The immediately releasable pool of mouse chromaffin cell vesicles is coupled to P/Q-type calcium channels via the synaptic protein interaction site.

It is generally accepted that the immediately releasable pool is a group of readily releasable vesicles that are closely associated with voltage dependent Ca(2+) channels. We have previously shown that exocytosis of this pool is specifically coupled to P/Q Ca(2+) current. Accordingly, in the present work we found that the Ca(2+) current flowing through P/Q-type Ca(2+) channels is 8 times more effective at inducing exocytosis in response to short stimuli than the current carried by L-type channels. To investigate the mechanism that underlies the coupling between the immediately releasable pool and P/Q-type channels we transiently expressed in mouse chromaffin cells peptides corresponding to the synaptic protein interaction site of Cav2.2 to competitively uncouple P/Q-type channels from the secretory vesicle release complex. This treatment reduced the efficiency of Ca(2+) current to induce exocytosis to similar values as direct inhibition of P/Q-type channels via ω-agatoxin-IVA. In addition, the same treatment markedly reduced immediately releasable pool exocytosis, but did not affect the exocytosis provoked by sustained electric or high K(+) stimulation. Together, our results indicate that the synaptic protein interaction site is a crucial factor for the establishment of the functional coupling between immediately releasable pool vesicles and P/Q-type Ca(2+) channels.

The Cavß subunit prevents RFP2-mediated ubiquitination and proteasomal degradation of L-type channels.

It is well established that the auxiliary Cavß subunit regulates calcium channel density in the plasma membrane, but the cellular mechanism by which this occurs has remained unclear. We found that the Cavß subunit increased membrane expression of Cav1.2 channels by preventing the entry of the channels into the endoplasmic reticulum-associated protein degradation (ERAD) complex. Without Cavß, Cav1.2 channels underwent robust ubiquitination by the RFP2 ubiquitin ligase and interacted with the ERAD complex proteins derlin-1 and p97, culminating in targeting of the channels to the proteasome for degradation. On treatment with the proteasomal inhibitor MG132, Cavß-free channels were rescued from degradation and trafficked to the plasma membrane. The coexpression of Cavß interfered with ubiquitination and targeting of the channel to the ERAD complex, thereby facilitating export from the endoplasmic reticulum and promoting expression on the cell surface. Thus, Cavßß regulates the ubiquitination and stability of the calcium channel complex.

Direct G protein modulation of Cav2 calcium channels.

The regulation of presynaptic, voltage-gated calcium channels by activation of heptahelical G protein-coupled receptors exerts a crucial influence on presynaptic calcium entry and hence on neurotransmitter release. Receptor activation subjects presynaptic N- and P/Q-type calcium channels to a rapid, membrane-delimited inhibition-mediated by direct, voltage-dependent interactions between G protein betagamma subunits and the channels-and to a slower, voltage-independent modulation involving soluble second messenger molecules. In turn, the direct inhibition of the channels is regulated as a function of many factors, including channel subtype, ancillary calcium channel subunits, and the types of G proteins and G protein regulatory factors involved. Twenty-five years after this mode of physiological regulation was first described, we review the investigations that have led to our current understanding of its molecular mechanisms.

Scanning mutagenesis reveals a role for serine 189 of the heterotrimeric G-protein beta 1 subunit in the inhibition of N-type calcium channels.

Direct interactions between the presynaptic N-type calcium channel and the beta subunit of the heterotrimeric G-protein complex cause voltage-dependent inhibition of N-type channel activity, crucially influencing neurotransmitter release and contributing to analgesia caused by opioid drugs. Previous work using chimeras of the G-protein beta subtypes Gbeta1 and Gbeta5 identified two 20-amino acid stretches of structurally contiguous residues on the Gbeta1 subunit as critical for inhibition of the N-type channel. To identify key modulation determinants within these two structural regions, we performed scanning mutagenesis in which individual residues of the Gbeta1 subunit were replaced by corresponding Gbeta5 residues. Our results show that Gbeta1 residue Ser189 is critical for N-type calcium channel modulation, whereas none of the other Gbeta1 mutations caused statistically significant effects on the ability of Gbeta1 to inhibit N-type channels. Structural modeling shows residue 189 is surface exposed, consistent with the idea that it may form a direct contact with the N-type calcium channel alpha1 subunit during binding interactions.