Cross-talk between G-protein and Protein Kinase C Modulation of N-type Calcium Channels Is Dependent on the G-protein β Subunit Isoform

Abstract

The modulation of N-type calcium current by protein kinases and G-proteins is a factor in the fine tuning of neurotransmitter release. We have previously shown that phosphorylation of threonine 422 in the ␣ 1B calcium channel domain I-II linker region resulted in a dramatic reduction in somatostatin receptor-mediated G-protein inhibition of the channels and that the I-II linker consequently serves as an integration center for cross-talk between protein kinase C (PKC) and G-proteins (Hamid, J., Nelson, D., Spaetgens, R., Dubel, S. J., Snutch, T. P., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 6195-6202). Here we show that opioid receptor-mediated inhibition of N-type channels is affected to a lesser extent compared with that seen with somatostatin receptors, hinting at the possibility that PKC/G-protein cross-talk might be dependent on the G-protein subtype. To address this issue, we have examined the effects of four different types of G-protein ␤ subunits on both wild type and mutant ␣ 1B calcium channels in which residue 422 has been replaced by glutamate to mimic PKC-dependent phosphorylation and on channels that have been directly phosphorylated by protein kinase C. Our data show that phosphorylation or mutation of residue 422 antagonizes the effect of G␤ 1 on channel activity, whereas G␤ 2 , G␤ 3 , and G␤ 4 are not affected. Our data therefore suggest that the observed cross-talk between G-proteins and protein kinase C modulation of N-type channels is a selective feature of the G␤ 1 subunit.The modulation of calcium channel activity by activation of intracellular messenger pathways is a key mechanism for fine tuning calcium entry into neurons. For example, the activation of protein kinase C has been shown to mediate an up-regulation of N-type calcium currents in intact neurons (1, 2) and in transient expression systems (3,4). In contrast, the direct 1:1 binding of G protein ␤␥ subunits to the domain I-II linker region of N-type, P/Q-type, and possibly R-type calcium channels results in a depression of current activity (5-8) (reviewed in Refs. 9 and 10), which can be reversed by strong membrane depolarization (10 -12). Different types of calcium channels are modulated by G-proteins to different extents, such that N-type channels are typically inhibited more effectively than P/Q-type channels (13-16). There is also increasing evidence that the degree of inhibition is dependent on the G-protein ␤ subunit species (16 -18). Finally, it has been shown that protein kinase C-dependent phosphorylation of the N-type calcium channel ␣ 1 subunit antagonizes receptor-mediated G-protein inhibition of the channel (1,2,12,19). This phenomenon (termed PKC 1 /Gprotein cross-talk) appears to be mediated by a single threonine residue in the ␣ 1B domain I-II linker region (4). For somatostatin receptor-induced G-protein inhibition of N-type calcium channels, mutation of Thr-422 to glutamic acid mimics the antagonistic effect of protein kinase C on G protein inhibition, whereas a switch to alanine precludes the occurrence of P...

G Protein Modulation of Voltage-Gated Calcium Channels

“…It has been proposed that phosphorylation by PKC of this threonine residue counteracts G␤␥ binding to the I-II linker and thus counters inhibitory modulation (Zamponi et al, 1997;Hamid et al, 1999). However, this was subsequently found only to hold true for G␤ 1 and not other G␤ subunits (Cooper et al, 2000). Furthermore, the sequence is not completely conserved in rabbit Ca V 2.2, which has alanine in place of threonine at the equivalent residue 422 (KRAAAKKSR in rabbit Ca V 2.2), although the same phenomenon of cross talk between G protein modulation and PKC activation occurs (Bertaso et al, 2003).…”

G protein inhibition of CaV2 calcium channels

“…109,110 Of note, phosphorylation of Thr 422 only disrupts inhibition mediated by Gβ 1 , but not other Gβ subunits. 111 Thus the with Gβγ demonstrated by the Yue group. 122 Moreover, Agler et al 122 also demonstrated another role for the N-terminus: residues 56-95 directly bind to the N-terminal one-third of the I-II linker (from Ca V 2.2 but not Ca V 1.2).…”

Direct G Protein Modulation of Cav2 Calcium Channels

“…5, B and C). The ability of PKC to antagonize G␤␥-mediated inhibition depends on the G protein ␤ subunit isoform, with only G␤ 1 being capable of sensing PKC-dependent phosphorylation of the channel (Cooper et al, 2000) (Fig. 5B).…”