Molecular determinants of cysteine string protein modulation of N-type calcium channels

Cysteine string proteins (CSPs) are secretory vesicle chaperones that are important for neurotransmitter release. We have previously reported an interaction of CSP with both heterotrimeric GTP-binding proteins (G proteins)and N-type calcium channels that results in a tonic G protein inhibition of the channels. In this report we directly demonstrate that two separate regions of CSP associate with G proteins. The N-terminal binding site of CSP, which includes the J domain, binds G_α subunits but not G_αβ subunits whereas the C terminal binding site of CSP associates with either free G_αβ subunits or G_αβ in complex with G_α. The interaction of either binding site of CSP (CSP_1-82 or CSP83-198) with G proteins elicits robust tonic inhibition of N-type calcium channel activity. However, CSP_1-82 inhibition and CSP83-198 inhibition of calcium channels occur through distinct mechanisms. Calcium channel inhibition by CSP83-198 (but not CSP_1-82) is completely blocked by co-expression of the synaptic protein interaction site (synprint) of the N-type channel,indicating that CSP83-198 inhibition is dependent on a physical interaction with the calcium channel. These results suggest that distinct binding sites of CSP can play a role in modulating G protein function and G protein inhibition of calcium channels.