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

Abstract

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 Cd 2؉ 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.

Relative Movements of Transmembrane Regions at the Outer Mouth of the Cystic Fibrosis Transmembrane Conductance Regulator Channel Pore during Channel Gating

“…The proximity of two cysteine side chains was also determined by tight binding of externally applied Cd 2ϩ ions, resulting in inhibition of channel function. The functional consequences of both CuPhe-induced cross-linking and Cd 2ϩ coordination have previously been used to infer distances between pairs of cysteine side chains of Ͻ10 Å in other channel types (31)(32)(33)(34)(35). Furthermore, both CuPhe-induced cysteine cross-linking (18,19) and Cd 2ϩ inhibition of single cysteine mutants (23) have been investigated in the CFTR channel pore using electrophysiological recording.…”

Outer Pore Topology of the ECaC-TRPV5 Channel by Cysteine Scan Mutagenesis

“…Experiments where nonspecific leaks developed during the experiment were simply discarded. The reversibility of covalent bonds was tested with 5 mM BMS (bis(2-mercaptoethylsulfone)) (Calbiochem, San Diego, CA) a water-soluble reagent considered to be a superior reducing agent than dithiothreitol (25,26). The percentage of currents remaining after MTS modification was computed at V m ϭ Ϫ150 mV using [(I wash )/(I ctrl )] ϫ 100 where I wash is the whole cell peak current remaining after MTS application and washout and I ctrl is the whole cell peak current measured before MTS application.…”

Structure, Function, and Modification of the Voltage Sensor in Voltage-Gated Ion Channels

“…In subsequent studies based on disulfide linkage between S4 and S5 the VSD of one subunit was shown to make close contact with the pore domain of a neighbouring subunit, not with its own subunit [60,[71][72][73][74]. This proposed interaction between two neighbouring subunits was later on supported by the X-ray structure of Kv1.2 [37].…”