Novel consequences of voltage-dependence to G-protein-coupled P2Y1 receptors.

BACKGROUND AND PURPOSE: Emerging evidence suggests that activation of G-protein-coupled receptors (GPCRs) can be directly regulated by membrane voltage. However, the physiological and pharmacological relevance of this effect remains unclear. We have further examined this phenomenon for P2Y1 receptors in the non-excitable megakaryocyte using a range of agonists and antagonists. EXPERIMENTAL APPROACH: Simultaneous whole-cell patch clamp and fura-2 fluorescence recordings of rat megakaryocytes, which lack voltage-gated Ca2+ influx, were used to examine the voltage-dependence of P2Y1 receptor-evoked IP3-dependent Ca2+ mobilization. RESULTS: Depolarization transiently and repeatedly enhanced P2Y1 receptor-evoked Ca2+ mobilization across a wide concentration range of both weak, partial and full, potent agonists. Moreover, the amplitude of the depolarization-evoked [Ca2+]i increase displayed an inverse relationship with agonist concentration, such that the greatest potentiating effect of voltage was observed at near-threshold levels of agonist. Unexpectedly, depolarization also stimulated an [Ca2+]i increase in the absence of agonist during exposure to the competitive antagonists A3P5PS and MRS2179, or the allosteric enhancer 2,2'-pyridylisatogen tosylate. A further effect of some antagonists, particularly suramin, was to enhance the depolarization-evoked Ca2+ responses during co-application of an agonist. Of several P2Y1 receptor inhibitors, only SCH202676, which has a proposed allosteric mechanism of action, could block ADP-induced voltage-dependent Ca2+ release. CONCLUSIONS AND IMPLICATIONS: The ability of depolarization to potentiate GPCRs at near-threshold agonist concentrations represents a novel mechanism for coincidence detection. Furthermore, the induction and enhancement of voltage-dependent GPCR responses by antagonists has implications for the design of therapeutic compounds.

Distinct mechanisms for activation of Cl- and K+ currents by Ca2+ from different sources in mouse sympathetic neurones.

We have investigated the roles of different voltage-dependent Ca2+ channels in the activation of the Cl- and K+ channels responsible for the afterdepolarization (ADP) and slow afterhyperpolarization (AHP) in sympathetic neurones of the isolated mouse superior cervical ganglion in vitro. The ADP and its associated Ca2+-activated Cl- current were markedly decreased by omega-agatoxin IVA (40-200 nM) and nifedipine (1-10 microM), but not by omega-conotoxin GVIA (300 nM). In contrast, the AHP and the apamin-sensitive Ca2+-activated K+ current that underlies this potential were blocked by omega-conotoxin GVIA, but were not affected by omega-agatoxin IVA and were only slightly reduced by nifedipine. Ryanodine (20 microM) reduced the Ca2+-activated Cl- current following an action potential by 75% but on average did not affect the Ca2+-activated K+ current. Evidence that R-type channels provide a proportion of the Ca2+ activating both types of Ca2+-dependent channel was obtained. We conclude that Ca2+ entering through L- and P-type Ca2+ channels preferentially activates the Cl- current responsible for the ADP in mouse sympathetic neurones, predominantly via Ca2+-induced Ca2+ release, whereas the Ca2+ that activates the K+ channels responsible for the AHP enters predominantly through N-type channels. The data can be explained by the selective association of each type of Ca2+ channel with particular intracellular mechanisms for activating other membrane channels, one indirect and the other direct, probably located at discrete sites on the soma and dendrites.

Diversity of channels involved in Ca(2+) activation of K(+) channels during the prolonged AHP in guinea-pig sympathetic neurons.

The types of Ca(2+)-dependent K(+) channel involved in the prolonged afterhyperpolarization (AHP) in a subgroup of sympathetic neurons have been investigated in guinea pig celiac ganglia in vitro. The conductance underlying the prolonged AHP (gKCa2) was reduced to a variable extent in 100 nM apamin, an antagonist of SK-type Ca(2+)-dependent K(+) channels, and by about 55% in 20 nM iberiotoxin, an antagonist of BK-type Ca(2+)-dependent K(+) channels. The reductions in gKCa2 amplitude by apamin and iberiotoxin were not additive, and a resistant component with an amplitude of nearly 50% of control remained. These data imply that, as well as apamin- and iberiotoxin-sensitive channels, other unknown Ca(2+)-dependent K(+) channels participate in gKCa2. The resistant component of gKCa2 was not abolished by 0.5-10 mM tetraethylammonium, 1 mM 4-aminopyridine, or 5 mM glibenclamide. We also investigated which voltage-gated channels admitted Ca(2+) for the generation of gKCa2. Blockade of Ca(2+) entry through L-type Ca(2+) channels has previously been shown to reduce gKCa2 by about 40%. Blockade of N-type Ca(2+) channels (with 100 nM omega-conotoxin GVIA) and P-type Ca(2+) channels (with 40 nM omega-agatoxin IVA) each reduced the amplitude of gKCa2 by about 35%. Thus Ca(2+) influx through multiple types of voltage-gated Ca(2+) channel can activate the intracellular mechanisms that generate gKCa2. The slow time course of gKCa2 may be explained if activation of multiple K(+) channels results from Ca(2+) influx triggering a kinetically invariant release of Ca(2+) from intracellular stores located close to the membrane.

Electrophysiological roles of L-type channels in different classes of guinea pig sympathetic neuron.

The electrophysiological consequences of blocking Ca(2+) entry through L-type Ca(2+) channels have been examined in phasic (Ph), tonic (T), and long-afterhyperpolarizing (LAH) neurons of intact guinea pig sympathetic ganglia isolated in vitro. Block of Ca(2+) entry with Co(2+) or Cd(2+) depolarized T and LAH neurons, reduced action potential (AP) amplitude in Ph and LAH neurons, and increased AP half-width in Ph neurons. The afterhyperpolarization (AHP) and underlying Ca(2+)-dependent K(+) conductances (gKCa1 and gKCa2) were reduced markedly in all classes. Addition of 10 microM nifedipine increased input resistance in LAH neurons, raised AP threshold in Ph and LAH neurons, and caused a small increase in AP half-width in Ph neurons. AHP amplitude and the amplitude and decay time constant of gKCa1 were reduced by nifedipine in all classes; the slower conductance, gKCa2, which underlies the prolonged AHP in LAH neurons, was reduced by 40%. Surprisingly, AHP half-width was lengthened by nifedipine in a proportion of neurons in all classes; despite this, neuron excitability was increased during a maintained depolarization. Nifedipine's effects on AHP half-width were not mimicked by 2 mM Cs(+) or 2 mM anthracene-9-carboxylic acid, a blocker of Cl(-) channels, and it did not modify transient outward currents of the A or D types. The effects of 100 microM Ni(2+) differed from those of nifedipine. Thus in Ph neurons, Ca(2+) entry through L-type channels during a single action potential contributes to activation of K(+) conductances involved in both the AP and AHP, whereas in T and LAH neurons, it acts only on gKCa1 and gKCa2. These results differ from the results in rat superior cervical ganglion neurons, in which L-type channels are selectively coupled to BK channels, and in hippocampal neurons, in which L-type channels are selectively coupled to SK channels. We conclude that the sources of Ca(2+) for activating the various Ca(2+)-activated K(+) conductances are distinct in different types of neuron.