GPCR Regulation of Voltage-Gated Ca2<sup>+</sup> Channels

The most prominent and by far best studied mechanism is the so-called voltage-dependent inhibition (Figure 6). This is found in Ca*V*2.x channels, where the G-protein G*βγ* subunits can directly bind to the calcium channel *α*1 subunit. This binding event leads to a shift in the gating mode from *"wiilling"* to a *"reluctant"* one that manifests itself mainly in a marked slowing of activation [172–174]. The term "voltage dependent" refers to the fact that depolarization can relief the channels from inhibition and restore normal gating. As this type of inhibition is mostly exerted by G*αi*/*o*-coupled receptors it can be abolished by treating the cells with pertussis toxin (PTX) that ribosilates G*αi*/*o*-proteins and renders them inactive.

**Figure 6.** Ca*<sup>V</sup>* channels are activated by depolarizing voltages (as indicated). Activation of a G*αi*/*o*-coupled receptor (**right**) leads to a phenomenon called "voltage-dependent inhibition". This involves direct binding of the G*βγ* dimer to Ca*V*2.x channels. Activation of G*αs*-coupled receptors (**center**) leads to activation of adenylyl cyclase (AC), which forms cAMP (cyclic adenosine monophosphate) to activate protein kinase A (PKA). PKA-mediated phosphorylation of Ca*V*1.x channels was shown to increase their currents. Activation of a G*αq*/11-coupled receptor (**left**) stimulates phospholipase C (PLC), which hydrolyzes phosphatitylinositol 4,5 bisphosphate (PIP2). Depletion of PIP2 is sufficient to decrease currents through Ca*<sup>V</sup>* channels.

One of the best studied examples for voltage dependent calcium channel inhibition is the action of opioid receptors. All three types of opioid receptors, *μ* (MOP), *κ* (KOP) and *δ* (DOP), are found in DRG neurons, with the exact expression pattern depending on the cell type [175,176]. Initially, it was found that exposure of nociceptive neurons to opioids leads to a shortening of action potential durations [177–181]. It was demonstrated that application of [D-Ala2]-enkephalin (DADLE), an unspecific opioid receptor agonist, not only reduced action potential duration, but also diminished substance P release in these neurons [177]. As became clear later on, both effects were caused mainly by the inhibition of Ca2<sup>+</sup> channels [172,182]. The major target for opioid modulation are Ca*V*2.x channels [183–188]. These channels are found at the presynapse and govern neurotransmitter release [170], thus inhibition of Ca*V*2.x channels leads to reduced Ca2<sup>+</sup> influx and concomitantly reduced transmitter release from peripheral nociceptive neurons onto second-order neurons of the pain pathway located in the spinal dorsal horn. In line with the fact that opioid receptors couple predominantly to G*αi*/*<sup>o</sup>* G proteins [110], opioid induced calcium channel inhibition was found to be PTX sensitive [188], and voltage dependent [172]. These findings were corroborated by intracellular administration of G*α<sup>o</sup>* antiserum, that strongly reduced opioid receptor mediated I*Ca* inhibition [189], unequivocally demonstrating the mechanism of action.

Besides opioid receptors, many other GPCRs expressed in DRG neurons were found to lead to a similar kind of calcium channel modulation. For example GABA*<sup>B</sup>* [190], adenosine A1 [191], 5-HT [163,192–195], P2Y [196], cannabinoid [197], neuropeptide Y Y2 receptors [198–200], somatostatin SST4 [201] or *α*<sup>2</sup> adrenoceptors [202].

Besides the well-studied G*βγ* mediated voltage dependent inhibition, other mechanisms have also been described [174]. Most prominently, phospholipase C (PLC) mediated PIP2 depletion can lead to inhibition of calcium channels [203–205]. Similar to K*V*7 and K*ir*3 channels (see below), PIP2 stabilizes the open state of calcium channels. Thus, a reduction in membrane PIP2 leads to a voltage independent decrease in channel open probability.

While several reports about this kind of inhibition exist from sympathetic neurons [174], there are few data from nociceptive neurons. However, given the similarity of receptor and channel expression between sympathetic and sensory neurons, it is reasonable to assume that these findings will also hold true in DRG neurons. Only recently, the Mas-related G protein coupled receptor type C (MrgC) was found to inhibit high voltage-gated calcium channels in a PLC dependent manner [206].

Recently, a potentially novel form of calcium channel inhibition has been described. Huang et al. [207] found that GABA*<sup>B</sup>* receptors not only inhibit HVA channels but also LVA channels. Inhibition of both channel types was PTX sensitive, however the LVA inhibition was strongly reduced by application of DTT, pointing to a novel mechanism that involves redox processes.

GPCRs cannot only inhibit Ca2<sup>+</sup> channels but they are also known to be able to facilitate their function. A classic example would be PKA which phosphorylates Ca*V*1.x channels and increases their currents [208]. In line with this, a broadening of the action potential upon application of noradrenaline to DRG neurons was reported. This increase in action potential duration could finally be attributed to an increase in Ca*V*1.x currents [202].
