Fast activation of dihydropyridine-sensitive calcium channels of skeletal muscle. Multiple pathways of channel gating.

Abstract

Dihydropyridine (DHP) receptors of the transverse tubule membrane play two roles in excitationcontraction coupling in skeletal muscle: (a) they function as the voltage sensor which undergoes fast transition to control release of calcium from sarcoplasmic reticulum, and (b)they provide the conducting unit of a slowly activating L-type calcium channel. To understand this dual function of the DHP receptor, we studied the effect of depolarizing conditioning pulse on the activation kinetics of the skeletal muscle DHP-sensitive calcium channels reconstituted into lipid bilayer membranes. Activation of the incorporated calcium channel was imposed by depolarizing test pulses from a holding potential of -80 mV. The gating kinetics of the channel was studied with ensemble averages of repeated episodes. Based on a first latency analysis, two distinct classes of channel openings occurred after depolarization: most had delayed latencies, distributed with a mode at 70 ms (slow gating); a small number of openings had short first latencies, <12 ms (fast gating). A depolarizing conditioning pulse to +20 mV placed 200 ms before the test pulse (-10 mV), led to a significant increase in the activation rate of the ensemble averaged-current; the time constant of activation went from "fin = 110 ms (reference) to T,,~ = 45 ms after conditioning. This enhanced activation by the conditioning pulse was due to the increase in frequency of fast open events, which was a steep function of the intermediate voltage and the interval between the conditioning pulse and the test pulse. Additional analysis demonstrated that fast gating is the property of the same individual channels that normally gate slowly and that the channels adopt this property after a sojourn in the open state. The rapid secondary activation seen after depolarizing prepulses is not compatible with a linear activation model for the calcium channel, but is highly consistent with a cyclical model. A six-state cyclical model is proposed for the DHP-sensitive Ca channel, which pictures the normal pathway of activation of the calcium channel as two voltagedependent steps in sequence, plus a voltage-independent step which is rate limiting. The model reproduced well the fast and slow gating modes of the calcium channel, and the effects of conditioning pulses. It is possible that the voltage-sensitive gating transitions of the DHP receptor, which occur early in the calcium channel activation sequence, could underlie the role of the voltage sensor and yield the rapid excitation-contraction coupling in skeletal muscle, through either electrostatic or allosteric linkage to the ryanodine receptors/calcium release channels.

Inactivation of L-type calcium channels is determined by the length of the N terminus of mutant β1 subunits

“…Selection of the gating model for the single-channel data Different gating schemes were proposed to describe behavior of L-type calcium channels [6,18,21,27,29]. Here, we sought a simple model, which adequately fits the following single-channel data: As other voltage-gated ion channels, L-type calcium channels proceed through the sequence of closed states before they open.…”

Gating of the HypoPP-1 mutations: I. Mutant-specific effects and cooperativity

“…Common questions arising are (1) an existence of multiple open states [9,14,42] and (2) an evidence for multiple closed states [5,11,17,30]. To answer these questions, we tested several models for their suitability in fitting ionic currents.…”

“…Calcium channels, as other voltage-gated cation channels, were suggested to pass a series of non-conducting (closed) states before channel opening; however, different numbers of closed states were proposed for Ca 2+ channel gating [5,11,17,30]. Considering channel structure where four voltage sensors are thought to move upon membrane depolarisation [3], analogously to other voltage-gated cation channels [24] and as the model with four closed states (Fig.…”

“…The most prominent effect of HypoPP-1 mutations was a reduction of current density [26,28,31,32]. Changes of steady-state and kinetic parameters of activation in HypoPP-1 mutants were controversial among different groups [23,[29][30][31][32] and even within the same group [31,32]. The main reason for controversies was a different analysis of calcium channel gating.…”

“…In contrast to other voltage-gated cation channels, calcium channels have a very pronounced second component of activation due to an existence of the second open state, characterised by long-lasting openings [9,10,14,42]. However, all previous works on HypoPP-1 mutants did not take into account this characteristic feature of calcium channel gating, analyzing only peak amplitudes and singleexponential activation [23,26,[29][30][31][32].…”