Role of extracellular Ca2+ in gating of CaV1.2 channels

Abstract

We examined changes in ionic and gating currents in Ca(V)1.2 channels when extracellular Ca(2+) was reduced from 10 mm to 0.1 microm. Saturating gating currents decreased by two-thirds (K(D) approximately 40 microm) and ionic currents increased 5-fold (K(D) approximately 0.5 microm) due to increasing Na(+) conductance. A biphasic time dependence for the activation of ionic currents was observed at low [Ca(2+)], which appeared to reflect the rapid activation of channels that were not blocked by Ca(2+) and a slower reversal of Ca(2+) blockade of the remaining channels. Removal of Ca(2+) following inactivation of Ca(2+) currents showed that Na(+) currents were not affected by Ca(2+)-dependent inactivation. Ca(2+)-dependent inactivation also induced a negative shift of the reversal potential for ionic currents suggesting that inactivation alters channel selectivity. Our findings suggest that activation of Ca(2+) conductance and Ca(2+)-dependent inactivation depend on extracellular Ca(2+) and are linked to changes in selectivity.

Figure 1. Two components of activation of Na⁺ currents

A, ionic currents with 150 mm Na⁺ and different [Ca²⁺] (indicated) in the extracellular solution. Voltage pulses were applied from −90 mV holding potential to −60 to +50 mV. Red lines illustrate fits by I=A(1 − exp(−k_At)) +B(1 − exp(−k_Bt)). Traces at 40 μm and 10 mm Ca²⁺ were fitted by a single exponential. B, rates of the fast component of a bi-exponential fit to activation of currents at submicromolar Ca²⁺ (coloured symbols) and rates of a single exponential fit to activation of currents at 10 mm Ca²⁺ (black symbols). The dashed line shows the rates for 10 mm Ca²⁺ shifted by −40 mV. C, rates of the slow component of a bi-exponential fit to activation of currents at submicromolar Ca²⁺ (coloured symbols) and rates of a single exponential fit to activation of currents at 40 μm Ca²⁺ (dark green symbols). The dashed line is the same as in B. D, relative contributions of the fast component.E, magnitudes of the fit. The magnitudes were normalized to the value at 20 mV and 10 mm Ca²⁺ in each cell. n = 4–7.

Figure 2. Effect of extracellular Ca²⁺ on gating currents

A, changes of gating currents at low Ca²⁺. Traces b are asymmetric currents recorded at 10 mm Ca²⁺. Traces c are for 1.35 μm Ca²⁺. Traces shown by thin lines were elicited at −150 mV, thick lines show traces elicited at the reversal potential for ionic currents (70 mV in b; and 25 mV in c). B, dependencies of the peak amplitude of ionic current (I_max, open symbols) and of the Q_max values (filled symbols) on [Ca²⁺]. Q_max values were determined as described in Supplemental material, Fig. 2. Before averaging, I_max and Q_max were normalized to their values obtained at 10 mm Ca²⁺ in the same cell. n = 6–16. The I_max values (except the points at 10 and 0.2 mm Ca²⁺) were fitted by the function I=I_∞+ΔIK_D/(K_D+[Ca²⁺]) with parameters: I_∞= 0.33 ± 0.06, ΔI= 5.51 ± 0.93, K_D= 0.49 ± 0.08 μm. The Q_max values were fitted by the function Q_max= 1 −ΔQ_maxK_D/(K_D+[Ca²⁺]) with parameters: ΔQ_max= 0.68 ± 0.11, K_D= 39 ± 14 μm. C, intramembrane charge movements recorded from 0 mV holding potential at 10 mm Ca²⁺ (b) by pulses ranging from −160 to −10 mV, and at 0.1 μm Ca²⁺ (c) by pulses ranging from −160 to −40 mV. Thick lines highlight traces at −160 mV. D, charge transfer functions for transients recorded as in C. Charges obtained by integration of the ON transients at different voltages were normalized in each cell to the Q_max value obtained by pulses to 70 mV from −90 mV holding potential at 10 mm Ca²⁺. n = 5. Parameters of the fits by the Boltzmann function at 10 mm Ca²⁺ are: Q_−∞=−0.77 ± 0.11, Q_max= 0.97 ± 0.08, V_1/2=−95 ± 7 mV, K= 27 ± 4 mV.

Figure 3. Na⁺ current was not affected by inactivation at normal Ca²⁺

A, restoration of Na⁺ current after inactivation at normal Ca²⁺. Traces a and c were elicited at 150 mm Na⁺ and 1.35 mm Ca²⁺. The red trace b was recorded starting at 150 mm Na⁺ and 10 mm Ca²⁺, and then Ca²⁺ was reduced to 1.35 μm. The dashed line is trace a scaled to the peak of trace b. B, recovery from inactivation at normal Ca²⁺ was not accelerated by the absence of Ca²⁺. Trace a was obtained at 10 mm Ca²⁺. [Ca²⁺] was changed during the pulses for trace b at the times indicated. Ca²⁺ current during the second pulse was not affected. Trace c was obtained at 150 mm Na⁺ and 1.35 μm Ca²⁺.

Figure 4. Differential effect of Ca²⁺-dependent inactivation at 10 mm Ca²⁺ on inward Ca²⁺ and outward Cs⁺ currents

A, comparison of outward currents obtained after 20 mV pre-pulses of different duration. The dotted line in b was obtained by scaling the inward current at 20 mV to go through the peak current at 100 mV after a 50 ms-long pre-pulse. B, determination of the reversal potential after inactivating pre-pulses of different duration. Tracings are shown for the second step ranging from 20 to 90 mV. The dotted line shows current elicited by a 250 ms long pre-pulse to 20 mV and the second step to 90 mV after addition of 10 μm GdCl₃ to the bath. C, instantaneous current–voltage relationships after inactivating pre-pulses of different duration as shown in B. Circles plot current magnitudes measured 2 ms after the step from a 12.5 ms-long pre-pulse to 20 mV; triangles plot current magnitudes measured 2 ms after the step from a 250 ms-long pre-pulse to 20 mV. Before averaging, the values were normalized in each cell to current amplitude at 20 mV. n = 6.