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Deletion of the synaptic protein interaction site of the N-type (CaV2.2) calcium channel inhibits secretion in mouse pheochromocytoma cells.

ABSTRACT: Presynaptic N-type Ca2+ channels (CaV2.2, alpha1B) are thought to bind to SNARE (SNAP-25 receptor) complex proteins through a synaptic protein interaction (synprint) site on the intracellular loop between domains II and III of the alpha1B subunit. Whether binding of syntaxin to the N-type Ca2+ channels is required for coupling Ca2+ ion influx to rapid exocytosis has been the subject of considerable investigation. In this study, we deleted the synprint site from a recombinant alpha1B Ca2+ channel subunit and transiently transfected either the wild-type alpha1B or the synprint deletion mutant into mouse pheochromocytoma (MPC) cell line 9/3L, a cell line that has the machinery required for rapid stimulated exocytosis but lacks endogenous voltage-dependent Ca2+ channels. Secretion was elicited by activation of exogenously transfected Ca2+ channel subunits. The current-voltage relationship was similar for the wild-type and mutant alpha1B-containing Ca2+ channels. Although total Ca2+ entry was slightly larger for the synprint deletion channel, compared with the wild-type channel, when Ca2+ entry was normalized to cell size and limited to cells with similar Ca2+ entry (approximately 150 x 10(6) Ca2+ ions/pF cell size), total secretion and the rate of secretion, determined by capacitance measurements, were significantly reduced in cells expressing the synprint deletion mutant channels, compared with wild-type channels. Furthermore, the amount of endocytosis was significantly reduced in cells with the alpha1B synprint deletion mutant, compared with the wild-type subunit. These results suggest that the synprint site is necessary for efficient coupling of Ca2+ influx through alpha1B-containing Ca2+ channels to exocytosis.

SUBMITTER: Harkins AB

PROVIDER: S-EPMC524046 | biostudies-literature | 2004 Oct

REPOSITORIES: biostudies-literature

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Deletion of the synaptic protein interaction site of the N-type (CaV2.2) calcium channel inhibits secretion in mouse pheochromocytoma cells.

Harkins Amy B AB Cahill Anne L AL Powers James F JF Tischler Arthur S AS Fox Aaron P AP

Proceedings of the National Academy of Sciences of the United States of America 20041007 42

Presynaptic N-type Ca2+ channels (CaV2.2, alpha1B) are thought to bind to SNARE (SNAP-25 receptor) complex proteins through a synaptic protein interaction (synprint) site on the intracellular loop between domains II and III of the alpha1B subunit. Whether binding of syntaxin to the N-type Ca2+ channels is required for coupling Ca2+ ion influx to rapid exocytosis has been the subject of considerable investigation. In this study, we deleted the synprint site from a recombinant alpha1B Ca2+ channel ...[more]

PMID: 15471993

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Project description:Structural studies with an α subunit fragment of voltage-gated calcium (CaV) channels in complex with the CaVβ subunits revealed a high homology between the various CaVα-β subunits, predicting that targeting of this interface would result in nonselective compounds. Despite this likelihood, my laboratory initiated a rational structure-based screening campaign focusing on "hot spots" on the alpha interacting domain (AID) of the CaVβ2a subunits and identified the small molecule 2-(3,5-dimethylisoxazol-4-yl)-N-((4-((3-phenylpropyl)amino)quinazolin-2-yl)methyl)acetamide ( IPPQ ) which selectively targeted the interface between the N-type calcium (CaV2.2) channel and CaVβ. IPPQ (i) specifically bound to CaVβ2a; (ii) inhibited CaVβ2 's interaction with CaV.2-AID; (iii) inhibited CaV2.2 currents in sensory neurons; (iv) inhibited pre-synaptic localization of CaV2.2 in vivo; and (v) inhibited spinal neurotransmission, which resulted in decreased neurotransmitter release. IPPQ was anti-nociceptive in naïve rats and reversed mechanical allodynia and thermal hyperalgesia in rodent models of acute, neuropathic, and genetic pain. In structure-activity relationship (SAR) studies focused on improving binding affinity of IPPQ , another compound (BTT-369), a benzoyl-3,4-dihydro-1'H,2 H-3,4'-bipyrazole class of compounds, was reported by Chen and colleagues, based on work conducted in my laboratory beginning in 2008. BTT-369 contains tetraaryldihydrobipyrazole scaffold - a chemotype featuring phenyl groups known to be significantly metabolized, lower the systemic half-life, and increase the potential for toxicity. Furthermore, the benzoylpyrazoline skeleton in BTT-369 is patented across multiple therapeutic indications. Prior to embarking on an extensive optimization campaign of IPPQ , we performed a head-to-head comparison of the two compounds. We conclude that IPPQ is superior to BTT-369 for on-target efficacy, setting the stage for SAR studies to improve on IPPQ for the development of novel pain therapeutics.

Dataset Information

Deletion of the synaptic protein interaction site of the N-type (CaV2.2) calcium channel inhibits secretion in mouse pheochromocytoma cells.

Publications

Deletion of the synaptic protein interaction site of the N-type (CaV2.2) calcium channel inhibits secretion in mouse pheochromocytoma cells.

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