Novel CaV2.1 clone replicates many properties of Purkinje cell CaV2.1 current.
The P-type calcium current is mediated by a voltage-sensing CaV2.1 alpha subunit in combination with modulatory auxiliary subunits. In Purkinje neurones, this current has distinctively slow inactivation kinetics that may depend on alternative splicing of the alpha subunit and/or association with different CaVbeta subunits. To better understand the molecular components of P-type calcium current, we cloned a CaV2.1 cDNA from total mouse brain. The full-length CaV2.1 isoform that we isolated (GenBank AY714490) contains sequences recently shown to be present in Purkinje neurones. In agreement with previously published work, the alternatively spliced amino acid V421, implicated in slow inactivation, was not encoded in AY714490 and was absent from reverse transcription-polymerase chain reaction products generated from single Purkinje cells. Next, we studied the expression of the four known mouse auxiliary CaVbeta2 isoforms in Purkinje neurones. Confirmation of the presence of CaVbeta2a in Purkinje cells, previously shown by others to slow CaV2.1 kinetics, led us to characterize its influence on current dynamics. We studied currents generated by the clone AY714490 coexpressed in tsA201 cells with four different CaVbeta subunits. In addition to the well-documented slowing of open-state inactivation kinetics, coexpression with the CaVbeta2a subunit also protected CaV2.1 channels from closed-state inactivation and prevented the channel from inactivating during physiological trains of action potential-like stimuli. This strong resistance to inactivation parallels the property of Purkinje neurone P-type currents and is suggestive of a role for CaVbeta2a in modulating the inactivation properties of P-type calcium currents in Purkinje neurones.
Richards
Kathryn S
KS
Department of Neuroscience, Brown University, Providence, RI, USA. kat@chups.jussieu.fr
Swensen
Andrew M
AM
Lipscombe
Diane
D
Bommert
Kurt
K
D000818:Animals; D001921:Brain / Q000166:cytology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000502:physiology*; D002461:Cell Line, Transformed; D003001:Cloning, Molecular / Q000379:methods; D004307:Dose-Response Relationship, Radiation; D004558:Electric Stimulation / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology* / Q000528:radiation effects; D051379:Mice; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011689:Purkinje Cells / Q000187:drug effects / Q000502:physiology*; D016415:Sequence Alignment / Q000379:methods; D014162:Transfection / Q000379:methods; D020867:omega-Agatoxin IVA / Q000494:pharmacology
D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D049452:Green Fluorescent Proteins; D020033:Protein Isoforms; D020867:omega-Agatoxin IVA