In this renewal we study three essential cellular mechanisms that modulate the activity of the voltage-gated N-type calcium channel CaValpah-1 subunit. Voltage-gated calcium channels are the gatekeepers of excitation-driven calcium entry in all excitable cells. As such they control a diverse array of cellular functions including neurotransmitter release, gene expression, synaptic plasticity, and neurite outgrowth. Cells use a number of ingenious methods to optimize the activity of voltage-gated calcium channels and to couple calcium channels to different cellular functions. Nine genes encode the major CaValpha-1 subunits that are expressed in the mammalian nervous system and each of these is subject to alternative splicing. A single CaValpha-1 gene has the capacity to generate over 1000 different splice isoforms, consistent with the theory that alternative splicing underlies the expansion of the proteome required to support complex brain functions. Alternatively spliced exons that are under cellular control and selected for during evolution, occur in critical domains of proteins that regulate function. We have used a combination of molecular, biochemical, and electrophysiological methods to analyze two such loci in the N-type CaV2.2 gene. We identify three novel protein interactions that control channel modulation, channel degradation, and association with novel a target protein implicated in exocytosis. In the previous funding period of this grant we discovered an alternatively spiced exon in the C-terminus of CaV2.2, exon 37a, that is preferentially expressed in nociceptive neurons of the dorsal root ganglia. We now show that regulated alternative splicing of exon 37a in the CaV2.2 channel triggers a dramatic increase change in the sensitivity of N-type channels to G-protein-dependent, voltage- independent inhibition (aim 1). This finding has major implications for understanding the cellular mechanisms that control N-type calcium channel function in nociception. The CaV2.2 channel is a major target for a number of analgesics. This same region in the C-terminus of CaV2.2 mediates interactions with an enzyme of the ubiquitin complex (aim 2) and represents a general cellular mechanism to control surface expression of N-type channels. Finally, we report a novel interaction between JFC1, a C2-domain containing synaptotagmin-like protein implicated in exocytosis, and the ll-lll intracellular loop of CaV2.2. This novel interaction is likely to mediate coupling of the CaV2.2 channel to the synaptic release machinery (aim 3).