Evidence for Intersubunit Interactions between S4 and S5 Transmembrane Segments of the Shaker Potassium Channel

• Published in: The Journal of biological chemistry 2003-08, Vol.278 (31), p.29079-29085
• Main Authors: Neale, Edward J., Elliott, David J.S., Hunter, Malcolm, Sivaprasadarao, Asipu
• Format: Article
• Language: English
• Subjects: Animals; Cadmium - pharmacology; Cell Membrane - chemistry; Cysteine - chemistry; Disulfides - chemistry; Electric Conductivity; Female; Gene Expression; Models, Molecular; Molecular Structure; Mutagenesis, Site-Directed; Oocytes - metabolism; Oxidation-Reduction; Phenanthrolines - pharmacology; Phosphines - pharmacology; Potassium - metabolism; Potassium Channels - chemistry; Potassium Channels - genetics; Potassium Channels - physiology; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - physiology; Reducing Agents - pharmacology; Shaker Superfamily of Potassium Channels; Structure-Activity Relationship; Xenopus; Xenopus laevis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M301991200

Voltage-gated potassium channels are transmembrane proteins made up of four subunits, each comprising six transmembrane (S1-S6) segments. S1-S4 form the voltage-sensing domain and S5-S6 the pore domain with its central pore. The sensor domain detects membrane depolarization and transmits the signal to the activation gates situated in the pore domain, thereby leading to channel opening. An understanding of the mechanism by which the sensor communicates the signal to the pore requires knowledge of the structure of the interface between the voltage-sensing and pore domains. Toward this end, we have introduced single cysteine mutations into the extracellular end of S4 (positions 356 and 357) in conjunction with a cysteine in S5 (position 418) of the Shaker channel and expressed the mutants in Xenopus oocytes. We then examined the propensity of each pair of engineered cysteines to form a metal bridge or a disulfide bridge, respectively, by examining the effect of Cd2+ ions and copper phenanthroline on the K+ conductance of a whole oocyte. Both reagents reduced currents through the S357C,E418C double mutant channel, presumably by restricting the movements necessary for coupling the voltage-sensing function to pore opening. This inhibitory effect was seen in the closed state of the channel and with heteromers composed of S357C and E418C single mutant subunits; no effect was seen with homomers of any of the single mutant channels. These data indicate that the extracellular end of S4 lies in close proximity to the extracellular end of the S5 of the neighboring subunit in closed channels.