Gating deficiency in a familial hemiplegic migraine type 1 mutant P/Q-type calcium channel.
Familial hemiplegic migraine type 1 (FHM1) arises from missense mutations in the gene encoding alpha1A, the pore-forming subunit of P/Q-type calcium channels. The nature of the channel disorder is fundamental to the disease, yet is not well understood. We studied how the most prevalent FHM1 mutation, a threonine to methionine substitution at position 666 (TM), affects both ionic current and gating current associated with channel activation, a previously unexplored feature of P/Q channels. Whole-cell currents were measured in HEK293 cells expressing channels containing either wild-type (WT) or TM alpha1A. Calcium currents were significantly smaller in cells expressing TM channels, consistent with previous reports. In contrast, surface expression of TM channels, measured by immunostaining against an extracellular epitope, was not decreased, and Western blots demonstrated that TM alpha1A subunits were expressed as full-length proteins. WT and TM gating currents were isolated by replacing Ca2+ with the nonpermeant cation La3+. The gating currents generated by the mutant channels were one-third that of WT, a deficiency sufficient to account for the observed attenuation in calcium current; the remaining gating current was no different in kinetics or voltage dependence. Thus, the decreased calcium influx seen with TM channels can be attributed to a reduced number of channels available to undergo the voltage-dependent conformational changes needed for channel opening, not to fewer channel proteins expressed on the cell surface. This identification of an intrinsic defect in FHM1 mutant channels helps explain their impact on neurotransmission when they occupy type-specific slots for P/Q channels at central nerve terminals.
Barrett
Curtis F
CF
Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305, USA.
Cao
Yu-Qing
YQ
Tsien
Richard W
RW
D000595:Amino Acid Sequence; D020865:Calcium Channels, P-Type / Q000502:physiology*; D020907:Calcium Channels, Q-Type / Q000502:physiology*; D002478:Cells, Cultured; D006801:Humans; D015640:Ion Channel Gating; D020325:Migraine with Aura / Q000503:physiopathology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D020865:Calcium Channels, P-Type; D020907:Calcium Channels, Q-Type