Voltage-dependent modulation of single N-Type Ca2+ channel kinetics by receptor agonists in IMR32 cells

Abstract

The voltage-dependent inhibition of single N-type Ca(2+) channels by noradrenaline (NA) and the delta-opioid agonist D-Pen(2)-D-Pen (5)-enkephalin (DPDPE) was investigated in cell-attached patches of human neuroblastoma IMR32 cells with 100 mM Ba(2+) and 5 microM nifedipine to block L-type channels. In 70% of patches, addition of 20 microM NA + 1 microM DPDPE delayed markedly the first channel openings, causing a four- to fivefold increase of the first latency at +20 mV. The two agonists or NA alone decreased also by 35% the open probability (P(o)), prolonged partially the mean closed time, and increased the number of null sweeps. In contrast, NA + DPDPE had little action on the single-channel conductance (19 versus 19.2 pS) and minor effects on the mean open time. Similarly to macroscopic Ba(2+) currents, the ensemble currents were fast activating at control but slowly activating and depressed with the two agonists. Inhibition of single N-type channels was effectively removed (facilitated) by short and large depolarizations. Facilitatory pre-pulses increased P(o) significantly and decreased fourfold the first latency. Ensemble currents were small and slowly activating before pre-pulses and became threefold larger and fast decaying after facilitation. Our data suggest that slowdown of Ca(2+) channel activation by transmitters is mostly due to delayed transitions from a modified to a normal (facilitated) gating mode. This single-channel gating modulation could be well simulated by a Monte Carlo method using previously proposed kinetic models predicting marked prolongation of first channel openings.

cGMP/Protein Kinase G-Dependent Inhibition of N-Type Ca2+Channels Induced by Nitric Oxide in Human Neuroblastoma IMR32 Cells

“…G-protein-mediated inhibition of these channels is characterized by a slowing of the current activation kinetics, which has been attributed to a time-dependent recovery from voltage-dependent inhibition (Bean, 1989). Strong membrane depolarizations can overcome this inhibitory effect by Ca 2ϩ channel facilitation consisting of changes in channel gating (Grassi and Lux, 1989;Elmslie et al, 1990;Delcour and Tsien, 1993;Carabelli et al, 1996;Lee and Elmslie, 2000). In light of these findings, it is conceivable that the nonconventional transmitter NO might contribute in some manner to the downregulation of N channel Ca 2ϩ influx induced by classical neurotransmitters.…”

“…In the presence of the L-channel blocker, N-channel activity could be recorded and clearly distinguished from that of other channels that might be found in the patch, using criteria reported in the literature (Carabelli et al, 1996;Elmslie, 1997). Data collected from patches in which non-N-type activity was present were excluded from analysis.…”

G protein inhibition of CaV2 calcium channels

“…These characteristics of the inhibition have been incorporated into models in which the channels exhibit two functional gating states, "willing" and "reluctant". [49][50][51][52][53] The "reluctant" channels are bound to Gβγ and display the voltage-dependent shifts in channel gating noted above. Two isoforms of Ca V 2.2 were identified in rat brain and one of these, Ca V 2.2a, was constitutively reluctant in the absence of Gβγ due to a single amino acid substitution in domain IS3.…”

L-type channel inhibition by CB1 cannabinoid receptors is mediated by PTX-sensitive G proteins and cAMP/PKA in GT1-7 hypothalamic neurons

“…The main effect of this "membrane-delimited" modulation is a delayed Ca 2+ channel activation at low voltages, which accelerates at higher potentials [42,43]. The slow activation derives from a prolonged latency of first channel opening [44] and is fully recovered by applying strong positive prepulses [45]. After prepulse, the N-and P/Q-type currents are facilitated: they recover their normal activation time course and increase their amplitude.…”