Dissection of the calcium channel domains responsible for modulation of neuronal voltage-dependent calcium channels by G proteins.
The molecular determinants for G-protein regulation of neuronal calcium channels remain controversial. We have generated a series of alpha 1B/alpha 1E chimeric channels, since rat brain alpha 1E (rbEII), unlike human alpha 1E, showed no G-protein modulation. The study, carried out in parallel using D2 receptor modulation of calcium currents in Xenopus oocytes of G beta gamma modulation of calcium currents in COS-7 cells, consistently showed an essential role for domain I (from the N terminus to the end of the I-II loop) of the alpha 1B Ca2+ channel in G-protein regulation, with no additional effect of the C terminal of alpha 1B. The I-II loop alone of alpha 1B, or the I-II loop together with the C-terminal tail, was insufficient to confer G-protein modulation of alpha 1E (rbEII). We have further observed that the alpha 1E clone rbEII is truncated at the N-terminus compared to other alpha 1 subunits, and we isolated a PCR product from rat brain equivalent to a longer N-terminal isoform. The long N-terminal alpha 1E, unlike the short form, showed G-protein modulation. Furthermore, the equivalent truncation of alpha 1B (delta N1-55) abolished G-protein modulation of alpha 1B. Thus, we propose that the N terminus of alpha 1B and alpha 1E calcium channels contains essential molecular determinants for membrane-delimited G-protein inhibition, and that other regions, including the I-II loop and the C terminus, do not play a conclusive role alone.
Dolphin
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Department of Pharmacology, University College London, United Kingdom. a.dolphin@ucl.ac.uk
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Berrow
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Stephens
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Cantí
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D000595:Amino Acid Sequence; D000818:Animals; D001418:Baclofen / Q000494:pharmacology; D001665:Binding Sites; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014981:Xenopus
D015220:Calcium Channels; D009419:Nerve Tissue Proteins; D016376:Oligonucleotides, Antisense; D011993:Recombinant Fusion Proteins; D019204:GTP-Binding Proteins; D001418:Baclofen