title: Orientations and proximities of the extracellular ends of transmembrane helices S0 and S4 in open and closed BK potassium channels.
abstract: The large-conductance potassium channel (BK) α subunit contains a transmembrane (TM) helix S0 preceding the canonical TM helices S1 through S6. S0 lies between S4 and the TM2 helix of the regulatory β1 subunit. Pairs of Cys were substituted in the first helical turns in the membrane of BK α S0 and S4 and in β1 TM2. One such pair, W22C in S0 and W203C in S4, was 95% crosslinked endogenously. Under voltage-clamp conditions in outside-out patches, this crosslink was reduced by DTT and reoxidized by a membrane-impermeant bis-quaternary ammonium derivative of diamide. The rate constants for this reoxidation were not significantly different in the open and closed states of the channel. Thus, these two residues are approximately equally close in the two states. In addition, 90% crosslinking of a second pair, R20C in S0 and W203C in S4, had no effect on the V50 for opening. Taken together, these findings indicate that separation between residues at the extracellular ends of S0 and S4 is not required for voltage-sensor activation. On the contrary, even though W22C and W203C were equally likely to form a disulfide in the activated and deactivated states, relative immobilization by crosslinking of these two residues favored the activated state. Furthermore, the efficiency of recrosslinking of W22C and W203C on the cell surface was greater in the presence of the β1 subunit than in its absence, consistent with β1 acting through S0 to stabilize its immobilization relative to α S4.
authors.affiliation: Center for Molecular Recognition, Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, New York, USA.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Niu
authors.forename: Guoxia
authors.initials: G
authors.lastname: Liu
authors.forename: Roland S
authors.initials: RS
authors.lastname: Wu
authors.forename: Neelesh
authors.initials: N
authors.lastname: Chudasama
authors.forename: Sergey I
authors.initials: SI
authors.lastname: Zakharov
authors.forename: Arthur
authors.initials: A
authors.lastname: Karlin
authors.forename: Steven O
authors.initials: SO
authors.lastname: Marx
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000737:chemistry*; D008564:Membrane Potentials; D051379:Mice; D009154:Mutation; D010100:Oxygen / Q000737:chemistry; D017433:Protein Structure, Secondary
chemical_list: D004220:Disulfides; D051036:Large-Conductance Calcium-Activated Potassium Channels; D003545:Cysteine; D010100:Oxygen