Differential Interactions of the C terminus and the Cytoplasmic I-II Loop of Neuronal Ca2+ Channels with G-protein α and βγ Subunits

Abstract

The present study was designed to obtain evidence for direct interactions of G-protein ␣ (G␣) and ␤␥ subunits (G␤␥) with N-(␣ 1B ) and P/Q-type (␣ 1A ) Ca 2؉ channels, using synthetic peptides and fusion proteins derived from loop 1 (cytoplasmic loop between repeat I and II) and the C terminus of these channels. For N-type, prepulse facilitation as mediated by G␤␥ was impaired when a synthetic loop 1 peptide was applied intracellularly. Receptor agonist-induced inhibition of N-type as mediated by G␣ was also impaired by the loop 1 peptide but only when applied in combination with a C-terminal peptide. For P/Q-type channels, by contrast, the G␣-mediated inhibition was diminished by application of a C-terminal peptide alone. Moreover, in vitro binding analysis for N-and P/Q-type channels revealed direct interaction of G␣ with C-terminal fusion proteins as well as direct interaction of G␤␥ with loop 1 fusion proteins. These findings define loop 1 of N-and P/Q-type Ca 2؉ channels as an interaction site for G␤␥ and the C termini for G␣.

Differential Interactions of the C terminus and the Cytoplasmic I-II Loop of Neuronal Ca2+ Channels with G-protein α and βγ Subunits

“…These results indicate that there are two distinct interaction sites, namely loop 1 and the C terminus, for G␣ and G␤␥ on N-type Ca 2ϩ channels and that the two sites regulate the channel activity independently when they receive inhibitory signals from G␣ and G␤␥. This independence of loop 1 and the C terminus in the ␣ 1B channel modulation is supported by the observation that the G i3 ␣-dependent potentiation of inhibition was not affected by single application of the loop 1 peptide (PL1) or a C-terminal peptide (PB3T4) inside the oocyte but abolished by simultaneous application of both of them (16).…”

“…In the case of the P/Q-type ␣ 1A channel, prepulse facilitation was not as prominent as observed in the ␣ 1B channel when G␤ 1 ␥ 2 was co-expressed (16). Nonetheless, the prepulse facilitation via G␤ 1 ␥ 2 appeared to be abolished in a mutant ␣ 1A channel (B1T⌬2) that contained a deletion of the C terminus.…”

“…When DOR was further co-expressed, ␣ 1B and ␣ 1A channel currents, but not ␣ 1C channel currents, were inhibited within seconds when stimulated by Leu-EK. It is likely that agonist-induced inhibitions of ␣ 1B and ␣ 1A channels are mediated by endogenous oocyte G-proteins that are coupled to the receptor, because the inhibitions of ␣ 1B and ␣ 1A channels were reduced when the antisense oligonucleotide AGO against Xenopus G o ␣ was injected (16).…”

“…Furthermore, G i3 ␣-dependent potentiation of inhibition of the ␣ 1A channel was impaired only when the C terminus was deleted (B1T⌬2). This is probably due to a minor contribution of G␤␥ to the potentiation of inhibition by agonist, despite the direct binding of G␤␥ to the loop 1 of ␣ 1A (11,16). In fact, the potentiation of inhibition via G␣ was abolished by intracellular application of a C-terminal peptide (PPQT1) alone (16).…”

“…In this paper, interactions of G␣ and G␤␥ with Ca 2ϩ channels were characterized using mutant and chimeric N-(␣ 1B ) and P/Q-type (␣ 1A ) Ca 2ϩ channels. The results, together with evidence for a direct binding provided by the companion paper (16), define the interaction sites of Ca 2ϩ channels for G␣ and G␤␥. EXPERIMENTAL …”