Ca2+- and voltage-dependent inactivation of Ca2+ channels in nerve terminals of the neurohypophysis

Abstract

Ca2+ channel inactivation was investigated in neurohypophysial nerve terminals by using patch-clamp techniques. The contribution of intracellular Ca2+ to inactivation was evaluated by replacing Ca2+ with Ba2+ or by including BAPTA in the internal recording solution. Ca2+ channel inactivation during depolarizing pulses was primarily voltage-dependent. A contribution of intracellular Ca2+ was revealed by comparing steady-state inactivation of Ca2+ channels with Ca2+ current and with intracellular [Ca2+]. However, this contribution was small compared to that of voltage. In contrast to voltage-gated Ca2+ channels in other preparations, in the neurohypophysis Ba2+ substitution or intracellular BAPTA increased the speed of inactivation while reducing the steady-state level of inactivation. Ca2+ channel recovery from inactivation was studied by using a paired-pulse protocol. The rate of Ca2+ channel recovery from inactivation at negative potentials was increased dramatically by Ba2+ substitution or intracellular BAPTA, indicating that intracellular Ca2+ inhibits recovery. Stimulation with trains of brief pulses designed to mimic physiological bursts of electrical activity showed that Ca2+ channel inactivation was much greater with 20 Hz trains than with 14 Hz trains. Inactivation induced by 20 Hz trains was reduced by intracellular BAPTA, suggesting an important role for Ca2+-dependent inactivation during physiologically relevant forms of electrical activity. Inhibitors of calmodulin and calcineurin had no effect on Ca2+ channel inactivation, arguing against a mechanism of inactivation involving these Ca2+-dependent proteins. The inactivation behavior described here, in which voltage effects on Ca2+ channel inactivation predominate at positive potentials and Ca2+ effects predominate at negative potentials, may be relevant to the regulation of neuropeptide release.

Fig. 1.

Calcium current inactivation. A, Calcium current recorded in response to a 500 msec pulse from −100 to 10 mV. The decay shown was fit with a biexponential function with the following parameters: τ_fast = 35 msec, τ_slow = 126 msec, A_fast = −400 pA, A_slow = −336 pA, andA₀ = −32 pA (see Materials and Methods, Eq. 2). B, Ca²⁺ currents were evoked by trains of 2 msec pulses from −100 to 50 mV. Peak currents measured during trains were normalized to the first current response, averaged, and plotted versus time. Error bars are plotted at 1 sec intervals to avoid obscuring the data. Inactivation was frequency-dependent and decayed monoexponentially at 14 Hz (τ₁ = 6.45 sec;n = 6) and biexponentially at 20 Hz (τ₁ = 0.96 and τ₂ = 5.79 sec;n = 11). The fitted exponential functions were drawn in both A and B, but they are nearly concealed by the data.

Fig. 2.

Current through Ca²⁺ channels with Ba²⁺ substitution and intracellular BAPTA.A, Extracellular Ca²⁺ was replaced by Ba²⁺ (left), or 10 mmBAPTA was included in the patch pipette filling solution (right). Current was activated by 500 msec pulses from −100 to 10 mV, as in Figure 1. Current traces were normalized to their peak values and displayed together with normalized control Ca²⁺ current from Figure 1. Inactivation was quantified by fitting the decay of the current to a sum of exponentials. This yielded the following parameters for the traces shown: τ_fast = 26 msec, τ_slow = 144 msec,A_fast = −192 pA,A_slow = −247 pA, andA₀ = −91 pA for Ba²⁺substitution; τ_fast = 38 msec, τ_slow = 152 msec, A_fast = −397 pA,A_slow = −171 pA, andA₀ = −29 pA for intracellular BAPTA (see Materials and Methods, Eq. 2). B, Time constants for inactivation of Ca²⁺ channels are shown for pulses to 0 mV. The values for Ba²⁺ substitution and intracellular BAPTA differ significantly from controls (p < 0.01 for each pairwise comparison). For controls, n = 16; for Ba²⁺,n = 12; for BAPTA, n = 11.C, Plots of peak current versus voltage for control Ca²⁺ current (▪), Ba²⁺ current through Ca²⁺ channels (•), and Ca²⁺ current with intracellular BAPTA (▴).

Fig. 6.

Recovery of Ca²⁺ current from inactivation. Ca²⁺ current was inactivated with 500 msec pulses from −100 to 10 mV. Then recovery was examined with 100 msec test pulses applied at various time intervals after the end of the inactivating pulse. A, Ba²⁺currents are shown, and in these traces the noise appears greater than it actually is because the four traces selected do not superimpose perfectly. B, Normalized peak current from traces such as those in A was plotted versus the interpulse interval to show the time course of recovery. The current at the end of the first inactivating pulse was subtracted from the peak current, and this difference was plotted versus interpulse interval. The best-fitting double-exponential functions were drawn through the data (see text for parameter values and n values). In these fits we imposed the constraint A_fast +A_slow = −A₀ and set t₀ = 0 (see Materials and Methods, Eq. 2). C, Data in B are replotted with an expanded time scale to show the rapid component of recovery more clearly.

Fig. 7.

Inverse correlation between recovery and [Ca²⁺]_i. The time course of recovery of Ca²⁺ current from inactivation (τ = 1.25 sec) was similar to that for recovery of [Ca²⁺]_i (τ = 2.20 sec). Data were pooled from five nerve terminals. Inset, [Ca²⁺]_i measured during a 300 msec pulse from −100 to 10 mV. Arrows indicate the times at which the test pulses were applied to produce points in the plot.

Fig. 3.

Steady-state inactivation. A, The current traces shown were recorded by using the steady-state inactivation protocol indicated schematically by theinset. The prepulses were −50 mV (a), −10 mV (b), and 50 mV (c). Control pulses were similar each time and therefore are not labeled. Note the decrease in inactivation with a prepulse of 50 mV (c) when compared with a prepulse of −10 mV (b).B, Peak test pulse currents were normalized to peak control pulse currents and plotted against the prepulse voltage (n = 21). Points from −120 to 10 mV were fit to a Boltzmann equation (I₁ = 0.98,I₂ = 0.07,V_½ = −30.5 mV, andk = 12.1 mV; see Materials and Methods, Eq. 3), and points from 10 to 70 mV were fit to a line (slope = 0.0040 ± 0.0003 mV⁻¹). Inactivation decreased at prepulse potentials more positive than 10 mV.

Fig. 4.

Steady-state inactivation is correlated with prepulse [Ca²⁺]_i. A, [Ca²⁺]_i was measured during the steady-state inactivation protocol illustrated in Figure 3. Prepulse steps were given at t = 0.5 sec for 500 msec to the following voltages: −50 mV (a), 10 mV (b), and 50 mV (c). Thearrow at the top indicates the [Ca²⁺]_i level at the end of the prepulse that is plotted in B. With no added Ca²⁺ buffer (other than 0.1 mm fura-2), resting [Ca²⁺]_i was 0.35 ± 0.04 μm (n = 7). B, Steady-state inactivation and [Ca²⁺]_iat the end of the prepulse (shown in A) are plotted as a function of prepulse voltage (n = 7). The fraction of noninactivated current and [Ca²⁺]_iis inversely related.

Fig. 5.

Dependence of steady-state inactivation on [Ca²⁺]_i. Steady-state inactivation of Ca²⁺ current was studied with the paired-pulse protocol shown in Figure 3, using Ba²⁺ substitution (A) and intracellular BAPTA (B). Points from −120 to −10 mV for Ba²⁺ and −110 to 10 mV for BAPTA were fit to a Boltzmann equation (see Materials and Methods, Eq. 3) to obtain the following parameters: for Ba²⁺,I₁ = 1.01, I₂ = 0.19, V_½ = −41.9 mV, andk = 10.0 mV; for BAPTA,I₁ = 0.98, I₂ = 0.11, V_½ = −27.4 mV, andk = 10.8 mV (see Materials and Methods, Eq. 3). The curves based on these fits were extended to 70 mV to emphasize the upward slope at positive potentials. Points from 10 to 70 mV were fit to a line (see text for slopes). Control data from Figure3B with 5 mm Ca²⁺ as the charge carrier are reproduced for comparison.

Fig. 8.

Ca²⁺ dependence of inactivation during trains. A, Ca²⁺ current was evoked by a train of 2 msec pulses, as in Figure1B, but with BAPTA included in the patch pipette solution. BAPTA reduced inactivation during a 20 Hz train to that seen with a 14 Hz train. Normalized peak Ca²⁺ currents were averaged and plotted versus time with error bars at 1 sec intervals. The time constants from fits of double exponential functions (see Materials and Methods, Eq. 2) were τ₁ = 0.69 and τ₂ = 26.3 sec at 14 Hz (n = 4), and τ₁ = 0.40 and τ₂ = 24.7 sec at 20 Hz (n = 5). B, [Ca²⁺]_i rises to a plateau during 14 and 20 Hz trains (with no intracellular BAPTA). The increases and decreases were both faster at 20 Hz. Baseline [Ca²⁺]_i was 300 nm in this terminal, and the plateau was 0.7 μm for both frequencies (see text for averages). The inset shows two selected [Ca²⁺]_i traces from different terminals on an expanded time scale to illustrate the different rates of rise for 14 and 20 Hz trains. The time constants in these two traces were 3.2 and 0.5 sec, respectively (see text for average time constants for both rises and falls in [Ca²⁺]_i).