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Extracellular Ca²⁺ Modulates the Effects of Protons on Gating and Conduction Properties of the T-type Ca²⁺ Channel _1G (Ca_V3.1)

¹ Laboratorium voor Fysiologie, Campus Gasthuisberg, KU Leuven, B-3000 Leuven, Belgium
² Institut für Pharmakologie und Toxikologie, Technische Universität München, D-80802 München, Germany

Address correspondence to Karel Talavera, Laboratorium voor Fysiologie, Campus Gasthuisberg, KU Leuven, B-3000 Leuven, Belgium. Fax: (32) 16 34 59 91; E-mail: karel.talavera{at}med.kuleuven.ac.be

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Since Ca²⁺ is a major competitor of protons for the modulation of high voltage–activated Ca²⁺ channels, we have studied the modulation by extracellular Ca²⁺ of the effects of proton on the T-type Ca²⁺ channel _1G (Ca_V3.1) expressed in HEK293 cells. At 2 mM extracellular Ca²⁺ concentration, extracellular acidification in the pH range from 9.1 to 6.2 induced a positive shift of the activation curve and increased its slope factor. Both effects were significantly reduced if the concentration was increased to 20 mM or enhanced in the absence of Ca²⁺. Extracellular protons shifted the voltage dependence of the time constant of activation and decreased its voltage sensitivity, which excludes a voltage-dependent open pore block by protons as the mechanism modifying the activation curve. Changes in the extracellular pH altered the voltage dependence of steady-state inactivation and deactivation kinetics in a Ca²⁺-dependent manner, but these effects were not strictly correlated with those on activation. Model simulations suggest that protons interact with intermediate closed states in the activation pathway, decreasing the gating charge and shifting the equilibrium between these states to less negative potentials, with these effects being inhibited by extracellular Ca²⁺. Extracellular acidification also induced an open pore block and a shift in selectivity toward monovalent cations, which were both modulated by extracellular Ca²⁺ and Na⁺. Mutation of the EEDD pore locus altered the Ca²⁺-dependent proton effects on channel selectivity and permeation. We conclude that Ca²⁺ modulates T-type channel function by competing with protons for binding to surface charges, by counteracting a proton-induced modification of channel activation and by competing with protons for binding to the selectivity filter of the channel.

Key Words: pH • activation • permeation • selectivity • kinetic model

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
At present, there are several lines of evidence that permeant ions modulate T-type Ca²⁺ channel gating. Shuba et al. (1991) showed that the mean open time of the T-type channel of mouse neuroblastoma cells depends on the type and concentration of the permeant ion and proposed that the channel region controlling channel closure senses a smaller fraction of the electric field due to a dynamic interaction between the ionic flux and the selectivity filter. In frog-atrial cardiomyocytes, Alvarez et al. (2000) showed that channel reopening after strong depolarizing prepulses was enhanced in low-Na⁺ extracellular solutions and proposed that a competition between Na⁺ and Ca²⁺ for binding sites within the channel modulates the amplitude of the voltage-facilitated T-type current. It is also known that the _1G clone (Ca_V3.1) shows faster macroscopic inactivation in the presence of Ba²⁺ than in Ca²⁺ (Klugbauer et al., 1999; Staes et al., 2001).

T-type Ca²⁺ channels are also modulated by extracellular protons. Tytgat et al. (1990) reported that, in guinea pig cardiomyocytes, low extracellular pH (pH_e) induced a positive shift in the voltages for half-maximal activation (V_act) and inactivation (V_inac) together with an increase in the slope factor of channel activation (s_act). In a recent paper, Delisle and Satin (2000) concluded that proton block of T-type currents (_1H, Ca_V3.2) under physiological conditions is due to gating modifications. These authors related the shift in the voltage for half-maximal activation by high extracellular proton concentrations to the titration of negative surface charges. To explain the increased slope factor of activation, they proposed that external acidification titrates some of the charges involved in the voltage sensing, but suggested already to test the influence of intrapore ions on the gating mechanisms.

Protons have also been shown to affect the open pore conduction of T-type channels. Tytgat et al. (1990) showed that extracellular acidification from pH 9.0 to 6 decreases the single channel conductance of the cardiac T-type channel of the guinea pig using 110 mM Ca²⁺ as charge carrier. For the _1H T-type clone, Delisle and Satin (2000) found that open pore block was not the main determinant for the inhibition of whole-cell currents by extracellular protons in the presence of 2.5 mM extracellular Ca²⁺ and 140 mM Na⁺. They reported a negative shift of the reversal potential after extracellular acidification and suggested that channel protonation shifts channel selectivity toward monovalent ions.

Protons have been shown to modulate the gating processes of high voltage–activated (HVA)^* Ca²⁺ channels (Pietrobon et al., 1989; Prod'hom et al., 1989; Kwan and Kass, 1993; Tombaugh and Somjen, 1996; Zhou and Jones, 1996; Smirnov et al., 2000; Shah et al., 2001). In these channels, Ca²⁺ has been shown to be a major competitor of protons for the neutralization of surface charges (Kwan and Kass, 1993) and for binding to pore residues that control ion permeation and selectivity (Chen et al., 1996; Chen and Tsien, 1997). To achieve further insight in the mechanisms of ionic modulation of T-type channel function, we investigated the influence of the extracellular Ca²⁺ concentration ([Ca²⁺]_e) on the proton-induced modifications of gating and permeation mechanisms of the _1G T-type subunit and the role of the selectivity filter (the EEDD pore locus) in the modulation of channel selectivity and permeation by protons. Our results suggest for three substrates of competition between Ca²⁺ and protons, resulting in modifications of channel gating and ionic conduction.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Solutions
Before current recordings, cells were rinsed with Krebs solution containing: 150 mM NaCl, 6 mM KCl, 1 mM MgCl₂, 1.5 mM CaCl₂, 10 mM glucose, 10 mM HEPES, and titrated to pH 7.4 with 1 N NaOH. All extracellular test solutions contained 5 mM CsCl, 5 mM glucose, a mixture of MES, HEPES, and TAPS (5 mM each) to extend the buffering range from pH 5.5 to 9.1, and were kept Mg²⁺ free to avoid extracellular Mg²⁺ block (Serrano et al., 2000). We used extracellular solutions of 2 or 20 mM CaCl₂ or a Ca²⁺ free solution (5 mM EDTA) with 20 mM NaCl, osmotically compensated with 150, 120, or 130 mM NMDG, respectively. To assess the influence of Na⁺ on conduction properties in the 2 mM Ca²⁺ solution, 150 mM NMDG was substituted by an equimolar amount of Na⁺. Test solutions containing NMDG were titrated with HCl and those containing 150 mM NaCl with CsOH. When 2 or 20 mM Ca²⁺ solutions were used, the intracellular (pipette) solution contained 102 mM CsCl, 10 mM HEPES, 5 mM MgCl₂, 5 mM Na₂-ATP, 10 mM TEA-Cl, 10 mM EGTA, titrated to pH 7.4 with 1 N CsOH. In the experiments in zero [Ca²⁺]_e the intracellular solution did not contain MgCl₂ and Na₂-ATP in order to avoid block by Mg²⁺ and to limit the size of outward currents, respectively. All chemicals were purchased from Sigma-Aldrich.

Electrophysiology
We used human embryonic kidney cells (HEK293) grown and transfected with the wild-type _1G (Klugbauer et al., 1999) or the EEED pore mutant as in previous works (Staes et al., 2001; Talavera et al., 2001). Currents were recorded in the whole-cell configuration of the patch-clamp technique using an EPC-7 (LIST Electronics) patch-clamp amplifier and filtered with an eight-pole Bessel-filter (Kemo). For control of voltage-clamp protocols and data acquisition, we used an IBM-compatible PC with a TL-1 DMA interface (Axon Instruments, Inc.) and the software pCLAMP (Axon Instruments, Inc.). Bath solutions were perfused by gravity via a multibarreled pipette. Patch pipettes were pulled from Vitrex capillary tubes (Modulohm) using a DMZ-Universal puller (Zeitz-instruments). An Ag-AgCl wire was used as reference electrode. Adequate voltage control was achieved by using low pipette resistances (1–2.5 M) and series resistance compensation to the maximum extent possible (50–70%). Membrane-capacitive transients were electronically compensated and the linear background components were digitally subtracted before data analysis. Current traces were filtered at 2.5–5 kHz and digitized at 5–10 kHz. All experiments were performed at room temperature (22–25°C).

Stimulation Protocols and Data Analysis
I-V curves were obtained from the peak amplitude of currents evoked by the application 200-ms lasting voltage steps from -90 to 60 mV. These curves were fitted with:

(1)

where I is the measured peak current, V the step potential and G(V) the conductance, which may be voltage dependent. The activation curves in 2 and 20 mM Ca²⁺ solutions were best described by the sum of two Boltzmann components (Serrano et al., 1999; Lacinova et al., 2002). V_act and V'_act are the potentials of half-maximal amplitude, s_act and s'_act are the slope parameters and A is the amplitude of the steeper component. In Ca²⁺ free solutions only one Boltzmann function was needed (A = 1). To account for the strong inward rectification in the 2 and 20 mM Ca²⁺ solutions, we have approximated G(V) from a fit of the amplitudes of the tail current after a depolarizing step to 100 mV in the voltage range from -70 to 110 mV for _1G and from -70 to 70 mV for the EEED mutant with the equation:

(2)

with . The I-V relation in the Ca²⁺ free, 20 mM Na⁺ extracellular solution is fairly linear, and hence G(V) = G.

The average values of the fit parameters b and c for each experimental condition were then used to fit the I-V curves with Eq. 1. We noticed that an adequate description of the open pore conduction was essential for an accurate estimation of the slope factor s_act of the activation curve, and that the use of the classical linear function for in Eq. 1 consistently overestimated the values for s_act for the currents in 2 or 20 mM Ca²⁺. Activation curves were calculated by dividing the experimental I-V curves by the appropriate function in each experimental condition.

The time constants of activation (_act) and inactivation (_inac) were determined from a fit of current traces with the equation:

(3)

where I_m is a normalization factor and I_b is a background component. Given the sampling rate used in the activation protocol, the values of _act at positive voltages (<0.5 ms) should be considered as an upper limit of the real values. Nevertheless, our estimates are equal or smaller than previously reported values (0.6 ms, Nilius, 1992; 0.3 ms, Chen and Hess, 1990). The decaying phase of the voltage dependence of _act was fitted with an exponential function of the form:

(4)

where s_act is the voltage sensitivity, _act() is the asymptotic value at positive potentials and V_act is the voltage at which _act is equal to 1 + _act().

Steady-state inactivation (h) was determined from the peak current recorded during a 160-ms test pulse to 0 mV after a 5,120-ms lasting prepulses to potentials between -100 and -25 mV. The voltage dependence of these peak currents, normalized to that following the prepulse to -100 mV, was fitted with the equation:

(5)

where V_inac is the potential of half-maximal inactivation and s_inac the slope factor for the inactivation.

To dissect the effects of ionic conditions on conduction properties from those on gating, tail currents were recorded during voltage steps between -120 and 110 mV after a 7.5-ms lasting depolarization to 100 mV to normalize for the positive voltage shift of the steady-state activation at low pH_e. Linear background components and capacitive transients were subtracted by the application of a -P/4 protocol. In the presence of 2 mM Ca²⁺ there was an 10% decrease in the estimated maximal open probability during the prepulse to 100 mV when the pH_e was changed from 9.1 to 6.2. However, at 7.5 ms the estimated open probability was not significantly different between these conditions (unpublished data). This ensured that the changes in the amplitude of the tail currents with the extracellular perfusion condition were determined by open pore properties and not by modifications in gating during the prepulse to 100 mV. We determined the amplitude (I_tail) and time constant of current decay (_decay) from a single-exponential fit of the time course of the tail currents. _decay corresponds to the time constant of deactivation (_deac) at negative potentials and to the time constant of the macroscopic inactivation (_inac) at potentials positive to the activation threshold. The usual way to characterize the voltage dependence of _deac is to fit _decay(V) with a growing exponential function in a range of very negative potentials. This approximation is expected to underestimate the steepness of the voltage dependency of _deac since it does not consider the contribution of the inactivation to current decay. Additional underestimation of the steepness may occur when fitting tail currents at very negative potentials given that the estimated values of _decay tend to be higher than the actual ones due to voltage clamp errors that are the consequence of large tail current amplitudes. When considering the current models of T-type channel gating (see Fig. 13), in a second order of approximation, the voltage dependence of _decay can be expressed by:

(6)

where V is the repolarization potential, V_deac the voltage at which _deac is equal to 1 ms, s_deac the voltage sensitivity of the time constant of deactivation, and k_OIo is the rate constant of the transition between the open state (O) and the closest inactivated state (I_O). This approximation is proved to be valid as the channels have low probability of being in the closed state (C₃) near the open state and the inactivation process is largely absorbing (k_OIo >> k_IoO) (see Fig. 13).

In all voltage protocols the holding potential was -100 mV and the stimulation frequency 0.5 Hz, with the exception of the inactivation protocol in which it was 0.2 Hz. Electrophysiological data were analyzed using the WinASCD software package (ftp://ftp.cc.kuleuven.ac.be/pub/droogmans/winascd.zip; G. Droogmans, Laboratory of Physiology, KU Leuven). For all measurements pooled data are given as mean ± SEM. We used ANOVA and Student's paired t test, taking P < 0.05 or P < 0.01 as the level of significance.

Data Simulation
We used MATLAB (MathWorks) to solve a Markov model for the gating of _1G in several pH_e, in the presence of 2 or 20 mM Ca²⁺. Parameter optimization and numerical solution of the differential equations were performed with the built-in functions fmin and expm, respectively.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Effects of pH_e and Extracellular Ca²⁺ on the Activation of _1G
We tested the modulation of the gating of _1G by extracellular Ca²⁺ and protons by studying the effects of pH_e in the range from 9.1 to 5.5 on I-V curves in the presence of 0, 2, or 20 mM Ca²⁺. Fig. 1, A–C , shows current traces at various pH_e values in the presence of 2 mM Ca²⁺ during voltage steps to -60, -40, and -20 mV. Current reduction at low pH_e was most pronounced at more negative potentials. As a consequence, the I-V curve, derived from the peak current amplitudes, was shifted in the positive direction at lower pH_e (Fig. 1 D). Fig. 1, E and F, shows that extracellular acidification also shifts the voltage dependence of the time constants of activation (_act) and macroscopic inactivation (_inac).

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Proton inhibition of 1G currents in the presence of 2 mM extracellular Ca2+. (A–C) Typical proton effects on current traces evoked by voltage steps to -60, -40, and -20 mV, respectively. The labels at the left of each trace indicate the pHe at which they were registered. (D) Voltage dependence of the amplitude of the traces shown in the top panels. Continuous lines are the best fit of the data with Eq. 1. (E) pHe modulation of the voltage dependence of the time constant of activation (act) (n = 5–6). The decaying phase of act(V) was fitted with Eq. 4. Data points of the raising phase of act(V) at pHe 7.4, 6.8, and 6.2 are not shown for clarity. (F) pHe effects on the voltage dependence of the time constant of macroscopic inactivation (inac). In D–F symbols apply to pHe values as follows: 9.1, ; 8.2, ; 7.4, ; 6.8, ; 6.2, ; 5.5, .

_act

_inac

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Proton inhibition of 1G currents in 20 mM extracellular Ca2+. (A–C) Typical proton effects on current traces evoked by voltage steps to -40, -20, and 0 mV, respectively. The pHe is indicated at the left of each trace. (D) I-V curves corresponding to the traces shown in the top panels. Continuous lines are the best fits of the data with Eq. 1. (E and F) pHe effects on the voltage dependencies of the time constants of activation (act) and inactivation (inac) (n = 5–7). Data points of the raising phase of act(V) at pHe values from 7.4 to 5.5 are not shown for clarity. The decaying phase of act(V) was fitted with Eq. 4. In D–F symbols apply to pHe values as follows: 9.1, ; 8.2, ; 7.4, ; 6.8, ; 6.2, ; 5.5, .

_act

_inac

View larger version (29K): [in this window] [in a new window]   FIGURE 3. Proton inhibition of 1G currents in zero Ca2+. (A–C) Typical proton effects on current traces evoked by voltage steps to -80, -60, and 0 mV, respectively. Numbers at the left of each trace indicate the pHe at which they were registered. (D) I-V curves corresponding to the traces shown in the top panels. Continuous lines are the best fits with Eq. 1. (E) pHe modulation of the voltage dependence of the time constant of activation (act) (n = 3–5). The decaying phase of act(V) was fitted with Eq. 4. Data points of the raising phase of act(V) at pHe values from 8.2 to 5.5 are not shown for clarity. (F) pHe effects on the voltage dependence of the time constant of macroscopic inactivation (inac). In D–F pHe values are symbolized as follows: 9.1, ; 8.2, ; 7.4, ; 6.8, ; 6.2, ; 5.5, .

View larger version (15K): [in this window] [in a new window]   FIGURE 4. Summary of proton effects on the activation curve of 1G in 0, 2, and 20 mM extracellular Ca2+. (A) Average activation curves obtained at pHe 9.1 (filled symbols) and 6.2 (open symbols), in 0 (, ), 2 (, ), or 20 (, ) mM Ca2+. Continuous lines are the activation curves calculated from the average values of the parameters obtained from the fit of I-V curves with Eq. 1 in each experimental condition. (B and C) Effects of pHe on Vact and sact. Labels (#), (*), and (+) indicate significant difference with P < 0.05 from the values of Vact and sact obtained at pHe 9.1 for 0 (), 2 (), and 20 () mM Ca2+ conditions, respectively. Double labels indicate significant difference with P < 0.01.

The slope factor of the steeper component of the activation s_act increased with extracellular acidification, an effect that is antagonized by increasing [Ca²⁺]_e (Fig. 4 C). Reducing pH_e from 9.1 to 7.4 significantly increased s_act in 2 mM Ca²⁺, whereas acidification to pH_e 6.2 and below was necessary to affect s_act in 20 mM Ca²⁺. Also the values of s_act in 2 and 20 mM Ca²⁺ seem to converge at low pH_e. The changes in s_act were much more dramatic in the absence of Ca²⁺, since this parameter increased about fourfold if pH_e was changed from 9.1 to 6.2.

The rightward shift and increased slope factor of the activation curve by extracellular acidification might result from a voltage-dependent open pore block (Woodhull, 1973). Such a mechanism would, however, not affect the kinetics of channel activation, and is in contrast with the observed shift of the voltage dependences of the time constants of activation _act and macroscopic inactivation _inac (Figs. 1–3). The rightward shift of the voltage dependence of _act at pH_e 9.1 by increasing [Ca²⁺]_e is consistent with a mechanism of screening of surface charges by Ca²⁺. However, the observation that a decrease of pH_e did not only shift the voltage dependence of _act to more positive voltages but also decreased its slope is inconsistent with simple screening by protons. To quantify these effects, we have estimated the position of the curve _act(V) along the voltage axis (V_act), and the steepness of the voltage dependence (s_act) from the fit of the data points of the decaying phase with Eq. 4 for each experimental condition. Fig. 5, A and B , show that the patterns of modulation of the pH_e dependence of V_act and s_act by extracellular Ca²⁺ are similar to those of V_act and s_act (compare with Fig. 4, B and C). This indicates that the Ca²⁺-dependent inhibition of _1G by protons is to a large extent due to an effect of protons on gating. The issue of open pore block by protons and its Ca²⁺ dependence is addressed below.

View larger version (14K): [in this window] [in a new window]   FIGURE 5. Proton effects on the time constant of activation of 1G are Ca2+ dependent. (A and B) pHe dependencies of Vact and sact, respectively. Labels (#), (*), and (+) indicate significant difference with P < 0.05 from the values obtained at pHe 9.1 for 0 (), 2 (), and 20 () mM Ca2+ conditions, respectively. Double labels indicate significant difference with P < 0.01.

Extracellular Ca²⁺ Modulates the Effect of Protons on Steady-state Inactivation of _1G

View larger version (18K): [in this window] [in a new window]   FIGURE 6. [Ca2+]e and pHe dependency of steady-state inactivation of 1G. (A) Average inactivation curves obtained in 2 mM (filled symbols, n = 4–10) or in 20 mM Ca2+ (open symbols, N = 5–9) at different pHe (9.1, ; 8.2, ; and 6.2, ,). Continuous lines are the inactivation curves calculated from Eq. 5 using the average values of Vinac and sinac determined for each experimental condition. (B) pHe dependence of the average Vinac in the presence of 2 () or 20 () mM Ca2+. (C) average shift of inactivation curves respect to the curves obtained at pHe 9.1 and 8.2, in 2 () or 20 () mM Ca2+, respectively. (D) average slope factor of the steady-state inactivation as function of pHe in 2 () or 20 () mM Ca2+. In B and D the label (*) indicates significant difference with P < 0.05 from the values obtained at pHe 9.1 (Ca2+ 2 mM) or pHe 8.2 (Ca2+ 20 mM). In C the labels (*) and (**) denote significant difference from zero with P < 0.05 and P < 0.01, respectively.

View larger version (15K): [in this window] [in a new window]   FIGURE 7. Proton-induced changes in activation and inactivation are not strictly coupled. Relation between the gating parameters describing steady-state activation and inactivation when pHe is changed from 9.1 to 6.2 in 2 mM Ca2+ () and from 8.2 to 5.5 in 20 mM Ca2+ (). (A) Correlation between the voltage for half-maximal inactivation Vinac and the voltage for half-maximal activation Vact. Continuous lines are the best fits obtained using the function Vinac = m·Vact + n. In 2 mM Ca2+ m and n were equal to 0.45 ± 0.09 and -54 ± 4 mV, respectively, while in 20 mM Ca2+ were 0.40 ± 0.07 and -41 ± 2 mV, respectively. (B) Changes in sact are not strictly correlated to changes in sinac. (C) The changes in Vact are accompanied by changes in sact. (D) Lack of strict correlation between the changes in Vinac and the voltage sensitivity of the steady-state inactivation (sinac).

The Effects of Extracellular Protons on Ion Permeation and Selectivity Depend on Extracellular Ca²⁺ and Na⁺. Comparison between 1G and the EEED Pore Mutant

We analyzed the effects of pH_e on the amplitude of _1G tail currents in 2 mM Ca²⁺ (150 mM Na⁺ or NMDG⁺) or 20 mM Ca²⁺ (120 mM NMDG⁺) (Fig. 8 , A–C). Decreasing pH_e from 9.1 to 6.2 in 2 mM Ca²⁺ and 150 mM NMDG⁺ significantly reduced inward tail currents, but did not affect outward currents (Fig. 8 A). The effects of pH_e on the inward currents were smaller if NMDG⁺ was replaced by Na⁺ (Fig. 8 B) or if [Ca²⁺]_e was increased to 20 mM (Fig. 8 C). To quantify the voltage dependence of this proton block, we have calculated at each potential and for each cell and Ca²⁺-Na⁺ condition the ratio of the current amplitudes at pH_e 6.2 and 9.1 (I(pH6.2)/I(pH9.1), Fig. 8 D). Extracellular protons exerted a significant (33%) block of the current in 2 mM Ca²⁺ in the absence of extracellular Na⁺. This block was voltage independent in the range from -120 to 20 mV and declined sharply around the reversal potential. The voltage range in which the block was voltage dependent was larger in 2 mM Ca²⁺ and 150 mM Na⁺, but the maximal block was the same as in the Na⁺ free solution. The proton block was significantly smaller at 20 mM Ca²⁺ (10%), indicating that extracellular Ca²⁺ antagonizes the effects of protons on ion conduction through _1G.

View larger version (31K): [in this window] [in a new window]   FIGURE 8. Open pore block by protons in 1G is dependent on extracellular Ca2+ and Na+ concentrations. Voltage dependence of the amplitude of tail currents through the wild-type channel 1G at pHe 9.1 () and 6.2 () in the presence of: A, 2 mM Ca2+ (150 NMDG+); B, 2 mM Ca2+ (150 Na+); and C, 20 mM Ca2+ (120 NMDG+). Continuous lines are the best fits with Eq. 2 in the voltage range from -70 to 110 mV. Insets in A–C show current traces at -80 mV in the corresponding perfusion conditions. The asterisks denote the traces obtained at pHe 9.1. The difference in current amplitude during the prepulse to 100 mV at pHe 6.2 with respect to that at pHe 9.1 (insets A and B) is mainly (90%) due to a background current. (D) Voltage dependence of the ratio between the amplitude of the tail currents recoded at pHe 6.2 respect to those determined at pHe 9.1 in the presence of 2 mM Ca2+ (150 NMDG+) (), 2 mM Ca2+ (150 Na+) () or 20 mM Ca2+ (120 NMDG+) () (n = 6–7).

View larger version (28K): [in this window] [in a new window]   FIGURE 9. Proton effects on the conduction properties of the 1G pore mutant EEED. Voltage dependence of the amplitude of tail currents in a cell expressing the EEED pore mutant at pHe 9.1 () and 6.2 () in the presence of: A, 2 mM Ca2+ (150 NMDG+); B, 2 mM Ca2+ (150 Na+) and C, 20 mM Ca2+ (120 NMDG+). Continuous lines are the best fits with Eq. 2 in the voltage range from -70 to 70 mV. Insets in A–C show current traces at -80 mV in the corresponding perfusion conditions. During the prepulse to 100 mV current values are out of the scale. The asterisks denote the traces obtained at pHe 9.1. Note the difference in the time scale from that used in Fig. 8. (D) Voltage dependence of the ratio between the amplitude of the tail currents recorded at pHe 6.2 respect to those in determined at pHe 9.1 in the presence of 2 mM Ca2+ (150 NMDG+) (), 2 mM Ca2+ (150 Na+) () or 20 mM Ca2+ (120 NMDG+) () (n = 7–10).

View larger version (20K): [in this window] [in a new window]   FIGURE 10. Proton effect on reversal potential of 1G and the EEED pore mutant depend on extracellular [Ca2+]. (A and B) Voltage dependence of average amplitudes of tail currents though the 1G wild-type and the EEED mutant channels, respectively, at pHe 9.1 (filled symbols) and 6.2 (open symbols), in the presence of 2 mM Ca2+ (150 NMDG+) (, ), 2 mM Ca2+ (150 Na+) (, ) or 20 mM Ca2+ (120 NMDG+) (, ). (C) Average reversal potentials (Vr) obtained form the fits of the voltage dependence of the amplitude of tail currents through 1G (empty bars) (n = 6–7) and the EEED mutant (dashed bars) (n = 7–10). The different extracellular perfusion conditions are indicated by the horizontal bars. For each channel, the symbols (*) and (**) indicate significant difference between the values at 9.1 and 6.2 with P < 0.05 and P < 0.01, respectively. Significant difference between the values in Na+ and NMDG+ with P < 0.01 is denoted by (##).

View larger version (18K): [in this window] [in a new window]   FIGURE 11. Proton-dependent contribution of Na+ to the permeation through 1G and the EEED pore mutant. Voltage dependence of the ratio between the amplitude of the tail currents recorded in 2 mM Ca2+ in the presence of 150 mM extracellular NMDG+ and those obtained in equimolar Na+ at pHe 9.1 (filled symbols) or at pHe 6.2 (empty symbols) (1G: , , N = 5; EEED: , , N = 4).

Modulation of Deactivation Kinetics of _1G by Extracellular Protons Is Dependent on Extracellular Ionic Conditions. Effects of Extracellular Ca²⁺ and Na⁺

_decay

_deac

_deac

_deac

View larger version (32K): [in this window] [in a new window]   FIGURE 12. Proton modulation of deactivation kinetics of 1G and the EEED pore mutant is dependent on extracellular Ca2+. (A and B) Voltage dependence of the average time constant of the decay (decay) of tail currents through 1G (N = 6–7) and the EEED mutant (N = 7–10), respectively, at pHe 9.1 (filled symbols) and 6.2 (open symbols), in the presence of 2 mM Ca2+ (150 NMDG+) (, ), 2 mM Ca2+ (150 Na+) (, ) or 20 mM Ca2+ (120 NMDG+) (, ). Continuous lines are functions of the form of Eq. 6 calculated using average values of Vdeac, sdeac shown in C and D, respectively, and kOIo. The symbols (**) and (++) indicate significant difference between the values at 9.1 and 6.2 with P < 0.01 for 1G (empty bars) and the EEED mutant (dashed bars), respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Ca²⁺ Inhibits the Effects of Protons on Channel Gating
Tytgat et al. (1990) reported that changes in pH_e from 9 to 6 did not shift the voltage for half-maximal activation V_act in the presence of 110 mM Ca²⁺, but significantly changed it in 5.4 mM Ca²⁺. In the present study, we show that extracellular acidification induced a shift of V_act and increased the slope factor of activation s_act in the presence of either 0, 2 or 20 mM Ca²⁺, and that the magnitudes of these effects were more prominent at lower [Ca²⁺]_e. Remarkably, protons shifted V_act to more positive values in Ca²⁺ free solutions than in 2 or 20 mM Ca²⁺ (Fig. 4), indicating that protons induce an extra positive shift in the voltage dependence of the activation process that cannot be explained by the neutralization of surface charges. The shift of V_act and increase of s_act could be interpreted as a voltage-dependent open pore block by protons (Woodhull, 1973). However, the finding that Ca²⁺-dependent proton effects on the voltage dependence of _act were similar to those on steady-state activation indicate that the proton effects on the shape of the current-voltage relationship are to a large extent due to modifications in channel gating. Moreover, the open pore block by protons was proven to be voltage independent when NMDG⁺ was present in the bath solution. In a more physiological condition (150 mM extracellular Na⁺), the voltage dependence of the proton block shows an opposite slope to that expected from a Woodhull-like open pore block (see below).

Extracellular protons also affected inactivation of T-type channels by shifting the voltage for half-maximal inactivation V_inac, as it has been reported previously for cardiac T-type Ca²⁺ channels (Tytgat et al., 1990) and the T-type _1H subunit (Delisle and Satin, 2000). Changes in the slope factor of the inactivation curve s_inac had not been reported so far. Our results show that extracellular Ca²⁺ antagonizes the effects of pH_e on both V_inac and s_inac of _1G, in the sense that significant changes in these parameters require larger changes in pH_e at higher Ca²⁺ concentrations. These effects of protons on channel inactivation might be indirect, due to a coupling between activation and inactivation, as discussed in the next section.

Extracellular protons shift the voltage dependence of the time constant of deactivation _deac of _1G along the voltage axis, in agreement with the results of Delisle and Satin (2000) for _1H. Furthermore, we show that this shift, as for the other gating processes, depends on [Ca²⁺]_e. Delisle and Satin (2000) found similar depolarizing shifts of the activation and deactivation processes for a pH_e change from 8.2 to 5.5 in 2.5 mM Ca²⁺, and suggested that these might be due to the neutralization of surface charges by extracellular protons. However, a closer examination of the Ca²⁺ effects reveals peculiar features of the proton modulation. With a milder acidification we observed that the proton-induced shift in V_deac was larger than that in V_act at high [Ca²⁺]_e. Extracellular acidification from pH 9.1 to 6.2 shifted V_deac and V_act, respectively, by 19 and 20.1 mV in 2 mM Ca²⁺, but by 13 and 7 mV in 20 mM Ca²⁺ (compare Figs. 4 B and 5 C with Fig. 12 C). We believe that these results are not in contradiction with those of Delisle and Satin (2000) because they worked in proton-favoring conditions that possibly override Ca²⁺ effects. However, our findings cannot be explained by the standard surface potential theory (Frankenhaeuser and Hodgkin, 1957), which implies that protons shift the voltage dependence of all gating parameters by the same amount at each [Ca²⁺]_e. To explain the different proton-induced shifts in voltage dependence of activation and deactivation by the neutralization of surface charges it has to be assumed that this effect is state dependent, in the way that due to structural rearrangements during gating, the channel structure present different substrates for protons and/or different sensitivity of the voltage sensors (or gating machinery) to proton effects (Hille, 2001).

The standard surface-potential theory also predicts that the effects of increasing extracellular proton and Ca²⁺ concentrations on the positive shift of the gating parameters are additive (Kwan and Kass, 1993). Our observation that protons shift the activation curve to more positive potentials in the absence of extracellular Ca²⁺ (Fig. 4, A and B) is therefore incompatible with neutralization of surface charges being the sole mechanism responsible for the shift of activation kinetics. Delisle and Satin (2000) proposed that the reduced voltage sensitivity of activation is due to a proton-induced slowing of voltage dependent transitions distally to channel opening. We have extended this idea to explain our results. First, we propose that protons not only decrease the voltage sensitivity of the activation (increase s_act) but also shift the voltage of half-maximal activation to more positive potentials independently of the neutralization of surface charges. Second, we postulate that Ca²⁺ inhibits these effects of protons on activation. On the other hand, in accord with Delisle and Satin (2000), we consider that the transition determining macroscopic deactivation is only modulated by the neutralization of negative surface charges.

We have reported previously that aspartate-to-glutamate mutations in the EEED pore locus of _1G induces changes in the activation curve of the channel (Talavera et al., 2001). The present result demonstrate that the EEED mutant shows alterations in the deactivation process, with less negative V_deac values and smaller voltage sensitivity for _deac than the wild-type channel. These and other gating modifications induced by pore mutations are discussed in the accompanying paper (Talavera et al., 2003, in this issue).

Activation-inactivation Coupling and Proton Effects on Channel Gating
The voltage dependence of the inactivation of T-type Ca²⁺ channels arises from voltage-dependent transitions occurring during channel activation (Droogmans and Nilius, 1989; Chen and Hess, 1990; Serrano et al., 1999; Burgess et al., 2002). Since protons modify the activation process it is interesting to study the possible correlation between the proton-induced changes in the parameters describing steady-state activation and inactivation. We have found that if pH_e is changed in the range of 9.1 to 6.2, for each 1 mV shift in V_act there was a 0.4–0.5 mV shift in V_inac in both 2 and 20 mM Ca²⁺. A similar correlation factor was calculated from the data of Delisle and Satin (2000) for _1H (their Figs. 1 D and 2 C). On the other hand, there was no strict correlation between the voltage sensitivity of inactivation (s_inac) and activation (s_act) and between s_inac and V_inac, in contrast with that observed between s_act and V_act (see Fig. 7). Thus, although proton-induced changes in the inactivation and activation processes seem to be linked, their degree of correlation is not absolute. As we discuss below, a kinetic model that includes state-dependent proton effects can explain these results.

A Kinetic Model Predicts State-dependent Effects of Extracellular Protons and Ca²⁺
Currently, there are several kinetic models of T-type channel gating (Droogmans and Nilius, 1989; Chen and Hess, 1990; Serrano et al., 1999; Burgess et al., 2002). The model of Burgess et al. (see Fig. 13) is particularly attractive since it accounts for the properties of the _1G and _1H channels in nearly physiological conditions and includes for the first time an explicit description of the gating charges associated with channel transitions. We adopted this model and determined which parameters had to be modified in order to describe our experimental data in the different pH_e in the presence of 2 or 20 mM Ca²⁺. First, we readjusted the parameters of the original model to describe our own experimental data at pH_e 7.4 in 2 mM Ca²⁺ (see Table I) . We then considered as working hypothesis that the modifications of gating induced by H⁺ and Ca²⁺ are due to: (a) the neutralization of surface charges, (b) state-dependent alterations of the electric field sensed by the gating charges, and (c) the modification of the gating charges. To evaluate these effects independently from each other we used the following approach. All voltage-dependent transitions, with the exception of the deactivation transitions (O C₃ and I_O I₃), were rewritten as:

where T = 25.4 mV is the thermal energy in electron-volts, q₁ and q₂ are the gating charges associated with the first and second boxes of the kinetic scheme, and ₁ and ₂ account for the coupling between the local electric potential sensed by q₁ and q₂ and the membrane potential V. We introduced a voltage offset (V_Shift) in all these rate constants to describe the neutralization of surface charges. To account for the state-dependent modification of the electric field sensed by the gating charges, we included the voltage offset V_O2 in the second box of the kinetic scheme (see Fig. 13). Starting from the set of parameters obtained ^</