title: The SK3 subunit of small conductance Ca2+-activated K+ channels interacts with both SK1 and SK2 subunits in a heterologous expression system.
abstract: The aim of this study was to determine whether functional heteromeric channels can be formed by co-assembly of rat SK3 (rSK3) potassium channel subunits with either SK1 or SK2 subunits. First, to determine whether rSK3 could co-assemble with rSK2 we created rSK3VK (an SK3 mutant insensitive to block by UCL 1848). When rSK3VK was co-expressed with rSK2 the resulting currents had an intermediate sensitivity to UCL 1848 (IC50 of approximately 5 nM compared with 120 pM for rSK2 and >300 nM for rSK3VK), suggesting that rSK3 and rSK2 can form functional heteromeric channels. To detect co-assembly of SK3 with SK1, we initially used a dominant negative construct of the human SK1 subunit (hSK1YP). hSK1YP dramatically reduced the SK3 current, supporting the idea that SK3 and SK1 subunits also interact. To determine whether these assemblies were functional we created rSK3VF, an rSK3 mutant with an enhanced affinity for tetraethylammonium chloride (TEA) (IC50 of 0.3 mM). Co-transfection of rSK3VF and hSK1 produced currents with a sensitivity to TEA not different from that of hSK1 alone (IC50 approximately 15 mM). These results suggest that hSK1 does not produce functional cell-surface assemblies with SK3. Antibody-staining experiments suggested that hSK1 may reduce the number of functional SK3 subunits reaching the cell surface. Additional experiments showed that co-expression of the rat SK1 gene with SK3 also dramatically suppressed SK current. The pharmacology of the residual current was consistent with that of homomeric SK3 assemblies. These results demonstrate interactions that cause changes in protein trafficking, cell surface expression, and channel pharmacology and strongly suggest heteromeric assembly of SK3 with the other SK channel subunits.
authors.affiliation: Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
authors.forename: Alan S
authors.initials: AS
authors.lastname: Monaghan
authors.forename: David C H
authors.initials: DC
authors.lastname: Benton
authors.forename: Parmvir K
authors.initials: PK
authors.lastname: Bahia
authors.forename: Ramine
authors.initials: R
authors.lastname: Hosseini
authors.forename: Yousaf A
authors.initials: YA
authors.lastname: Shah
authors.forename: Dennis G
authors.initials: DG
authors.lastname: Haylett
authors.forename: Guy W J
authors.initials: GW
authors.lastname: Moss
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D005799:Genes, Dominant; D006801:Humans; D007150:Immunohistochemistry; D020128:Inhibitory Concentration 50; D018613:Microscopy, Confocal; D009154:Mutation; D015221:Potassium Channels / Q000737:chemistry* / Q000378:metabolism; D024681:Potassium Channels, Calcium-Activated; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011806:Quinolinium Compounds / Q000494:pharmacology; D051381:Rats; D051640:Small-Conductance Calcium-Activated Potassium Channels; D019789:Tetraethylammonium / Q000494:pharmacology; D014162:Transfection
chemical_list: C414209:8,14-diaza-1,7(1, 4)-diquinolinacyclotetradecaphane; 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; D011806:Quinolinium Compounds; D051640:Small-Conductance Calcium-Activated Potassium Channels; D019789:Tetraethylammonium; D002118:Calcium