Cysteines in the loop between IS5 and the pore helix of Ca(V)3.1 are essential for channel gating.
The role of six cysteines of Ca(V)3.1 in channel gating was investigated. C241, C271, C282, C298, C313, and C323, located in the extracellular loop between segment IS5 and the pore helix, were each mutated to alanine; the resultant channels were expressed and studied by patch clamping in HEK293 cells. C298A and C313A conducted calcium currents, while the other mutants were not functional. C298A and C313A as well as double mutation C298/313A significantly reduced the amplitude of the calcium currents, shifted the activation curve in the depolarizing direction and slowed down channel inactivation. Redox agents dithiothreitol (DTT) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) shifted the current activation curve of wild-type channels in the hyperpolarizing direction. Activation curve for all mutated channels was shifted in hyperpolarizing direction by DTT while DTNB caused a depolarizing shift. Our study reveals that the cysteines we studied have an essential role in Ca(V)3.1 gating. We hypothesize that cysteines in the large extracellular loop of Ca(V)3.1 form bridges within the loop and/or neighboring channel segments that are essential for channel gating.
Karmazinova
Maria
M
Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovakia.
Beyl
Stanislav
S
Stary-Weinzinger
Anna
A
Suwattanasophon
Chonticha
C
Klugbauer
Norbert
N
Hering
Steffen
S
Lacinova
Lubica
L
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020747:Calcium Channels, T-Type / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D017434:Protein Structure, Tertiary
C498197:Cacna1g protein, mouse; D020747:Calcium Channels, T-Type; D004228:Dithionitrobenzoic Acid; D003545:Cysteine; D004229:Dithiothreitol
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