A highly conserved alanine in the S6 domain of the hERG1 K+ channel is required for normal gating.
The central cavity of K(+)-selective ion channels is lined by four S6 transmembrane alpha-helices. An Ala residue is located near the midpoint of each S6 and marks the narrowest point of the central cavity. In hERG1 channels, we determined the functional consequences of substituting this conserved Ala (Ala653) with other hydrophobic or charged amino acids. Mutant channels were expressed in Xenopus oocytes and ionic currents measured by using the two-microelectrode voltage clamp technique. Substitution of Ala653 with bulkier hydrophobic residues (Val, Leu, Ile, Met, Phe, Trp) did not prevent ion conduction, but the mutant channels activated at more negative potentials compared to wild-type channels. The half-point for voltage dependent activation was shifted by -54 mV for the most conservative hydrophobic mutation, A653V. Oxidation of A653C hERG1 channels induced a maintained current at negative membrane potentials. This effect was not reversible with dithiothreitol, indicating that the sulfhydryl side-chains of Cys653 were oxidized to a negatively charged sulfinic or sulfonic acid. Substitution of Ala653 with acidic (Asp, Glu) or basic (Arg, Lys) residues prevented channel deactivation. Thus, an Ala at position 653 in hERG1 is required for normal voltage dependence of channel gating and a charged residue in this position prevents channel closure.
Brown
Scott
S
Department of Physiology, Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, UT 84112, USA. sanguinetti@cvrti.utah.edu
Sonntag
David P
DP
Sanguinetti
Michael C
MC
D000409:Alanine / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D055592:Biophysical Phenomena; D017124:Conserved Sequence; D003545:Cysteine; D004229:Dithiothreitol; D051638:Ether-A-Go-Go Potassium Channels / Q000737:chemistry* / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D015640:Ion Channel Gating; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D051638:Ether-A-Go-Go Potassium Channels; C496884:KCNH1 protein, human; D050505:Mutant Proteins; D003545:Cysteine; D000409:Alanine; D004229:Dithiothreitol