Identification of the amino terminus of neuronal Ca2+ channel alpha1 subunits alpha1B and alpha1E as an essential determinant of G-protein modulation.
We have examined the basis for G-protein modulation of the neuronal voltage-dependent calcium channels (VDCCs) alpha1E and alpha1B. A novel PCR product of alpha1E was isolated from rat brain. This contained an extended 5' DNA sequence and was subcloned onto the previously cloned isoform rbEII, giving rise to alpha1Elong whose N terminus was extended by 50 amino acids. VDCC alpha1 subunit constructs were co-expressed with the accessory alpha2-delta and beta2a subunits in Xenopus oocytes and mammalian (COS-7) cells. The alpha1Elong showed biophysical properties similar to those of rbEII; however, when G-protein modulation of expressed alpha1 subunits was induced by activation of co-expressed dopamine (D2) receptors with quinpirole (100 nM) in oocytes, or by co-transfection of Gbeta1gamma2 subunits in COS-7 cells, alpha1Elong, unlike alpha1E(rbEII), was found to be G-protein-modulated, in terms of both a slowing of activation kinetics and a reduction in current amplitude. However, alpha1Elong showed less modulation than alpha1B, and substitution of the alpha1E1-50 with the corresponding region of alpha1B1-55 produced a chimera alpha1bEEEE, with G-protein modulation intermediate between alpha1Elong and alpha1B. Furthermore, deletion of the N-terminal 1-55 sequence from alpha1B produced alpha1BDeltaN1-55, which could not be modulated, thus identifying the N-terminal domain as essential for G-protein modulation. Taken together with previous studies, these results indicate that the intracellular N terminus of alpha1E1-50 and alpha1B1-55 is likely to contribute to a multicomponent site, together with the intracellular I-II loop and/or the C-terminal tail, which are involved in Gbetagamma binding and/or in subsequent modulation of channel gating.
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Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom.
Cantí
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Stephens
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Berrow
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Dolphin
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D000818:Animals; D001923:Brain Chemistry / Q000502:physiology; D019556:COS Cells; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics*; D020864:Calcium Channels, N-Type; D020908:Calcium Channels, R-Type; D027682:Cation Transport Proteins; D002417:Cattle; D018491:Dopamine Agonists / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015870:Gene Expression / Q000502:physiology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D007536:Isomerism; D007700:Kinetics; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics; D009474:Neurons / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019257:Quinpirole / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013873:Thionucleotides / Q000494:pharmacology
D015220:Calcium Channels; D020864:Calcium Channels, N-Type; D020908:Calcium Channels, R-Type; D027682:Cation Transport Proteins; D018491:Dopamine Agonists; D019204:GTP-Binding Proteins; D006153:Guanosine Diphosphate; D009419:Nerve Tissue Proteins; D019257:Quinpirole; D013873:Thionucleotides; C498917:Cacna1e protein, rat; C023427:guanosine 5'-O-(2-thiodiphosphate)