Electrostatic interactions between transmembrane segments mediate folding of Shaker K+ channel subunits

Abstract

In voltage-dependent Shaker K+ channels, charged residues E293 in transmembrane segment S2 and R365, R368, and R371 in S4 contribute significantly to the gating charge movement that accompanies activation. Using an intragenic suppression strategy, we have now probed for structural interaction between transmembrane segments S2, S3, and S4 in Shaker channels. Charge reversal mutations of E283 in S2 and K374 in S4 disrupt maturation of the protein. Maturation was specifically and efficiently rescued by second-site charge reversal mutations, indicating that electrostatic interactions exist between E283 in S2 and R368 and R371 in S4, and between K374 in S4 and E293 in S2 and D316 in S3. Rescued subunits were incorporated into functional channels, demonstrating that a native structure was restored. Our data indicate that K374 interacts with E293 and D316 within the same subunit. These electrostatic interactions mediate the proper folding of the protein and are likely to persist in the native structure. Our results raise the possibility that the S4 segment is tilted relative to S2 and S3 in the voltage-sensing domain of Shaker channels. Such an arrangement might provide solvent access to voltage-sensing residues, which we find to be highly tolerant of mutations.

Structural basis of two-stage voltage-dependent activation in K+channels

“…Previous work in Shaker channels suggests a structural basis for the novel effect of Ni 2ϩ on D274A channels (22,23). D274 is the homologue of E283 in the S2 segment of Shaker (Fig.…”

“…4A). Using a second-site suppressor strategy, we have presented evidence that E283 is involved in short-range electrostatic structural interactions with R368 and R371, two positively charged residues in the S4 segment that participate in the voltage-dependent conformational changes of S4 during activation gating of Shaker channels (22). Similar to D274A in eag, a neutralization mutation of E283, E283Q, does not eliminate functional expression of Shaker (15).…”

“…In contrast, the charge-reversal mutation, E283R, disrupts folding of the Shaker protein. The folding defect of E283R is suppressed specifically by R368E or R371E (22). Functional analysis suggests that during voltage-dependent activation of Shaker channels, first R368 and, subsequently, R371 enter an extracellular-facing gating pocket, where they interact with E283 (22,23).…”

Functional diversity of potassium channel voltage-sensing domains

“…49,[57][58][59][60][61] The rest of the transmembrane domains seem to remain mostly static as the S4 moves, providing a physical and electrostatic scaffold. 62,63 Given that the rest of the VSD remains static, as the charged residues in S4 move, they traverse its central portion, which contains an aromatic residue that has been dubbed the charge transfer center. 64 This residue is located at the region of the waist of the hourglass, and is the thinnest portion of the VSD structure, separating the outer and inner vestibules and keeping water and ions from traversing it.…”

Depolarization Induces Intersubunit Cross-linking in a S4 Cysteine Mutant of the Shaker Potassium Channel

“…The structure of the pore domain is thought to be similar to that of the bacterial potassium channel, KcsA, whose structure has been determined by x-ray diffraction (5-7). The remaining transmembrane segments, in particular the S2 to S4 segments, are thought to comprise the voltage-sensing domain of the channel (2,9,10). Of the four segments, S4 plays a pivotal role.…”