| Abstract No.: | B-B2068 |
| Country: | Canada |
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| Title: | REM2 MUST INTERACT WITH THE CALCIUM CHANNEL BETA SUBUNIT AT THE CELL MEMBRANE TO REDUCE CALCIUM CURRENTS |
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| Authors/Affiliations: | 1 Robyn Flynn*; 1 Lina Chen; 1 Shahid Hameed; 1 Gerald Zamponi;
1 University of Calgary, AB, Canada
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| Content: | Rem2 is a member of the RGK family of small GTPases, all of which are targeted to the cell membrane. There they interact with the calcium channel beta subunit as well as inhibit or abolish calcium currents; however, it is not known whether RGK proteins must interact with the beta subunit in order to knock down calcium currents, and what role the membrane targeting has in this process.
Objective: to explore the structural determinants of Rem2 that are responsible for its interactions and effects.
Materials and Methods: we created several N- or C-terminally truncated Rem2 proteins and examined (1) their subcellular localization using fluorescence imaging, (2) their ability to interact with a flag-tagged calcium channel beta3 subunit by co-precipitation, and (3) their effect on calcium currents using whole-cell patch clamp analysis of tsA201 cells expressing N-type calcium channels as well as the beta3 and alpha2delta subunits, in presence and absence of full length and truncated Rem2 constructs.
Results: (1) We found full-length Rem2 and all N-terminal truncations to be targeted to the cell membrane, while C-terminally truncated proteins were largely cytoplasmic in their distribution. (2) All C-terminally truncated Rem2 proteins co-precipitated with flag-beta3 subunit regardless of length. The longest N-terminally truncated protein, 83-341 Rem2, also co-precipitated with flag-beta3, however the next longest, 124-341 Rem2, and all successive truncations did not. (3) Electrophysiologically, full-length Rem2 and 83-341 Rem2 were both able to reduce or abolish calcium current. Both C-terminal truncation mutants that we tested, 1-282 Rem2 and 1-149 Rem2, as well as the N-terminal truncations 124-341 Rem2 and 179-341 Rem2 showed calcium currents similar to those of cells not transfected with Rem2.
Conclusions: These data suggest that (1) Rem2 contains a membrane-targeting sequence in its C-terminus. This is consistent with other reports. (2) Interaction with the beta subunit requires the N-terminal 123 residues, and (3) is not sufficient to affect calcium currents; Rem2 must also be present at the cell membrane to achieve its functional effect.
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