Calcium channels

The release of neurotransmitters from synaptic nerve terminals is triggered by the influx of calcium ions (Ca2+) through voltage-gated Ca2+ channels (VDCC) located near vesicle docking sites. Calcium ions act in concert with distinct components of the presynaptic docking machinery to facilitate the fusion of synaptic vesicles with the plasma membrane. Modulating the entry of Ca2+ into the nerve terminal thus represents a major means by which neurotransmitter secretion can be controlled. There is substantial evidence that most VDCC are influenced by metabotropic pathways in many neurons and secretory cells (Hille 1994;

Dolphin 1995). These channels are placed into discrete subfamilies based on their voltage-dependence of activation as well as their sensitivity to different pharmacological toxins. In both neurons and cardiomyocytes, high-threshold activated channels (L,N,P,Q,R-type) open in response to large depolarizations relative to the resting membrane potential. Multiple neurotransmitters including dopamine, GABA and glutamate act through G;/o-coupled receptors to inhibit the opening of N- and P/Q-type Ca2+ channels in the central nervous system (reviewed by Hille 1994). G proteins were first implicated in this inhibition by studies showing that PTX blocks the response to some transmitters while GTPyS causes channel inhibition in the absence of receptor activation (Holz et al. 1986; Dolphin and Scott 1987). Macroscopic measurements of Ca2+ currents have revealed that the receptor-induced inhibition is due to a shift in the speed and voltage-dependence of channel opening. Receptor stimulation shifts a fraction of the channels into a 'reluctant' gating mode which decreases their voltage-sensitivity and makes them less likely to open at moderate depolarizing potentials (Bean 1989). The agonist-induced inhibition can be transiently overcome by large depolarizing (facilitating) prepulses that counteract the voltage-dependent block of the channels (Hille 1994).

Results from many studies revealed that these changes in N-type currents are largely mediated by a membrane-delimited pathway that does not involve second messengers (Forscher et al. 1986; Lipscombe et al. 1989; Shapiro and Hille 1993). Instead, channel inhibition can be mimicked in rat sympathetic ganglion neurons by the transient overexpression of exogenous GPy subunits even in the absence of receptor activation (Ikeda 1996; Herlitze et al. 1996). Similar results have been obtained in cell lines expressing recombinant P/Q-type Ca2+ channel subunits as well as GPy dimers (Herlitze et al. 1996). Purified GPy protein is also able to reproduce the receptor-evoked inhibition of endogenous Ca2+ channels when directly introduced into chick dorsal root ganglion (DRG) cells as well as rat superior cervical ganglion cells (Diverse-Pierluissi et al. 1995; Herlitze et al. 1996). GPy subunits bind to a cytoplasmic linker region found between transmembrane domains I and II in the a1 subunit of the N-type Ca2+ channel. This region contains a Py-binding motif (QXXER) that is found in adenylyl cyclase and is required for G protein regulation of the enzyme (Chen et al. 1995). De Waard et al. (1997) found that fusion proteins encompassing this region of the a1A channel subunit bound specifically to in vitro translated as well as rat brain-solubilized GPy subunits, whereas no binding was detected with activated Ga-GTPyS. Mutations of certain amino acids in a segment containing the QXXER motif together with several flanking residues prevented the inhibitory effects of exogenous GPy subunits on Ca2+ channels expressed in Xenopus oocytes. In addition, peptides containing the QXXER sequence are able to interfere with GPy-induced inhibition of Ca2+ currents in HEK 293 cells presumably by acting as binding competitors (Zamponi et al. 1997). An interesting observation derives from the finding that the same cytoplasmic linker region that binds to GPy has also been shown to interact with the Ca2+ channel P-subunit (Pragnell et al. 1994; Witcher et al. 1995). This protein influences the voltage-sensitivity of the channel when co-expressed together with a1 subunits. The binding of G proteins to the channel may therefore alter the voltage-dependence of activation by controlling various aspects of this interaction.

Additional domains within the amino terminus, the cytoplasmic linker regions, as well as the C-terminus have all been purported to interact with G Protein Py subunits (Zhang et al. 1996; De Waard et al. 1997; Furukawa et al. 1998; Canti et al. 1999). Taken together, these results suggest that neurotransmitters bind to their cognate receptors to activate a PTX-sensitive G protein which goes on to depress both N- and P/Q-type Ca2+ currents via a membrane-delimited pathway (i.e. one that does not involve cytosolic transduction elements) that most likely results from direct interactions with the channel. G^y subunits presumably bind to several different regions of the channel protein to promote a shift in the voltage-dependence of activation, which causes fewer channels to open with moderate depolarizations (-50 to 0mV). The consequences of such regulation can have enormous impacts on the frequency with which nerve impulses are transmitted between cells, and they are likely to play important roles in the modulation of synaptic strength.

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