphal area was measured using ImageJ (34). Measurements for the wild type and the nca-2 mutant were normally interspersed. Free [Ca 2ϩ ] after flooding of the mycelium with OM ranged from 2 to 50 M and tended to be higher for measurements of the nca-2 mutant (33 Ϯ 17 M) (n ϭ 3) than for the wild type (11 Ϯ 8 M) (n ϭ 3), possibly due to the reported elevated Ca 2ϩ concentration in the nca-2 mutant (6). Imaging of mitochondria. Tip-localized mitochondria were imaged using MitoFluor Red 589 (18). Overnight cultures grown on YPD plus agar were flooded with 2 ml of OM (containing the dye), and growing hyphae at the colony edge were imaged with a 40ϫ water immersion objective on a Zeiss Axioskop 2 microscope. Fluorescence was viewed with Zeiss filter set 15 (excitation band-pass filter, 546 nm; emission long-pass filter, 590 nm). Images were taken every 10 s with an Orca C-4742-95 camera (Hamamatsu Photonics KK, Japan) using OpenLab software (version 3.1.7; Improvision, Coventry, United Kingdom). The growing hyphae were treated with 125 mM CaCl 2 in OM by perfusing 2 ml of the solution through the dish, and imaging was recommenced as soon as possible (within 45 s of the beginning of perfusion). Statistical analysis. Statistics are shown as means Ϯ standard deviations (SD) (with the sample size in parentheses) (SD was used as an estimator of population variance) unless stated otherwise. Independent two-tailed or one-tailed t tests were performed in Excel (Mi-crosoft). Nonlinear regressions were performed in KaleidaGraph (Synergy Software, Reading, PA).

RESULTS
Much of our phenotypic analysis focused on electrical characterization of the calcium transport mutants, but we extended our characterization to include some aspects of growth and morphology. In general, all of the knockout mutants exhibited normal morphology both for leading hyphae at the colony edge and for the hyphal network within the mycelium. No differences in cell size or in hyphal cytology were observed.
Growth of calcium transport mutants. The effects of Ca 2ϩ depletion (by addition of the Ca 2ϩ chelator BAPTA at 1 mM) and excess Ca 2ϩ on colony growth were examined for all single and double mutants ( Fig. 2A). Under normal conditions (YPD), the nca-2 nca-3 and nca-2 cax double mutants grew more slowly than other mutant strains and the wild type. Ca 2ϩ depletion with BAPTA caused slight growth inhibition for all strains (including the wild type). Growth recovered when 1.1 mM CaCl 2 was added to ensure that the effect was due to Ca 2ϩ depletion rather than to some other effect of BAPTA addition. The growth of the nca-2 mutant and of all double mutants that included the nca-2 deletion was strongly inhibited by elevated Ca 2ϩ concentrations, confirming the results of Bowman et al. (5).
To determine if the inhibition of growth at elevated Ca 2ϩ concentrations was an osmotic effect, the effect of NaCl was examined for a subset of the mutants (the cax, MscS, nca-1, nca-2, and nca-3 mutants) (Fig. 2B). All exhibited osmotic sensitivity similar to that of the wild type up to 1.2 M NaCl. Thus, the Ca 2ϩ sensitivity of the nca-2 mutant is not due to an osmotic effect. Furthermore, any disruptions of Ca 2ϩ transport and homeostasis in these mutants does not affect their response to osmotic stress.
Given the Ca 2ϩ sensitivity of the nca-2 mutant, the effects of other ions on its growth were examined (Fig. 2C). Its Li ϩ and Zn 2ϩ sensitivities were similar to those of the wild type. There was a notable (and significant [P ϭ 0.016]) increase in the growth of the nca-2 mutant at 25 mM Mg 2ϩ that was not observed in the wild type.
Inhibitors of various components of Ca 2ϩ signaling were examined (Fig. 3). Inhibition of the nca-2 mutant by an inhibitor of inositol 1,4,5-triphosphate (IP 3 )-activated Ca 2ϩ channels (2-APB) and an inhibitor of Ca 2ϩ sequestration (cyclopiazonic acid) was similar to that of the wild type. Both 2-APB and cyclopiazonic acid are known to affect Ca 2ϩ gradients at growing hyphal tips, by decreasing apical [Ca 2ϩ ] or increasing basal [Ca 2ϩ ], respectively (37). The nca-2 mutant (but not the wild type) was inhibited by thapsigargin (a Ca 2ϩ -ATPase inhibitor) (P ϭ 0.0005) and exhibited less sensitivity to TMB-8 (a Ca 2ϩ antagonist) than did the wild type (P ϭ 0.0011).
Elevated cytoplasmic Ca 2؉ sensitivity of calcium transport mutants. To assess the ability of the Ca 2ϩ transport mutants to regulate cytoplasmic Ca 2ϩ , we examined the effect of the calcium ionophore A23187 on the growth of the knockout mutants (the MscS, cax, nca-1, nca-2, and nca-3 mutants). In Aspergillus awamori, A23187 is reported to increase cytoplasmic Ca 2ϩ levels, as monitored by aequorin fluorescence (30). The techniquegrowth into an A23187-containing zone-has been used to demonstrate the sensitivity of a mechanosensitive channel mutant (the mid-1 mutant) to the Ca 2ϩ ionophore in N. crassa (25).
The mutant strain phenotypes were the same as the wild-type phenotype except for the nca-2 mutant. For all mutants (in contrast to the mid-1 mutant), growth was not arrested when the colony edge grew into the A23187-containing zone. Except for the nca-2 mutant (which grew more sparsely than the other strains), aerial hyphae were absent in a zone extending back from the A23187-containing region (Fig. 4). Thus, the nca-2 mutant exhibits no response to elevated cytoplasmic Ca 2ϩ levels, in contrast to the other Ca 2ϩ transport mutants examined.
To explore whether the lesions in transport for the various mutants were reflected in ion transport, the current-voltage properties of the mutants were examined.
Electrical properties of calcium transport mutants. To determine the relative contributions of active transport (the electrogenic H ϩ -ATPase) and passive ion distributions (the Goldman-Hodgkin-Katz potential for permeant ions), the hyphae were treated with cyanide to deplete cytoplasmic ATP, thereby inhibiting H ϩ -ATPase activity. The use of cyanide as a respiratory inhibitor in N. crassa to deplete ATP and determine the activity of the electrogenic pump (H ϩ -ATPase) at the plasma membrane is well established (13,41). Measurements of each mutant strain were interspersed with measurements of a wild-type control. The current-voltage relations are shown in Fig. 5. The resting potential and conductance measurements are shown in Fig. 6 and 7, respectively.
The current-voltage relations were fairly similar for all mutants examined (Fig. 5). Two mutants (the MscS and nca-2 mutants) had significantly depolarized resting potentials compared to that of the wild type (Fig. 6). The MscS mutant potential was Ϫ107 Ϯ 18 mV (n ϭ 16), compared to the wild-type control potential of Ϫ131 Ϯ 20 mV (n ϭ 13) (P Ͻ 0.002). The nca-2 mutant potential was Ϫ116 Ϯ 13 mV (n ϭ 15), compared to the wild-type control potential of Ϫ137 Ϯ 13 mV (P Ͻ 0.0002). The resting potential was also significantly depolarized in the nca-2 nca-3 and nca-2 cax Growth rates of individual hyphae (usually 4 to 5 per treatment) for 10 min before and after inhibitor treatment were measured using ImageJ. Data are shown as percentages of the control value (before inhibitor treatment). An inhibitor of IP 3 -activated Ca 2ϩ channels (2-APB) and an inhibitor of Ca 2ϩ sequestration (cyclopiazonic acid) are known to affect Ca 2ϩ gradients at growing hyphal tips (37) but had similar inhibitory effects on the wild type and the nca-2 mutant. The nca-2 mutant exhibited less sensitivity to TMB-8 (P ϭ 0.0011), but greater sensitivity to the Ca 2ϩ -ATPase inhibitor thapsigargin, than the wild type (P ϭ 0.0005). plugs were placed at one side of the petri dishes (arrows), and growth was recorded using a time lapse camera (under room lighting and temperature). The growth of the colony edge was not affected as it entered the A23187containing zone (circles) (squares mark the location of an ethanol-spotted control zone) for the wild type and all mutants (not shown). After the strains had grown through the A23187-containing zone, an aconidiate zone (devoid of aerial hyphae) was observed in all strains, extending radially from the A23187 zone, except for the nca-2 mutant. double mutants but not in the nca-3 cax mutant, indicating that the nca-2 knockout was responsible. The cyanide-induced change in the potential was significantly smaller for the nca-2 mutant (84 Ϯ 15 mV) than for the wild-type control (100 Ϯ 11 mV) (P Ͻ 0.003), suggesting that the cause is lower electrogenic ATPase activity (either electrogenic Ca 2ϩ -ATPase or H ϩ -ATPase [see Discussion]).
The conductances of the mutants versus that of the wild type were very similar before and after cyanide treatment. Only the nca-2 cax and nca-3 cax double mutants exhibited statistically significant differences from the wild type (P, Ͻ0.007 and Ͻ0.002, respectively), but only in the cyanide-induced change in conductance (Fig. 7).
Because the growth of the nca-2 mutant was sensitive to elevated Ca 2ϩ concentrations, the effect of elevated extracellular Ca 2ϩ concentrations on its potential was explored in greater detail. Hyphae were impaled with a single-barrel micropipette so that only the potential was monitored. Extracellular [Ca 2ϩ ] was increased by adding 0.5 ml of 0.5 M CaCl 2 (in BS) dropwise around the water immersion objective (final concentration, 71 mM). Under this elevated-Ca 2ϩ challenge, the wild type and the nca-2 mutant depolarized from initial potentials of Ϫ166 Ϯ 11 mV and Ϫ157 Ϯ 13 mV to Ϫ58 Ϯ 49 and Ϫ99 Ϯ 28 mV, respectively (n ϭ 8). For both strains, the potential then repolarized, to a level slightly more negative than the initial potential (Ϫ178 Ϯ 18 mV for the WT [n ϭ 8]; Ϫ162 Ϯ 16 mV for the nca-2 mutant [n ϭ 8]). The electrical changes (transient depolarization followed by hyperpolarization) were reminiscent of those observed after hy-perosmotic treatments with sucrose at higher concentrations (500 mM) (26), suggesting that the effect was due to CaCl 2 causing hyperosmotic stress, rather than to a Ca 2ϩ -specific effect.
Mitochondrial distributions in the wild type and the nca-2 mutant. To assess the impact of Ca 2ϩ hypersensitivity of the nca-2 mutant in greater detail, the effects of elevated extracellular Ca 2ϩ concentrations on tip growth and mitochondrial distributions at growing tips were examined. Tip-localized mitochondria play a role in Ca 2ϩ sequestration (18); therefore, mitochondrial distributions at growing hyphal tips were examined before and after treatment with elevated Ca 2ϩ concentrations (Fig. 8). Mitochondria were normally vermiform, with a higher density just behind the hyphal tip. There were no dramatic differences in the tip localization of mitochondria between the nca-2 mutant and the wild type, and elevated Ca 2ϩ concentrations did not affect distributions in either strain.
Ca 2؉ fluxes in the nca-2 mutant. One possible phenotype of the nca-2 mutant is a lesion in Ca 2ϩ export from the hyphae, since this Ca 2ϩ -ATPase is located on the plasma membrane as well as the vacuole (5). This was tested directly by measuring net ion fluxes with Ca 2ϩ -selective electrodes (scanning ion-selective electrode technique [SIET]) (31) (Fig. 9). Previous flux measurements did not identify localized regions of net Ca 2ϩ flux along growing hyphae (20). By use of OM rather than BS for flux measurements,  The P values obtained by two-tailed t tests are shown for experimental runs for which statistically significant differences were obtained. The initial potentials (circles), potentials after cyanide treatment (squares), and difference in potential (potential after cyanide minus potential before cyanide) (triangles) are shown.
background [Ca 2ϩ ] was low, increasing the signal-to-noise ratio for flux measurements. In glucose-replete OM, the potentials of the WT and the nca-2 mutant were more positive than those measured in BS (Ϫ83 Ϯ 23 and Ϫ103 Ϯ 21 mV, respectively) (n ϭ 24). The net Ca 2ϩ fluxes were corrected for Ca 2ϩ reservoirs in the cellophane used to "sandwich" the mycelium by subtracting fluxes over the cellophane layers from fluxes over the mycelium. There was no significant difference in net Ca 2ϩ flux out of the hyphae between the WT (1.11 Ϯ 0.85 pmol cm Ϫ2 s Ϫ1 ) (n ϭ 3) and the nca-2 mutant (1.76 Ϯ 1.38 pmol cm Ϫ2 s Ϫ1 ) (n ϭ 3) (P ϭ 0.534, 2-tailed t test).

DISCUSSION
The complete genomic sequence of Neurospora crassa (11) and the creation of knockout mutants (9) have revolutionized our ability to explore the roles of practically all genes in the life of the fungus. With these tools in hand, we undertook an electrophysiological characterization of Ca 2ϩ transporter mutants in Neurospora crassa-the cax, nca-1, nca-2, and nca-3 mutants-for which localization and functional phenotypes are available (5,6), as well as the mechanosensitive channel MscS mutant.
Of the four Ca 2ϩ transporter mutants, the nca-2 mutant is sensitive to extracellular Ca 2ϩ levels and contains elevated intracellular Ca 2ϩ concentrations (6). The sensitivity to extracellular [Ca 2ϩ ] was confirmed for the nca-2 mutant and for double mutants containing the nca-2 mutation (Fig. 2). Elevated Mg 2ϩ levels increased the growth of the nca-2 mutant, suggesting that divalent cation replacement alleviates the impact of elevated cytoplasmic calcium concentrations. None of the mutants exhibited a delete- mutant. Hyphae were incubated with MitoFluor Red 589 and were imaged every 10 s before and after treatment with 125 mM CaCl 2 by use of epifluorescence with a 40ϫ water immersion objective. The same hypha was imaged, but the time required for addition of the CaCl 2 and repositioning differed (8 min for the wildtype image; 2.5 min for the nca-2 mutant). For all experiments, growth rates were 9.0 Ϯ 3.3 (wild type) and 8.7 Ϯ 2.3 (nca-2 mutant) m min Ϫ1 before Ca 2ϩ treatment and 15.0 (wild type) and 3.4 (nca-2 mutant) m min Ϫ1 after Ca 2ϩ treatment. After Ca 2ϩ treatment, growth rates were 9.0 Ϯ 4.2 (n ϭ 8) (wild type) and 5.5 Ϯ 2.0 m min Ϫ1 (n ϭ 7) (nca-2 mutant). Mitochondrial distributions were similar, and elevated Ca 2ϩ treatment did not affect the distributions. type (open symbols) as a control. The P values obtained by two-tailed t tests are shown for experimental runs for which statistically significant differences were obtained. The initial conductances (circles), the conductances after cyanide treatment (squares), and the difference in conductance (conductance after cyanide minus conductance before cyanide) (triangles) are shown. rious response to hyperosmotic treatment, implying that Ca 2ϩ does not play a role in hyperosmotic responses. Inhibitors known to affect the tip-high Ca 2ϩ gradient (2-APB and cyclopiazonic acid) (37) inhibited the growth of the nca-2 mutant similarly to that of the wild type, suggesting that the mutant phenotype does not impact the Ca 2ϩ gradient requirements for hyphal tip growth. However, the mutant is more sensitive to inhibition of Ca 2ϩ -ATPase by thapsigargin, probably a consequence of even more elevation of cytoplasmic [Ca 2ϩ ]. The Ca 2ϩ antagonist TMB-8 inhibits an IP 3 -activated large conductance Ca 2ϩ channel in N. crassa (38), so the reason why it inhibits the growth of the nca-2 mutant less than that of the wild type is unknown, but this phenomenon may be due to preexisting elevated intracellular Ca 2ϩ concentrations in the nca-2 mutant.
Our research on the electrical properties of the Ca 2ϩ transport mutants provides an analysis of mutant phenotypes that complements the work of Bowman et al. (5,6). For all of the mutant strains, the effects on the electrical properties of the hyphae could be direct or indirect. If the transporter contributed to electrogenic transport at the plasma membrane, its absence could affect the resting potential and/or conductance. Indirect effects would be due to modifications in calcium signaling that, in turn, modulate electrogenic transport at the plasma membrane. It has been reported that elevated cytoplasmic Ca 2ϩ concentrations activate the plasma membrane H ϩ -pump (19). Ca 2ϩ signaling pathways (mediated, for example, by IP 3 , calmodulin, and calcineurin) that regulate electrogenic transport could be affected in the calcium transport mutants due to dysfunctional Ca 2ϩ homeostasis. The overall electrical potential (E m ) of the hypha depends on contributions from both the H ϩ -ATPase current and passive ion distributions across the membrane. This can be summarized by the equation E m ϭ I pump /g m ϩ E G , where I pump /g m is the H ϩ -ATPase current divided by the conductance and E G is the Goldman-Hodgkin-Katz potential for permeant ions (43). The conductance (g m ) is a measure of the overall electrogenic flux across the plasma membrane, which was unaffected in all of the mutants. We tested directly for an effect on the Goldman-Hodgkin-Katz potential caused by ion distributions across the membrane versus an effect on the H ϩ -ATPase current by using the metabolic inhibitor cyanide to deplete ATP (8). Cyanide causes the potential to depolarize to the level of the Goldman-Hodgkin-Katz potential.
For the two mutants with aberrant electrical properties, the MscS and nca-2 mutants, the normal membrane potential is depolarized relative to that of the wild type, but conductance is unaffected. The net Ca 2ϩ efflux measured in the nca-2 mutant (nca-2 encodes a Ca 2ϩ -ATPase) was similar to that for the wild type, so a direct effect on Ca 2ϩ transport at the plasma membrane is unlikely. The depolarized potential after cyanide treatment was the same for the nca-2 mutant as for the wild type and was significantly more positive for the MscS mutant. Thus, there is less ATPdependent electrogenic pumping in the nca-2 mutant. Since Ca 2ϩ efflux is unaffected, the phenotypic effect of the nca-2 deletion on electrical properties is an effect on H ϩ -ATPase activity rather than a direct effect of the absence of the Ca 2ϩ -ATPase. For the MscS mutant, the phenotypic effect is likely an effect on passive ion distributions across the plasma membrane that would affect the Goldman-Hodgkin-Katz potential.
Tip growth in Neurospora relies on a tip-high Ca 2ϩ gradient (39). The elevated Ca 2ϩ level at the tip is generated from internal stores and is sequestered behind the tip by tip-localized mitochondria (18), among other mechanisms. In the nca-2 mutant, hyphal tip morphology and tip-localized mitochondrial distributions are unaffected, even by elevated external Ca 2ϩ concentrations. Bowman et al. (6) reported that the internal Ca 2ϩ concentration in the nca-2 mutant is elevated, but it appears that the tip-high Ca 2ϩ gradient can still be maintained, allowing growth, albeit at a rate slightly lower than that of the wild type.
Overall, in combination with the results of Bowman et al. (5,6), our results reveal a more complicated view of Ca 2ϩ regulation than would be expected, one in which Ca 2ϩ transport mutants affect other transport processes at the plasma membrane, but this is true only for the nca-2 mutant, which exhibits lower H ϩ -ATPase activity, and the MscS mutant, which exhibits a depolarized Goldman-Hodgkin-Katz potential. To what extent these phenotypes are due to modulation of the expression of other genes in the knockout mutants, or to other, unknown mechanisms regulating Ca 2ϩ homeostasis, remains to be explored.

ACKNOWLEDGMENTS
We are grateful to Sandra Khine, an undergraduate biophysics student who assisted us both with experiments and with cable analysis. We thank Andrew Donini for his generous willingness to let us perform the ion flux measurements in his laboratory; they were crucial to the successful completion of our research project. We thank Barry J. Bowman for providing us with the double mutants.
This project was funded with a Discovery Grant (to R.R.L.) from the Natural Sciences and Engineering Research Council of Canada. . This was corrected for by subtracting the "flux" measured above the cellophane in an area without mycelium from the fluxes measured above the mycelial network (the correction ranged from 8 to 29% of mycelial flux). The fluxes were normalized to the relative area filled with hyphae in the regions being sampled. (C) The net fluxes of Ca 2ϩ from the hyphae were outward (22) and were similar for the wild type and the nca-2 mutant.