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. 2012 Jun 11:3:109.
doi: 10.3389/fphar.2012.00109. eCollection 2012.

Acidosis differentially modulates inactivation in na(v)1.2, na(v)1.4, and na(v)1.5 channels

Yury Y Vilin  1 , Colin H Peters, Peter C Ruben
Affiliations

Acidosis differentially modulates inactivation in na(v)1.2, na(v)1.4, and na(v)1.5 channels

Yury Y Vilin et al. Front Pharmacol. .

Abstract

Na(V) channels play a crucial role in neuronal and muscle excitability. Using whole-cell recordings we studied effects of low extracellular pH on the biophysical properties of Na(V)1.2, Na(V)1.4, and Na(V)1.5, expressed in cultured mammalian cells. Low pH produced different effects on different channel subtypes. Whereas Na(V)1.4 exhibited very low sensitivity to acidosis, primarily limited to partial block of macroscopic currents, the effects of low pH on gating in Na(V)1.2 and Na(V)1.5 were profound. In Na(V)1.2 low pH reduced apparent valence of steady-state fast inactivation, shifted the τ(V) to depolarizing potentials and decreased channels availability during onset to slow and use-dependent inactivation (UDI). In contrast, low pH delayed open-state inactivation in Na(V)1.5, right-shifted the voltage-dependence of window current, and increased channel availability during onset to slow and UDI. These results suggest that protons affect channel availability in an isoform-specific manner. A computer model incorporating these results demonstrates their effects on membrane excitability.

Keywords: activation; fast inactivation; gating; patch-clamp; slow inactivation; sodium channels.

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Figures

Figure 1
Figure 1
Effects of pH on macroscopic ionic currents and window currents in NaV1.2, NaV1.4, and NaV1.5. (A,Ai) Families of NaV1.2 currents in response to the pulse protocol (Ai, inset) at pH 7.4 (A) and pH 6.0 (Ai). (B) NaV1.2 G(V) curves (triangles) and steady-state FI curves (circles) are plotted as a function of membrane potential at pH 7.4 (filled symbols) and at pH 6.0 (open symbols). (C) Shows magnified window current area at pH 7.4 (solid lines) and at pH 6.0 (dotted lines). Values in (C) were converted to percents. (D,Di) Show families of NaV1.4 currents in response to the pulse protocol (Di, inset) at pH 7.4 (D) and pH 6.0 (Di). (E) NaV1.4 G(V) curves (triangles) and steady-state FI curves (circles) are plotted vs. membrane potential at pH 7.4 (filled symbols) and at pH 6.0 (open symbols). (F) Shows magnified window current area at pH = 7.4 (solid lines) and at pH 6.0 (dotted lines). Values in (F) are in percents. (G) Gi show families of NaV1.5 currents in response to the pulse protocol (Gi, inset) at pH 7.4 (G) and pH 6.0 (Gi). (H) NaV1.5 G(V) curves (triangles) and steady-state FI curves (circles) are plotted vs. membrane potential at pH 7.4 (filled symbols) and at pH 6.0 (open symbols). (I) Shows magnified window current area at pH 7.4 (solid lines) and at pH 6.0 (dotted lines). Values in (I) are in percents. Solid lines in (B,E,H) are Boltzmann fits to corresponding datapoints in (B,E,H) (Eq. 2 in Materials and Methods). Data represent mean ± SEM (n = 9–12).
Figure 2
Figure 2
Low pH decelerates kinetics of open-state inactivation in hNaV1.5. (A) Open-state inactivation time constants in NaV1.2 at pH 7.4 (filled circles) and pH 6.0 (open circles) are derived from single exponential fits (not shown) to the decay of currents in (Ai,Aii). (B) Open-state inactivation time constants in NaV1.4 at pH 7.4 (filled circles) and pH 6.0 (open circles) are derived from single exponential fits (not shown) to the decay of currents in (Bi,Bii). (C) Open-state inactivation time constants in NaV1.5 at pH 7.4 (filled circles) and pH 6.0 (open circles) are derived from single exponential fits (not shown) to the decay of currents in (Ci,Cii). Arrow in Cii denotes deceleration of current decay in NaV1.5 at pH 6.0. Asterisks denote p < 0.05 between open states FI at pH 7.4 (filled circles) and pH 6.0 (open circles). Data in (A–C) are fitted with single exponential for visual guidance. Data represent mean ± SEM (n = 15).
Figure 3
Figure 3
Low pH accelerates recovery from FI. (A–C) The time course of recovery from FI at pH 7.4 (filled circles) and at pH 6.0 (open circles) for NaV1.2, NaV1.4, and NaV1.5, respectively. The averaged, normalized currents obtained during 0 mV test pulse, following an interpulse of increasing duration over a range of potentials (−130 to −80 mV, pulse protocol is shown at the bottom) are plotted vs. interpulse duration and fit with single exponential. Solid lines are single exponential fits to datapoints (Equation, Materials and Methods). Data for only −80 mV prepulse are shown. Data represent mean ± SEM (n = 10–12).
Figure 4
Figure 4
Low pH slows the rate of onset to FI in NaV1.2 and NaV1.5. (A) The time course of onset to FI in NaV1.2 at pH 7.4 (filled circles) and at pH 6.0 (open circles). Data represent averaged and normalized current peaks recorded during test potential following the −30 mV prepulse of variable duration (0–500 ms) plotted vs. prepulse potential. Diagram of used pulse protocols shown in (A), inset. (B) The time course of onset to FI in NaV1.5 at pH 7.4 (filled circles) and at pH 6.0 (open circles). Data represent averaged and normalized current peaks recorded during test potential following the −60-mV prepulse of variable duration (0–500 ms) plotted vs. prepulse potential. Diagram of used pulse protocols shown in (B), inset. Solid lines are single exponential fits (Eq. 3, Materials and Methods). Data represent mean ± SEM (n = 10–12)
Figure 5
Figure 5
Low pH alters the voltage dependency of FI time constants in NaV1.2, NaV1.4, and NaV1.5. Time constants of FI in NaV1.2 (A), NaV1.4 (B), and NaV1.5 (C) were derived from single exponential fits to recovery, onset, and decay of macroscopic currents in response to pulse protocols shown at the bottom (also see Materials and Methods) and plotted vs. membrane voltage. Circles represent recovery time constants, squires represent time constants of closed-state inactivation and triangles denote time constants for the open-state inactivation. Filled symbols represent time constants obtained at pH = 7.4 and open symbols represent time constants at pH 6.0. The solid lines are predictions of a first-order reaction model (inactivated ↔ not inactivated, Materials and Methods). Diagrams at the bottom of Figure depict pulse protocols used in this series of experiments. Data represent mean ± SEM (n = 10–12).
Figure 6
Figure 6
Low pH alters properties of slow inactivation in NaV1.2 and in NaV1.5. (A) Steady-state slow inactivation in NaV1.2 at pH 7.4 (filled circles) and at pH 6.0 (open circles) is plotted as averaged normalized current amplitude vs. 60-s prepulse voltage. Asymptotic values for NaV1.2 derived from double exponential fit to slow inactivation onset (B) at pH 7.4 (filled triangles) and at pH 6.0 (open triangles) are superimposed with steady-state slow inactivation data at corresponding prepulse voltage (−50 mV). (B) Steady-state slow inactivation in NaV1.5 at pH 7.4 (filled squares) and at pH 6.0 (open squares) is plotted as averaged normalized current amplitudes vs. 60-s prepulse voltage. Asymptotic values for NaV1.5 derived from double exponential fits to slow inactivation onset (B) at pH 7.4 (filled triangles) and at pH 6.0 (open triangles) are superimposed with steady-state slow inactivation data at corresponding prepulse voltage (−50 mV). Data were obtained with pulse protocol shown in (B) inset and fit with a modified Boltzmann function (Eq. 6, Material and Methods). Data represent mean ± SEM (n = 7–10).
Figure 7
Figure 7
Low pH alters kinetics of slow inactivation in NaV1.2 and in NaV1.5. (A) The time course of slow inactivation onset in NaV1.2 at pH 7.4 (filled circles) and at pH 6.0 (open circles) is plotted vs. prepulse voltage as averaged and normalized currents, obtained with pulse protocol shown in (C) inset. (B) The time course of recovery from slow inactivation in NaV1.2 at pH 7.4 (filled circles) and at pH 6.0 (open circles) is plotted vs. interpulse voltage as averaged and normalized currents, obtained with pulse protocol in (D) inset. (C) The time course of slow inactivation onset in NaV1.5 at pH 7.4 (filled squares) and at pH 6.0 (open squares). (D) The time course of recovery from slow inactivation in NaV1.5 at pH 7.4 (filled squares) and at pH 6.0 (open squares). Data represent mean ± SEM (n = 7–10). Asterisks denote statistical difference (p < 0.05).
Figure 8
Figure 8
Low pH differentially affects use-dependent inactivation. (A,B,C) Use-dependent inactivation at pH 7.4 (filled symbols) and at pH 6.0 (open symbols) in NaV1.2, NaV1.4 and NaV1.5, respectively. Cells were held at −60 mV and repetitively stimulated either with one thousand 20 ms test pulses to 0 mV (for NaV1.2 and NaV1.4) or with 120 500 ms test pulses to 0 mV (for NaV1.5). Corresponding pulse protocol diagrams are shown in (A,B,C) insets. Peak currents from test pulses were normalized to the amplitude of the first current in the series and values were plotted vs. number of pulses. (A) Use-dependent inactivation in NaV1.2 at pH 7.4 (filled symbols) and at pH 6.0 (open symbols). Ai time constants of double exponential use-dependent inactivation in NaV1.2 at pH 7.4 (filled histograms) and at pH 6.0 (open histograms). (B) Use-dependent inactivation in NaV1.4 at pH 7.4 (filled symbols) and at pH 6.0 (open symbols). Bi time constants of double exponential use-dependent inactivation in NaV1.4 at pH 7.4 (filled histograms) and at pH 6.0 (open histograms). (C) Use-dependent inactivation in NaV1.5 at pH 7.4 (filled symbols) and at pH 6.0 (open symbols). Ci time constants of double exponential use-dependent inactivation in NaV1.5 at pH 7.4 (filled histograms) and at pH 6.0 (open histograms). Data represent mean ± SEM (n = 9–12). Asterisks denote statistical difference (p < .05).
Figure 9
Figure 9
Effects of pH on neuronal action potential. (A) An overlap of a two neuronal APs with sodium currents at pH 7.4 (solid line) and pH 6.0 (dashed line). The APs were elicited by a continuous stimulus current of 1 pA/pF. (B) Continuous APs with sodium currents at pH 7.4 (solid line) and pH 6.0 (dashed line). The continuous firing was induced with a continuous current of 20 pA/pF. (C) Continuous APs of a neuron expressing a sodium channel “pH chimera” whose parameters of conductance and FI correspond to those at pH 6.0 and the parameters of slow inactivation correspond to those at pH 7.4. The APs were stimulated with a continuous current of 20 pA/pF. (D) Continuous APs of a neuron expressing a sodium channel “pH chimera” whose parameters of conductance and FI correspond to those at pH 7.4 and the parameters of slow inactivation correspond to those at pH 6.0. The APs were stimulated with a continuous current of 20 pA/pF.
Figure 10
Figure 10
Effects of pH on cardiac action potential. (A) The fast sodium current of a cardiac AP at pH 7.4 (solid line) and pH 6.0 (dashed line). The sodium current is recorded on the 15th AP in an endocardial myocyte stimulated at a frequency of 2.5 Hz. (B) The fast sodium current of a cardiac AP at pH 7.4 (solid line) and pH 6.0 (dashed line). The sodium current is recorded on the 200th AP in a endocardial myocyte stimulated at a frequency of 3.33 Hz. (C) The membrane potential over the time of one endocardial myocyte AP with sodium currents at pH 7.4 (solid line) and pH 6.0 (dashed line). The AP is the 15th produced in a model ventricular endocardial myocyte stimulated at 2.5 Hz. (Ci) The first 25 ms of the 15th endocardial AP stimulated at 2.5 Hz with sodium currents at pH 7.4 (solid line) and pH 6.0 (dashed line). (Cii) The last 50 ms of the 15th endocardial AP stimulated at 2.5 Hz with sodium currents at pH 7.4 (solid line) and pH 6.0 (dashed line). (D) The membrane potential over the time of one endocardial myocyte AP with sodium currents at pH 7.4 (solid line) and pH 6.0 (dashed line). The AP is the 200th produced in a model ventricular endocardial myocyte stimulated at 3.33 Hz. (Di) The first 25ms of the 200th endocardial AP stimulated at 3.33 Hz with sodium currents at pH 7.4 (solid line) and pH 6.0 (dashed line). (Dii) The last 50 ms of the 200th endocardial AP stimulated at 3.33 Hz with sodium currents at pH 7.4 (solid line) and pH 6.0 (dashed line).
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