Location of KCNE1 relative to KCNQ1 in the I(KS) potassium channel by disulfide cross-linking of substituted cysteines.
The cardiac-delayed rectifier K(+) current (I(KS)) is carried by a complex of KCNQ1 (Q1) subunits, containing the voltage-sensor domains and the pore, and auxiliary KCNE1 (E1) subunits, required for the characteristic I(KS) voltage dependence and kinetics. To locate the transmembrane helix of E1 (E1-TM) relative to the Q1 TM helices (S1-S6), we mutated, one at a time, the first four residues flanking the extracellular ends of S1-S6 and E1-TM to Cys, coexpressed all combinations of Q1 and E1 Cys-substituted mutants in CHO cells, and determined the extents of spontaneous disulfide-bond formation. Cys-flanking E1-TM readily formed disulfides with Cys-flanking S1 and S6, much less so with the S3-S4 linker, and not at all with S2 or S5. These results imply that the extracellular flank of the E1-TM is located between S1 and S6 on different subunits of Q1. The salient functional effects of selected cross-links were as follows. A disulfide from E1 K41C to S1 I145C strongly slowed deactivation, and one from E1 L42C to S6 V324C eliminated deactivation. Given that E1-TM is between S1 and S6 and that K41C and L42C are likely to point approximately oppositely, these two cross-links are likely to favor similar axial rotations of E1-TM. In the opposite orientation, a disulfide from E1 K41C to S6 V324C slightly slowed activation, and one from E1 L42C to S1 I145C slightly speeded deactivation. Thus, the first E1 orientation strongly favors the open state, while the approximately opposite orientation favors the closed state.
Chung
David Y
DY
Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
Chan
Priscilla J
PJ
Bankston
John R
JR
Yang
Lin
L
Liu
Guoxia
G
Marx
Steven O
SO
Karlin
Arthur
A
Kass
Robert S
RS
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D006801:Humans; D051657:KCNQ1 Potassium Channel / Q000737:chemistry* / Q000502:physiology; D008969:Molecular Sequence Data; D024642:Potassium Channels, Voltage-Gated / Q000737:chemistry* / Q000502:physiology; D017433:Protein Structure, Secondary
D004220:Disulfides; C489622:KCNE1 protein, human; D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; D024642:Potassium Channels, Voltage-Gated; D003545:Cysteine; D004229:Dithiothreitol