Proton Sensors in the Pore Domain of the Cardiac Voltage-gated Sodium Channel^*

Molecular Biology

The human variant of the pore-forming α-subunit, hNa_V1.5, in SP64T was graciously donated by Dr. Chris Ahern (University of British Columbia). hNa_V1.5 and H880Q DNA was linearized using XbaI (Invitrogen). Transcription was completed using a Sp6 mMESSAGE mMACHINE high yield capped RNA transcription kit (Applied Biosystems, Carlsbad, CA). The C373F mutant was graciously donated by Dr. Mohamed Chahine (Laval University, Canada). C373F DNA was linearized using NotI (New England BioLabs, Pickering, Canada). C373F transcription was completed using a T7 mMESSAGE mMACHINE high yield capped RNA transcription kit (Applied Biosystems). The H880Q mutant construct was generated using the QuikChange method (Stratagene, Mississauga, Canada) with primers synthesized by Sigma Genosys (Oakville, Canada). All constructs were sequenced with the use of Eurofins MWG Operon (Huntsville, AL).

Oocyte Preparation

Oocyte preparation was completed as described previously (34). Briefly, female Xenopus laevis were terminally anesthetized using tricaine solution (2 g/liter). Oocytes were surgically removed, and theca and follicular layers were enzymatically removed by ∼1-h agitation of semi-intact lobes in a calcium-free solution, containing 96 mm NaCl, 2 mm KCl, 20 mm MgCl₂, 5 mm HEPES and supplemented with 1 mg/ml type 1a collagenase. Collagenased oocytes were then washed and sorted in calcium-free solution. Stage V-VI oocytes were injected with 50 nl of cRNA encoding either WT or mutant hNa_V1.5. Injected oocytes were incubated at 19 °C in SOS+ medium containing 96 mm NaCl, 2 mm KCl, 1.8 mm CaCl₂, 1 mm MgCl₂, 5 mm HEPES, 2.5 mm sodium pyruvate, supplemented with 100 mg/liter gentamicin sulfate and 5% horse serum for 3–10 days prior to recording. All surgical and animal care procedures were completed in accordance with the policies and procedures of the Simon Fraser University Animal Care Committee and the Canadian Council of Animal Care (35).

Data Acquisition

Data were acquired as described previously (34). Briefly, recordings were made using a CA-1B amplifier in the cut-open mode. Data were low pass-filtered at 10 kHz, digitized at 50 kHz, and then recorded using Patchmaster (HEKA Electronics, Mahone Bay, Canada) running on an iMac. Cells were permeabilized via bottom bath perfusion with intracellular solution containing 9.6 mm NaCl, 88 mm KCl, 5 mm HEPES, 11 mm EGTA, titrated to pH 7.4, and supplemented with 0.1% saponin. After 1–2-min exposure, saponin-free intracellular solution was washed in. Extracellular solution of the top and middle chambers contained 96 mm NaCl, 4 mm KCl, 1.8 mm CaCl₂, 1 mm MgCl₂, and 5 mm HEPES. For recordings completed below extracellular pH 6.5, HEPES was substituted with 5 mm MES. Bath chambers were temperature-controlled at 22 °C using a Peltier device run by a TC-10 temperature controller (Dagan, Minneapolis, MN).

Pulse Protocols

Cells were maintained at a holding potential of −100 mV between all protocols. Unless otherwise stated, leak subtraction was completed online, using a −p/4 protocol from a holding potential of −100 mV. Current/voltage relationships, steady-state FI (SSFI), and FI recovery and onset were measured as described previously (34).

Prolonged versions of the FI protocols were used to measure SI. Steady-state SI (SSSI) was measured by holding the membrane potential at −150 mV for 30 s to recover channels from the slow inactivated state and then stepping to 60-s conditioning pulses between −150 and −10 mV in 20-mV increments. The membrane potential was then hyperpolarized to −150 mV for 20 ms to allow recovery of fast inactivated but not slow inactivated channels and then depolarized to a 0-mV test pulse to measure the remaining channel availability. Several cells were administered a −130 mV holding potential rather than −150 mV. Changing the holding potential from −150 to −130 mV had no effect on parameters of SSSI in any of the constructs. The rate of recovery from SI was measured by depolarizing the membrane to 0 mV for 60 s to fully slow inactivate channels. The membrane potential was then stepped to an interpulse potential (−150 mV), 0.2–30 s in duration, before measuring the recovered current with a 0-mV test pulse. To measure the rate of SI onset, cells were held at −150 mV for 30 s, stepped to an prepulse potential (0 mV) for 0–60 s, hyperpolarized to −150 mV for 20 ms to allow recovery of fast inactivated but not slow inactivated channels, and then available current was measured with a 0-mV test pulse.

Use-dependent inactivation was recorded as described previously (34). Briefly, the membrane was repetitively depolarized to 0 mV for 230 ms and hyperpolarized to −90 mV for 150 ms 500 times at a frequency of ∼2.6 Hz. The frequency, which coincides with a 156-beat/min heart rate, was sufficient to generate a measurable reduction in current due to inactivation while remaining within the range of physiological depolarization and frequency values. The ratio of depolarization to repolarization was chosen based on Bazett's formula to generate a healthy QT duration, calculated QTc = 371 (36). No leak subtraction was used in use-dependent inactivation (UDI) recordings to ensure appropriate pulse frequency.

Data Analysis

Analysis of activation, FI, and SI data were completed using Fitmaster version 2x32 (HEKA Electronics) and Igor Pro version 5.01 (Wavemetrics, Lake Oswego, OR) run on an iMac. Conductance curves were computed from current/voltage relationships using the equation,

where G represents conductance, I_max represents the peak test pulse current, V_m is the test pulse voltage, and E_rev is the measured reversal potential. Values were plotted as a function of test potential and then fitted with the Boltzmann equation,

where z represents the apparent valence, e₀ is the elementary charge, V_½ is the midpoint, T is the recording temperature in kelvin, and k is the Boltzmann constant. SSFI and SSSI data were fitted with a modified Boltzmann equation,

where I_Max and I_Min represent the maximum and minimum values of the fit, respectively. All other values are identical to those in Equation 2. The time constants of the recovery and onset of FI were calculated from the single exponential equation,

where Y₀ represents the asymptote of the fit, A is the relative component of the exponent, τ is the time constant, and x is time. The time constants of FI onset and recovery were plotted as a function of prepulse and interpulse voltage, respectively, and fitted with the Eyring model,

where τ_M represents the inverse of the maximum time constant, w is the reaction velocity, b is the barrier distance, V is the voltage, V_P is the voltage at the peak of the fit, and T is the temperature. SI onset and recovery as well as UDI data were fitted with the double exponential equation,

where Y₀ represents the asymptote of the fit, A₁ is the relative component of the first exponent, τ₁ is the slow time constant, A₂ is the relative component of the second exponent, τ₂ is the fast time constant, and x is time. Proton block data were plotted as a function of pH and were fitted with the Hill equation,

where Y_M and Y₀ represent the maximum and minimum values of the fit, respectively, X_½ is the midpoint of the curve, X is the pH, and b is the rate. Statistical analysis was completed using Student's t test and an analysis of variance, where appropriate, using JMP (SAS Institute Inc., Cary, NC). All data are reported as mean ± S.E., and statistical significance was taken at p < 0.05.

Activation and Proton Block

Reducing extracellular pH from pH 7.4 to pH 6.0 reduces single channel conductance and significantly depolarizes the midpoint (V_½) and reduces the apparent valence (z) of activation of Na_V1.5 channels (32–34). In this study, the H880Q and C373F mutants displayed a significant reduction in proton block at pH 6.0 relative to WT channels (Fig. 1). At pH 6.0, the maximum channel conductance was reduced by 38.7 ± 2.0, 27.0 ± 2.6, and 19.4 ± 2.2% relative to pH 7.4 for WT, H880Q, and C373F channels, respectively (Fig. 1, B, C, and D). To assess the profile of proton block for the three constructs, maximum conductance is plotted as a function of pH and fitted with a Hill curve (Equation 7) (Fig. 1E). The pK_a values were not significantly different between the three constructs: WT, pK_a = 5.8 ± 0.7; H880Q, pK_a = 5.7 ± 0.04; C373F, pK_a = 5.9 ± 0.1. The asymptotes of proton block, however, differed significantly from zero for H880Q and C373F (6.2 ± 1.6 and 8.5 ± 1.7%, respectively, p < 0.05, n = 4) but not WT channels (0.2 ± 2.9%).

We have previously shown that protons shift the voltage dependence of WT Na_V1.5 channel activation and SSFI and reduce the apparent valence of activation. Fig. 2 and Table 1 show that C373F and H880Q did not abolish this effect of protons. Surprisingly, the V_½ of activation and SSFI in the H880Q mutant was significantly depolarized compared with C373F and WT channels, and the apparent valence of activation in both mutants was significantly greater than that in WT channels (Fig. 2). Window currents were estimated from the overlay of paired activation and SSFI curves. Peak window current occurred over a more depolarized voltage range in H880Q than C373F and WT channels: peak window current occurred at −51.3 ± 1.8, −56.9 ± 1.1, and −60.3 ± 1.0 mV for H880Q, C373F, and WT, respectively (Fig. 2, C, D, and E). We also measured proton modulation of FI recovery (−150 through −70 mV), closed state FI (−90 through −40 mV), and open state FI (−40 through +30 mV) in all three channel types (data not shown). In each case, the results reflected the previously described trends, where acidic pH causes a depolarizing shift in gating, comparable with previous findings (34, 39), and H880Q gating is depolarized relative to WT and C373F channels. These data suggest that proton modulation of channel activation and slow inactivation are two independent processes.

Steady-state Slow Inactivation

Protons bind within the extracellular Na_V channel pore (32, 33). Given the mutual association of protons and SI at the outer turret, we hypothesized that residues within the pore mediate proton modulation of SI. We therefore measured the steady-state and kinetic parameters of SI in all three constructs with extracellular solution titrated to either pH 7.4 or pH 6.0. Fig. 3 displays SSSI recorded in WT, C373F, and H880Q channels at pH 7.4. Normalized current was plotted as a function of prepulse potential and fitted with a Boltzmann function (Equation 3). Both H880Q and C373F channels displayed an altered response to reduced extracellular pH compared with WT channels. Extracellular perfusion of pH 6.0 triggered a significant (albeit small) increase in the maximum probability of SSSI in H880Q and C373F channels (Fig. 3, C and D, and Table 2). pH 6.0 also reduced the apparent valence of SSSI in C373F but had no effect on the apparent valence of H880Q SSSI (Fig. 3, C and D, and Table 2). In contrast, reducing extracellular pH from pH 7.4 to pH 6.0 had no effect on the SSSI curve for WT channels (Fig. 3B). This correlates with previous recordings from Na_V1.5 channels expressed in oocytes (34). H880Q and C373F channels also displayed a reduced maximum probability of SSSI relative to WT channels, measured at pH 7.4 (Table 2).

Slow Inactivation Recovery

We measured proton modulation of SI recovery at −90 mV because it correlated with the hyperpolarizing pulse of our UDI protocol (discussed below) and could therefore help explain changes in UDI between the three channel constructs. In each channel, SI recovery kinetics were biexponential. Protons accelerated SI recovery kinetics in WT but not H880Q and C373F channels (Fig. 4). In WT channels, pH 6.0 significantly increased τ_slow and reduced the relative contribution of the slow component. Protons did not significantly affect τ_fast but significantly increased the amplitude of the τ_fast component (Fig. 4, A (inset) and B, and Table 3). Overall, the effects on the relative amplitudes of fast and slow decay were dominant to the changes in the fast and slow time constants, and extracellular acidosis accelerated SI recovery in WT channels (Fig. 4B). In H880Q channels, protons significantly increased τ_fast as well as its relative component but did not affect τ_slow (Fig. 4, A (inset) and C, and Table 3). The C373F mutation abolished proton modulation of SI recovery (Fig. 4, A (inset) and D, and Table 3). These data demonstrate that His-880 and Cys-373 both mediate modulation of SI recovery by protons and show that Cys-373 plays a greater role in mediating the response of SI to protons than His-880. Last, the apparent differences in recovery kinetics between constructs at pH 7.4 were not statistically significant (Fig. 4A and Table 3).

Slow Inactivation Onset

We assessed proton modulation of SI onset at 0 mV to coincide with the depolarizing pulse of our UDI protocol and could therefore help explain changes in UDI between the three channel constructs. Protons displayed differential modulation of SI onset in WT, H880Q, and C373F channels (Fig. 5 and Table 4). SI onset was slowed in WT channels at reduced extracellular pH (Fig. 5B). WT channels displayed significantly increased τ_fast and τ_slow at pH 6.0 relative to pH 7.4 (Fig. 5, B and E, and Table 4). Extracellular perfusion of pH 6.0 did not affect the relative components of SI onset (Fig. 5F and Table 4). SI onset was also slowed at pH 6.0 in the H880Q mutant, although the effect on τ_fast was significantly reduced relative to WT (Fig. 5E). pH 6.0 increased τ_fast in WT channels by 8.0 ± 2.4 ms but increased τ_fast by only 1.1 ± 0.4 ms in H880Q channels. Conversely, C373F abolished proton modulation of τ_fast and τ_slow; however, pH 6.0 significantly increased the relative component of τ_slow (Fig. 5, D–F, and Table 4). Protons did not significantly affect the asymptote of SI onset in any of the constructs. These data demonstrate that His-880 and Cys-373 both mediate proton modulation of SI onset, and, as is the case for SI recovery, Cys-373 plays a greater role in mediating the response of SI to protons than His-880.

The asymptote of SI onset at pH 7.4 was significantly different between the WT, H880Q, and C373F constructs (Fig. 5A and Table 4). Additionally, H880Q and C373F channels displayed a reduced τ_slow component relative to WT channels (Table 4). The differences in the SI onset asymptote between channel constructs are probably attributable to the observed reductions in the components of τ_slow in H880Q and C373F.

Use-dependent Inactivation

Protons display profound isoform-specific modulation of UDI (39). UDI in Na_V1.4 is pH-insensitive, whereas in Na_V1.5, UDI is destabilized by protons. Inserting the p-loops of the skeletal muscle isoform, Na_V1.4, into the Na_V1.5 channel backbone abolishes proton modulation of UDI (40). We measured UDI using a pulse protocol designed to mimic a human ventricular action potential, as described previously (34). Cells were depolarized to 0 mV for 230 ms and then hyperpolarized to −90 mV for 150 ms to simulate an elevated heart rate of ∼160 beats/min. This stimulation frequency is high enough to induce readily measurable levels of UDI but low enough to remain physiologically relevant. Peak currents from 500 consecutive pulses were recorded and normalized to the first pulse and then plotted as a function of time and fitted with a double exponential equation (Equation 6) as in Fig. 6.

In WT channels (Fig. 6A), we observed the previously described destabilization of UDI in response to reduced extracellular pH (34, 39). WT channels displayed a significant increase in the asymptote of UDI at pH 6.0 relative to pH 7.4 (Fig. 6A and Table 5). The C373F mutation removed the effects of protons on the asymptote of UDI but preserved proton modulation of τ_Fast (Fig. 6C). C373F channels also displayed a significantly reduced τ_Fast compared with WT (Table 5). H880Q completely abolished the effects of protons on UDI (Fig. 6, B, D, and E, and Table 5).

Last, both C373F and H880Q significantly reduced the relative components of τ_Slow compared with WT under control conditions (Table 5). The C373F mutant also displayed a significantly elevated UDI asymptote compared with WT and H880Q channels (Fig. 6 and Table 3). These data correlate well with the reduced maximum probability of SI observed in C373F and H880Q channels (Figs. 4 and 5).

Molecular Determinants of Proton Block

Unlike Na_V1.5, the skeletal muscle isoform, Na_V1.4, is not fully blocked by protons (32, 33). Na_V1.4 channels display a pH-insensitive current that is roughly ∼14% of maximum conductance (33). In Na_V1.4, replacement of Tyr-402 with a cysteine (the homologous residue in Na_V1.5) abolishes the proton-insensitive current (32). The reverse mutation in Na_V1.5, C373Y, imparts a proton-insensitive current similar to that seen in Na_V1.4 (32). Replacement of Tyr-402 with a phenylalanine or a serine, the equivalent residues in Na_V1.2 and Na_V1.8, respectively, does not alter the pH-insensitive current, suggesting that Na_V1.2 and Na_V1.8 would have a proton block asymptote similar to that of Na_V1.4 (32, 39). Kahn et al. (32) suggested that protonation of Cys-373 along with the outer pore carboxylates Glu-375, Glu-901, Asp-1423, and Asp-1714 imparts proton block in Na_V1.5 and that the absence of this pore cysteine in other Na_V channels results in a pH-insensitive current.

Here we report that two novel mutations, C373F and H880Q, alter Na_V1.5 channel proton block. Both mutations significantly reduced the degree of proton block observed at pH 6.0 relative to WT channels and displayed asymptotes of proton block that represented a pH-insensitive component of current (Fig. 1). C373F proton block data fitted with a Hill curve (Equation 7) displayed a pH-insensitive conductance that was roughly 9% of maximum conductance (Fig. 1E), consistent with previous results from the C373Y Na_V1.5 mutant (32). H880Q also displayed a proton block asymptote that was significantly different from zero at ∼6% of maximum conductance (Fig. 1E). His-880 has not been implicated previously in Na_V channel proton block, and it is interesting that H880Q proton block was intermediate between that observed in C373F and WT channels. Kahn et al. (33) hypothesized that protonation at the p-loop increases the electrostatic potential within the Na_V channel outer vestibule, thereby repelling Na⁺ ions. Our results further support that hypothesis. Cys-373 is adjacent to the selectivity filter, where the electrostatic potential is the most negative. His-880, however, is predicted to be at the top of the Na_V channel P1 helix of DII (Fig. 7). Based on their relative locations, it is understandable that the effect of the H880Q mutation would be less dramatic than that observed by the C373F mutation.

Pore Mutants Destabilize Slow Inactivation

Na_V1.2 channels inactivate more completely than Na_V1.5; during prolonged depolarization, more than 65% of Na_V1.2 channels slow inactivate compared with ∼50% in Na_V1.5 (8, 14, 34, 42). Because the homologous position to Na_V1.5 Cys-373 is Phe-385 in Na_V1.2 channels, and because SI is known to be sensitive to mutations within the external pore, we predicted that the C373F mutant would confer a stabilization of slow inactivated states in Nav1.5 (Fig. 7) (8, 14, 32). Because His-880 is highly conserved (Fig. 7), we also predicted that the H880Q mutation would disrupt Na_V1.5 SI. Interestingly, both H880Q and C373F destabilized the slow inactivated state. The maximum probability of SSSI was reduced by ∼10% in H880Q and C373F channels relative to WT (Table 2). Additionally, compared with WT channels, the asymptote of SI onset measured at 0 mV was increased in H880Q and C373F channels by ∼10 and ∼19%, respectively (Table 4). Destabilization of SI by C373F was further implicated by an observed increase in the asymptote of UDI, ∼12% (Table 5). The asymptote of H880Q UDI displayed an elevated trend (∼4%), but the difference from WT was not statistically significant (Table 5).

Although alterations to SI by H880Q were predicted because His-880 is highly conserved throughout mammalian Na_V isoforms, destabilized SI in C373F channels was surprising. As mentioned previously, Cys-373 in Na_V1.5 is homologous to Phe-385 in Na_V1.2 (Fig. 7), and, because Na_V1.2 has more complete SI than Na_V1.5, C373F was expected to enhance SI (8, 14, 34, 42). In contrast to expectations, C373F reduced the maximum probability of SI. When Ile-891, positioned in the Na_V1.5 p-loop (Fig. 7), is replaced with a valine, the homologous residue in Na_V1.4, the mutant channel displays Na_V1.4-like SI (14). The reverse mutation in Na_V1.4 channels confers Na_V1.5-like SI (14). Na_V1.2 also has a valine at this position (Val-935; Fig. 7). Our data suggest that the differences in maximum probability of SI between Na_V1.5 and Na_V1.2 channels may not be isolated to a single residue as is the case between Na_V1.5 and Na_V1.4.

Proton Sensors and Slow Inactivation

We measured UDI using a pulse protocol designed to mimic a human ventricular action potential cycling at 2.6 Hz or ∼160 beats/min, as described previously (34). Given the length of the depolarizing and hyperpolarizing pulses (230 and 150 ms, respectively), this protocol is an effective tool to assess the dynamic equilibrium of slow inactivated states, which have onset and recovery time constants in the range of seconds to tens of seconds (Tables 3 and 4). Fast inactivation, with onset and recovery time constants less than 30 ms, has a negligible contribution to the decay of current during this experimental protocol (34). Our data demonstrate that p-loop residues mediate proton modulation of UDI of Na_V1.5 channels.

Both H880Q and C373F abolished proton modulation of UDI. Protons increase, reduce, or have no effect on the asymptote of UDI in Na_V1.5, Na_V1.2, and Na_V1.4 channels, respectively (34, 39). Inserting the p-loops of Na_V1.4 into the Na_V1.5 channel backbone imparts Na_V1.4 channel-like proton modulation of UDI (40). The p-loops between isoforms are highly conserved, and Cys-373 is one of only a few positions that differ between the three channels. As mentioned previously, Cys-373 is responsible for isoform-specific proton block between cardiac and skeletal channels (32). The homologous residues in Na_V1.2, Na_V1.4, and Na_V1.8 channels (phenylalanine, tyrosine, and serine, respectively) impart a proton-insensitive current in Na_V1.4, whereas cysteine abolishes it (32). We therefore postulate that protonation of Cys-373 modulates UDI in Na_V1.5 and contributes to the differences in UDI modulation between cardiac and skeletal channel isoforms. Further, our results with H880Q and C373F suggest that accumulation of positive charge around the Na_V1.5 channel pore mediates destabilization of UDI and that Cys-373 protonation represents a “tipping point” of positive charge that induces the reduction in Na_V1.5 UDI. These hypotheses require the assumption that SI disruption under control conditions in H880Q and C373F relative to WT is allosteric and not electrostatic.

These data paired well with our data on the kinetics of SI. C373F was more effective than H880Q at abolishing the pH-dependent modulation of SI kinetics (Figs. 4 and 5 and Tables 3 and 4). SSSI in C373F also displayed a greater response to protons than H880Q, whereas WT channels showed no change (Fig. 3 and Table 2). Overall, H880Q showed intermediate responses to protons compared with WT and C373F channels (Tables 2–4). Like their role in proton block, the relative positions of His-880 and Cys-373 may underlie the differential contribution to SI proton modulation. Because slow inactivation is thought to involve a structural rearrangement at or around the selectivity filter, protonation of Cys-373 might be predicted to have a greater effect on SI than His-880, consistent with our results.

Conclusion

Our results demonstrate that p-loop residues His-880 and Cys-373 mediate Na_V1.5 sensitivity to protons. These results also identified Cys-373 as a residue responsible for isoform-specific proton modulation of UDI. It seems likely that proton block and SI modulation occur in tandem, given the strong overlap in results between the two processes. Additionally, these results suggest that His-880 and Cys-373 contribute to the stability of SI in Na_V1.5 channels.