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

Abstract

IntroductionCSPs (cysteine string proteins) are secretory vesicle chaperone proteins that are evolutionarily conserved. Deletion of CSP in Drosophila is semi-lethal; only 4% of the flies develop into adulthood . Adult survivors exhibit uncoordinated motor behavior that progresses to paralysis. Recordings from mutant neuromuscular junctions reveal that neurotransmission is reduced by 50% at 22°C and completely abolished above 29°C, indicating that the function of CSP is critical. Spontaneous vesicle release in these CSP-null mutants is not temperature sensitive Saitoe et al., 2001). CSPs have three domains: an N-terminal J domain, a linker-domain and a cysteine string domain. The J domain of CSP is a 70 amino-acid region homologous to the well-characterized bacterial chaperone protein DnaJ and many otherwise unrelated eukaryotic proteins. Although CSP is thought to be important in synaptic transmission, the exact details regarding the role of this synaptic chaperone in neurotransmission are not yet defined. Conflicting reports support either (i) a role for CSP in exocytosis or (ii) a role for CSP in the regulation of transmembrane Ca 2+ fluxes (reviewed by Zinsmaier and Bronk, 2001) (Chamberlain and Burgoyne, 2000).We have recently shown that CSP is capable of binding to both the N-type calcium channel and to Gβγ in vitro and that the interaction between CSP and the N-type calcium channel results in a robust tonic inhibition of channel activity by G protein βγ subunits . The CSP/G protein interaction was confirmed by co-immunoprecipitation, GST pull-down assays, crosslinking in intact brain slices as well as evaluation of functional proteins in HEK cells and has recently been confirmed by others (Evans et al., 2001). Numerous synaptic proteins were absent from the CSP immunoprecipitations and GST pull-down assays, demonstrating the specificity of the CSP/G protein interaction. Interestingly, CSP and G proteins have been shown to coenrich in detergent-insoluble lipid raft fractions from rat hippocampus (Magga et al., 2002). Binding of G proteins appears to involve two separate regions of CSP, such that Gα interacts with the J domain of CSP in an ATP-dependent manner, whereas Gβγ associates with full-length CSP but not the J domain of CSP in an ATP-independent fashion. Although CSP interacts with G proteins, it is not clear exactly how CSP affects G protein function. In particular, it is unclear exactly which regions of CSP associate with G protein subunits. Furthermore, it is unknown whether CSP's interaction with Gα proteins is direct or requires an additional component. Understanding the nature of the interaction between CSP, G proteins and calcium channels is crucial in understanding the molecular role of CSP. In the present study we have therefore analyzed the association of CSP with G proteins and N-type calcium channels. Materials and MethodsPreparation of rat hippocampal homogenate Rat hippocampi were hand homogenized with a teflon-coated

Cysteine String Protein (CSP) Inhibition of N-type Calcium Channels Is Blocked by Mutant Huntingtin

“…Upon application of a strong depolarizing prepulse, peak current amplitude was increased. This is consistent with removal of a G protein-mediated inhibitory effect, which we have characterized in detail (18,19). When N-type channels were co-transfected with HDQ25, HDQ47, or HDQ72 no effect on channel function (i.e.…”

“…Preparation of Fusion Proteins-Glutathione S-transferase (GST) fusion proteins of CSP and CSP deletion mutants were prepared as described previously (9,12,19). The SGT construct (␣-SGT) was prepared by subcloning SGT PCR fragments into pGEX-KG (20) and was expressed as a GST fusion protein in AB1899 cells.…”

“…Huntingtin exon1/exp Blocks CSP Regulation of N-type Calcium Channels-Previous work in our laboratory has shown that CSP modulates G protein-mediated inhibition of N-type calcium channels (18,19). Thus, the N-type calcium channel can be used as a functional readout of CSP-G protein interactions in live cells.…”

“…We have recently shown that CSP is capable of binding to both the N-type calcium channel and to G␤␥ in vitro, and that the interaction between CSP and the N-type calcium channel results in a robust tonic inhibition of channel activity by G protein ␤␥ subunits (18,19). Given that proteins with expanded polyglutamine repeats have been proposed to interfere with the chaperone balance of the cell, we have analyzed the effects of huntingtin on CSP modulation of N-type channels.…”

Old proteins, developing roles: The regulation of calcium channels by synaptic proteins

“…2), and in case of N-type channels, appears to trigger a tonic G protein inhibition that involves on one hand a colocalization of the channel with Gβγ, and on the other, a GEF-like activity that leads to activation of Gα subunits. 112 In addition, CSP may enhance channel function via a recruitment of channels to the plasma membrane. 113 CSP also indirectly regulates channel activity by virtue of its competition with syntaxin 1A for the channel, 114,115 and perhaps by its direct interactions with syntaxin 1A.…”