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First published online 3 June 2003
doi: 10.1242/jcs.00595

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

Linda C. Miller^1,*, Leigh Anne Swayne^1,*, Jason G. Kay¹, Zhong-Ping Feng^1,2, Scott E. Jarvis¹, Gerald W. Zamponi¹ and Janice E. A. Braun^1,

¹ Neuroscience Research Group, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, T2N 4N1, Canada
² NeuroMed Technologies Inc., Suite 301, Don Rix Building, 2389 Health Sciences Mall, UBC, Vancouver, BC, V6T 1Z4, Canada

View larger version (31K): [in a new window]   Fig. 1. CSP interacts with G indirectly. Immunoblot analysis showing binding of G to CSP-GST fusion protein or J domain GST-fusion protein immobilized on agarose. Fusion proteins were incubated in the presence of hippocampal homogenate (H) or purified Gß proteins (Calbiochem) (G). The beads were washed, and bound proteins were eluted in sample buffer, fractionated by SDS-PAGE and subjected to western blot analysis. The nitrocellulose membrane was probed with an anti-G polyclonal antibody. These results are representative of five independent experiments.

View larger version (33K): [in a new window]   Fig. 2. Nucleotide dependency of G protein interaction with recombinant CSP and J domain. Immunoblot analysis showing binding of G, Gß and Hsc70 to J-domain-GST fusion protein immobilized on agarose. Fusion proteins were incubated in the absence (-) or presence of rat hippocampal homogenate and 2 mm ATP, ATPS, ADP, GTP, GTPS or GDP at 37°C for 30 minutes. Lane 2 shows fusion proteins incubated with hippocampal homogenate in the absence of nucleotides. Lane 9 shows 15 µg of hippocampal homogenate loaded directly on the gel. The beads were washed, and bound proteins were eluted in sample buffer, fractionated by SDS-PAGE and subjected to western blot analysis. The nitrocellulose membrane was probed with anti-G polyclonal, anti-Gß monoclonal and anti-Hsc70 monoclonal antibodies. Gß does not interact with the J domain in the absence or presence of any nucleotide tested. G binds to the J domain in the presence of ATP. These results are representative of six independent experiments.

View larger version (77K): [in a new window]   Fig. 3. pH dependency of G protein association with CSP. Immunoblot analysis showing binding of Gß and G to CSP-GST and Jdomain-GST immobilized on agarose. Fusion proteins were incubated with hippocampal homogenate (in the absence of ATP) at the indicated pH for 1 hour. The beads were washed, and bound proteins were eluted in sample buffer, fractionated by SDS-PAGE and subjected to western blot analysis. The nitrocellulose membrane was probed with anti-Gß monoclonal and anti-G polyclonal (Calbiochem). These results are representative of five experiments. Gß binding to CSP is very stable over a broad pH range. In contrast, Gß does not bind to the J domain under any of the conditions examined.

View larger version (39K): [in a new window]   Fig. 4. Identification of interacting domains of CSP and G proteins. (A) Schematic representation of CSP and its deletion mutants encoded by the GST fusion cDNA constructs. (B) These fusion proteins were immobilized on glutathione-agarose beads and incubated with homogenate at 37°C for 30 minutes. The beads were washed, and bound proteins were eluted in sample buffer, fractionated by SDS-PAGE and subjected to western blot analysis. The nitrocellulose membrane was probed with anti-Gß monoclonal and anti-G polyclonal (Calbiochem) and anti-Gi polyclonal (Santa Cruz) antibodies. (C) Coomassie staining profile of CSP-GST and its truncation mutants. Note that CSP truncation mutants 83-198 and 137-198 migrate anomalously on SDS-PAGE. These results are representative of four independent experiments.

View larger version (36K): [in a new window]   Fig. 5. Identification of the CSP regions that associate with N-type calcium channels. CSP deletion mutants were immobilized on glutathione-agarose beads and incubated with recombinant 1B synprint His6 at 37°C for 1 hour. The beads were washed, and bound proteins were eluted in sample buffer, fractionated by SDS-PAGE and subjected to western blot analysis. The monoclonal antibody used recognizes a sequence Thr-Leu-Tyr-Asp-Asp-Asp-Asp-Lys (Anti Express, Invitrogen) in the fusion proteins. These results are representative of 10 independent experiments.

View larger version (22K): [in a new window]   Fig. 6. Regulation of N-type channel activity by individual CSP regions. (A) Current records obtained with N-type (1B + 2 - + ß1b) calcium channels expressed in tsa-201 cells before and after application of a 50 millisecond prepulse (pp) to +150 mV. Currents were elicited by stepping from a holding potential of -100 mV to a test potential of +20 mV. In the absence of CSP (left traces), the prepulses do not affect peak current amplitude. Following coexpression of either the cysteine string domain (middle traces) or the J domain (right traces), the channels undergo a tonic G protein inhibition that is reversed by the prepulses. In all traces, the vertical and horizontal bars indicate, respectively, a current amplitude of 200 pA and a 20 milliseond duration. (B) Bar graph illustrating the degree of prepulse relief in the absence and presence of CSP fragments. Additional coexpression of the C-terminal fragment (residues 495-689) of the ß-adrenergic receptor kinase (ßARK) reduces the CSP-mediated current inhibition as shown by the reduced PP relief. The numbers in parentheses reflect the number of experiments; the asterisks indicate statistical significance relative to control conditions at P<0.05 level.

View larger version (18K): [in a new window]   Fig. 7. Effect of synprint peptides on CSP action. (A) Current records obtained with transiently expressed N-type calcium channels following co-expression of either the cysteine string domain (left traces) or the J domain (right traces) and a cDNA construct corresponding to the synprint region of the rat N-type calcium channel. Note that synprint blocks the effect of the cysteine string region but not that mediated by the J domain. The experimental conditions were as outlined in Fig. 6. (B) Bar graphs summarizing the effect of synprint on the G protein effect mediated by the cysteine string and J domain regions. The numbers in parentheses indicate the number of experiments; the asterisk indicates statistical significance relative to control conditions. The data obtained with the J domain in the presence of synprint did not differ significantly from those shown in Fig. 6 in the absence of synprint.

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