No abstract

The functional roles subserved by G(alpha)z, a G protein alpha subunit found predominantly in neuronal tissues, have remained largely undefined. Here, we report that G(alpha)z coupled neurotransmitter receptors to N-type Ca2+ channels when transiently overexpressed in rat sympathetic neurons. The G(alpha)z-mediated inhibition was voltage dependent and PTX insensitive. Recovery from G(alpha)z-mediated inhibition was extremely slow but accelerated by coexpression with RGS proteins. G(alpha)z selectively interacted with a subset of receptors that ordinarily couple to N-type Ca2+ channels via PTX-sensitive Go/i proteins. In addition, G(alpha)z rescued the activation of heterologously expressed GIRK channels in PTX-treated neurons. These results suggest that G(alpha)z is capable of coupling receptors to ion channels and might underlie PTX-insensitive ion channel modulation observed in neurons under physiological and pathological conditions.

Voltage-dependent (VD) inhibition of N-type Ca 2؉ channels is mediated primarily by neurotransmitter receptors that couple to pertussis toxin (PTX)-sensitive G proteins (such as G o and Gi). To date, however, the composition of heterotrimeric complexes, i.e., specific G␣␤␥ combinations, capable of coupling receptors to N-type Ca 2؉ channels has not been defined. We addressed this question by heterologously expressing identified G␣␤␥ combinations in PTX-treated rat sympathetic neurons and testing for reconstitution of agonist-mediated VD inhibition. The heterologously expressed G␣ subunits were rendered PTX-insensitive by mutating the codon specifying the ADP ribosylation site. The following results were obtained from this approach. (i) Expression of G␣ oA, G␣oB, and G␣i2 (along with G␤1␥2) reconstituted VD inhibition mediated by ␣2-adrenergic, adenosine, somatostatin, and prostaglandin E 2 receptors. Conversely, expression of G␣i1 and G␣ i3 was ineffective at restoring coupling. (ii) Coupling efficiency, as determined from the magnitude of reconstituted Ca 2؉ current inhibition, depended on both the receptor and G␣ subtype. The following rank order of coupling efficiency was observed: G␣ oA ‫؍‬ G␣ oB > G␣i2 for ␣2-adrenergic receptor; G␣i2 > G␣oA ‫؍‬ G␣oB for adenosine and prostaglandin E 2 receptors; and G␣oB ‫؍‬ G␣i2 > G␣oA for the somatostatin receptor. (iii) In general, varying the G␤␥ composition of G␣oA-containing heterotrimers had little effect on the coupling of ␣2-adrenergic receptors to the VD pathway. Taken together, these results suggest that multiple, diverse G␣␤␥ combinations are capable of coupling neurotransmitter receptors to VD inhibition of N-type Ca 2؉ channels. Thus, if exquisite G␣␤␥-coupling specificity exists in situ, it cannot arise solely from the inherent inability of other G␣␤␥ combinations to form functional signaling complexes.

Experiments using heterologous overexpression indicate that regulator of G-protein signaling (RGS) proteins play important roles in Gbetagamma-mediated ion channel modulation. However, the roles subserved by endogenous RGS proteins have not been extensively examined because tools for functionally inhibiting natively expressed RGS proteins are lacking. To address this void, we used a strategy in which Galpha(oA) was rendered insensitive to pertussis toxin (PTX) and RGS proteins by site-directed mutagenesis. Either PTX-insensitive (PTX-i) or both PTX- and RGS-insensitive (PTX/RGS-i) mutants of Galpha(oA) were expressed along with Gbeta(1) and Ggamma(2) subunits in rat sympathetic neurons. After overnight treatment with PTX to suppress natively expressed Galpha subunits, voltage-dependent Ca(2+) current inhibition by norepinephrine (NE) (10 microm) was reconstituted in neurons expressing either PTX-i or PTX/RGS-i Galpha(oA). When compared with neurons expressing PTX-i Galpha(oA), the steady-state concentration-response relationships for NE-induced Ca(2+) current inhibition were shifted to lower concentrations in neurons expressing PTX/RGS-i Galpha(oA). In addition to an increase in agonist potency, the expression of PTX/RGS-i Galpha(oA) dramatically retarded the current recovery after agonist removal. Interestingly, the alteration in current recovery was accompanied by a slowing in the onset of current inhibition. Together, our data suggest that endogenous RGS proteins contribute to membrane-delimited Ca(2+) channel modulation by regulating agonist potency and kinetics of G-protein-mediated signaling in neuronal cells.

T-type Ca2+ channels play essential roles in numerous cellular processes. Recently, we reported that phorbol-12-myristate-13-acetate (PMA) potently enhanced the current amplitude of Ca v 3.2 T-type channels reconstituted in Xenopus oocytes. Here, we have compared PMA modulation of the activities of Ca v 3.1, Ca v 3.2 and Ca v 3.3 channels, and have investigated the underlying mechanism. PMA augmented the current amplitudes of the three T-type channel isoforms, but the fold stimulations and time courses differed. The augmentation effects were not mimicked by 4α-PMA, an inactive stereoisomer of PMA, but were abolished by preincubation with protein kinase C (PKC) inhibitors, indicating that PMA augmented T-type channel currents via activation of oocyte PKC. The stimulation effect on Ca v 3.1 channel activity by PKC was mimicked by endothelin when endothelin receptor type A was coexpressed with Ca v 3.1 in the Xenopus oocyte system. Pharmacological studies combined with fluorescence imaging revealed that the surface density of Ca v 3.1 T-type channels was not significantly changed by activation of PKC. The PKC effect on Ca v 3.1 was localized to the cytoplasmic II-III loop using chimeric channels with individual cytoplasmic loops of Ca v 3.1 replaced by those of Ca v 2.1.