Ca2+-dependent inactivation of CaV1.2 channels prevents Gd3+ block: does Ca2+ block the pore of inactivated channels?

Abstract

Lanthanide gadolinium (Gd(3+)) blocks Ca(V)1.2 channels at the selectivity filter. Here we investigated whether Gd(3+) block interferes with Ca(2+)-dependent inactivation, which requires Ca(2+) entry through the same site. Using brief pulses to 200 mV that relieve Gd(3+) block but not inactivation, we monitored how the proportions of open and open-blocked channels change during inactivation. We found that blocked channels inactivate much less. This is expected for Gd(3+) block of the Ca(2+) influx that enhances inactivation. However, we also found that the extent of Gd(3+) block did not change when inactivation was reduced by abolition of Ca(2+)/calmodulin interaction, showing that Gd(3+) does not block the inactivated channel. Thus, Gd(3+) block and inactivation are mutually exclusive, suggesting action at a common site. These observations suggest that inactivation causes a change at the selectivity filter that either hides the Gd(3+) site or reduces its affinity, or that Ca(2+) occupies the binding site at the selectivity filter in inactivated channels. The latter possibility is supported by previous findings that the EEQE mutation of the selectivity EEEE locus is void of Ca(2+)-dependent inactivation (Zong Z.Q., J.Y. Zhou, and T. Tanabe. 1994. Biochem. Biophys. Res. Commun. 201:1117-11123), and that Ca(2+)-inactivated channels conduct Na(+) when Ca(2+) is removed from the extracellular medium (Babich O., D. Isaev, and R. Shirokov. 2005. J. Physiol. 565:709-717). Based on these results, we propose that inactivation increases affinity of the selectivity filter for Ca(2+) so that Ca(2+) ion blocks the pore. A minimal model, in which the inactivation "gate" is an increase in affinity of the selectivity filter for permeating ions, successfully simulates the characteristic U-shaped voltage dependence of inactivation in Ca(2+).

Figure 1.

Tail currents reveal that Gd³⁺ reduces inactivation in Ca²⁺. (A) Voltage-pulse protocol used. 20-ms step to 200 mV was applied to relieve Gd³⁺ block at different times of the pulse to 20 mV. (B) Currents from a cell bathed in solution with 10 mM Ca²⁺ and 0 Gd³⁺. The peaks of tail currents in response to stepping from 200 to 20 mV followed the time course of inactivation. The dashed line through the peaks is the best fit by an exponential: I = −I₀ + ΔI (1 − e^−kt), where I₀ = 979 pA, ΔI = 669 pA, and k = 0.010 ms⁻¹. The ratio between the numbers of inactivated and noninactivated channels after 500 ms at 20 mV can be estimated by ΔI/(I₀ − ΔI). On average, it was 2.1 ± 0.17 (n = 6). (C) Currents from the same cell bathed in solution with 10 mM Ca²⁺ and 50 nM Gd³⁺. The dashed line through the peaks is the best fit with I₀ = 986 pA, ΔI = 552 pA, and k = 0.011 ms⁻¹. The averaged ΔI/(I₀ − ΔI) ratio was 1.24 ± 0.14 (n = 6). (D) Currents from the same cell bathed in solution with 10 mM Ca²⁺ and 1 μM Gd³⁺. The dashed line through the peaks is the best fit with I₀ = 948 pA, ΔI = 328 pA, and k = 0.007 ms⁻¹. The averaged ΔI/(I₀ − ΔI) ratio was 0.51 ± 0.13 (n = 6).

Figure 2.

Inactivation of Ba²⁺ currents in the presence of Gd³⁺. Currents were elicited similar to that in Fig 1. The 20-ms step to 200 mV was applied after 5 ms at 0 mV (traces a and c), or after 500 ms at 0 mV (traces b and d). Without the blocker (traces a and b), the peaks of the tails differ because of inactivation. With 25 nM Gd³⁺ (traces c and d), currents elicited by the step from −90 to 0 mV were smaller and decayed more rapidly. However, the 200-mV pulse after 5ms at 0 mV relieved the tonic Gd³⁺ block to reveal the magnitude of the unblocked current (compare traces a and c). Gd³⁺ did not change the magnitude of tail currents after 500 ms at 0 mV (compare traces b and d). The ratio between the numbers of inactivated and noninactivated channels after 500 ms at 0 mV was estimated directly from the traces as indicated. On average, it was 0.43 ± 0.11 (n = 5) without Gd³⁺ and 0.35 ± 0.09 after addition of 25 nM of Gd³⁺.

Figure 3.

The CAM1234 mutation of calmodulin reduced inactivation of Ca²⁺ currents, but it did not alter open-channel block by Gd³⁺. Currents were elicited by the same pulse protocol as in Fig. 1 A. Only traces with 500-ms pulse from −90 to 20 mV are shown. Although in the absence of Gd³⁺ (black traces) inactivation of currents at 20 mV was much less in the cell with CAM1234, application of 0.1 μM Gd³⁺ (gray traces) reduced the peak and accelerated the decay of currents similarly in the wild-type cell and in the cell with the mutated calmodulin. Since the step to 200 mV did not relieve inactivation (Fig. 1 B), but relieved Gd³⁺ block (see accompanying paper Babich et al., 2007), the ratio between the numbers of open and open-blocked channels after 500 ms at 20 mV can be simply estimated from the current before the step to 200 mV and from the peak of the tail current on the return from 200 to 20 mV, as indicated. On average, the ratio was 0.161 ± 0.012 in the wild-type cells (n = 3) and 0.156 ± 0.014 (n = 4) in cells with CAM1234.

Figure 4.

A model of ion-dependent inactivation.

Figure 5.

Simulated Ca²⁺ currents using the single-site approximation to calculate single-channel currents. The “inactivation-binding” coupling factor was γ = 50. (A) Total currents. Voltage steps were from the holding potential −100 mV to −60–90 mV, increment 10 mV. (B) Currents through inactivated channels. (C) Peak current–voltage relationship. (D) Rates of the best fits by the sum of an exponential and a constant to the decay phase of currents in A.