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.

Molecular Dynamics Simulations of Voltage-Gated Cation Channels: Insights on Voltage-Sensor Domain Function and Modulation

“…The S1–S4 bundles form the VSDs and are organized in a manner that agree with previous experimental results (Broomand et al, 2003; Neale, 2003): the VSD of one subunit sits next to the pore domain of the adjacent subunit and the connection between the VSD and the pore of the same subunit is insured by an S4–S5 helical linker (Figure 1). The VSD, of which all the segments are oriented roughly perpendicular to the membrane plane, shows rather tight packing.…”

Movement of the S4 segment in the hERG potassium channel during membrane depolarization

“…Thus the origin of the difference in the e o values between Shaker and hERG remains to be established. Structural differences in other parts of the voltage sensor, including the additional negative charges, or configuration of water crevices surrounding the cytoplasmic end of the sensor [16,28] might contribute to the differences in e o values between hERG and Shaker. One intriguing possibility is that the aqueous crevice at the intracellular phase of S4 could be smaller in hERG compared with other Kv channels, as this would increase the size of the electric field across which S4 charges need to move, and thereby reduce the net gating charge translocated.…”

Probing S4 and S5 segment proximity in mammalian hyperpolarization-activated HCN channels by disulfide bridging and Cd2+ coordination

“…The state-dependent changes in the S4-S5 arrangement seen here for HCN1 channels show intriguing similarities and differences with Shaker Kv channels (8, 18, 25, 36). Thus, certain pairs of Shaker residues near the extracellular ends of S4 and S5 form intersubunit disulfide bonds and Cd 2+ coordination sites (8, 25, 36).…”

“…Thus, certain pairs of Shaker residues near the extracellular ends of S4 and S5 form intersubunit disulfide bonds and Cd 2+ coordination sites (8, 25, 36). In contrast, Elliott et al (18) found that a different pair of Shaker residues near the external ends of S4 and S5 form an intrasubunit Cd 2+ coordination site preferentially when the channels are activated by depolarization.…”

“…Depending on the thermal energy present, Cys-Cys disulfide bridges only arise if the Cys residues are within 15 Ǻ of each other (10), while Cd 2+ coordination between 2 or more Cys residues further restricts the molecular proximity to <6 Ǻ (11, 42). With this approach, it has been shown that the extracellular end of the Kv S4 lies within 6 Ǻ of the extracellular end of S5 of a neighboring subunit, suggesting a close intersubunit proximity (8, 25, 36). However, there is also evidence that similar residue positions on Kv S4 and S5 within the same subunit interact to coordinate Cd 2+ , suggesting a close intrasubunit proximity.…”