Domain analysis of the calcium-activated potassium channel SK1 from rat brain. Functional expression and toxin sensitivity.
Two small conductance, calcium-activated potassium channels (SK channels), SK2 and SK3, have been shown to contribute to the afterhyperpolarization (AHP) and to shape the firing behavior in neurons for example in the hippocampal formation, the dorsal vagal nucleus, the subthalamic nucleus, and the cerebellum. In heterologous expression systems, SK2 and SK3 currents are blocked by the bee venom toxin apamin, just as well as the corresponding neuronal AHP currents. However, the functional role and pharmacological profile of SK1 channels from rat brain (rSK1) is still largely unknown, as so far rSK1 homomeric channels could not be functionally expressed. We have performed a domain analysis to elucidate the pharmacological profile and the molecular determinants of rSK1 channel expression by using channel chimeras in combination with immunocytochemistry, immunoblot analysis, and electrophysiology. Our results reveal that the rSK1 subunit is synthesized in cells but does not form functional homomeric channels. Exchanging the carboxyl terminus of rSK1 for that of hSK1 or rSK2 is sufficient to rescue the functional expression of rSK1 channels. Additionally, transplantation of both amino and carboxyl termini of rSK1 onto hSK1 subunits, normally forming functional homomeric channel, hinders their functional expression, while hSK1 channels containing only the rSK1 carboxyl terminus are functional. These results suggest that the lack of functional expression of rSK1 channels is probably due to problems in their assembly and tetramerization but not in their calmodulin-dependent gating. Finally, we show that chimeric channels containing the core domain (S1-S6) of rSK1, unlike hSK1, are apamin-insensitive.
D'hoedt
Dieter
D
Wellcome Laboratory for Molecular Pharmacology, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, U.K.
Hirzel
Klaus
K
Pedarzani
Paola
P
Stocker
Martin
M
D000595:Amino Acid Sequence; D000818:Animals; D001030:Apamin / Q000494:pharmacology; D001921:Brain / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D002460:Cell Line; D004247:DNA / Q000737:chemistry; D004594:Electrophysiology; D006801:Humans; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D015221:Potassium Channels / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D024681:Potassium Channels, Calcium-Activated; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051640:Small-Conductance Calcium-Activated Potassium Channels; D014162:Transfection; D014403:Tubocurarine / Q000494:pharmacology
D002147:Calmodulin; C496894:KCNN1 protein, human; C496897:KCNN2 protein, human; C496896:Kcnn1 protein, rat; C496899:Kcnn2 protein, rat; C496901:Kcnn3 protein, rat; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D011993:Recombinant Fusion Proteins; D051640:Small-Conductance Calcium-Activated Potassium Channels; D001030:Apamin; D004247:DNA; D014403:Tubocurarine