Mapping the Membrane-aqueous Border for the Voltage-sensing Domain of a Potassium Channel

Abstract

Voltage-sensing domains (VSDs) play diverse roles in biology. As integral components, they can detect changes in the membrane potential of a cell and couple these changes to activity of ion channels and enzymes. As independent proteins, homologues of the VSD can function as voltage-dependent proton channels. To sense voltage changes, the positively charged fourth transmembrane segment, S4, must move across the energetically unfavorable hydrophobic core of the bilayer, which presents a barrier to movement of both charged species and protons. To reduce the barrier to S4 movement, it has been suggested that aqueous crevices may penetrate the protein, reducing the extent of total movement. To investigate this hypothesis in a system containing fully functional channels in a native environment with an intact membrane potential, we have determined the contour of the membrane-aqueous border of the VSD of KvAP in Escherichia coli by examining the chemical accessibility of introduced cysteines. The results revealed the contour of the membrane-aqueous border of the VSD in its activated conformation. The water-inaccessible regions of S1 and S2 correspond to the standard width of the membrane bilayer (ϳ28 Å ), but those of S3 and S4 are considerably shorter (>40%), consistent with aqueous crevices pervading both the extracellular and intracellular ends. One face of S3b and the entire S3a were water-accessible, reducing the water-inaccessible region of S3 to just 10 residues, significantly shorter than for S4. The results suggest a key role for S3 in reducing the distance S4 needs to move to elicit gating.

Functionality of the voltage-gated proton channel truncated in S4

“…We performed site-directed cysteine-scanning of mVSOP proteins heterologously expressed in tsA201 cells in which the accessibility of cysteines to maleimide-containing reagent was determined by Western blotting. This "PEGylation-protection" assay was originally developed by Deutsch and colleagues (24) and used to study the topology and folding of Shaker-like potassium channels in microsomes and the KvAP channel in the bacterial membrane (32). With this method, target proteins are exposed to two maleimide-containing reagents, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS) and maleimide-conjugated polyethylene glycol (mal-PEG).…”

YidC Protein, a Molecular Chaperone for LacY Protein Folding via the SecYEG Protein Machinery

“…To do so, we used a gel shift alkylation assay (44), which has an advantage over protease accessibility assays because chemical modification can monitor translocation of individual loops of multispanning membrane proteins regardless of loop size. Moreover, these studies and others were facilitated using rifampicin in the labeling reaction to inhibit RNA polymerase and do not affect membrane insertion of LacY under YidC/Sec plus conditions, although they could potentially have unrelated effects because of shutting down endogenous protein synthesis.…”

“…Gel Shift Assay Monitors Translocation of Periplasmic LoopsSite-directed alkylation (55) was utilized to test translocation of the periplasmic loops and to assess the topology of LacY (44). Single Cys residues were introduced into the periplasmic TM or cytoplasmic region of a functional LacY mutant devoid of the native Cys residues (56).…”

“…To define a precise role of YidC in the insertion and folding of the galactoside/H ϩ symporter LacY, we examined the translocation of each of the six periplasmic loops of LacY utilizing a Cys-based alkylation method (44). The findings demonstrate that YidC is not required for translocation of the periplasmic loops of Sec-dependent LacY but is necessary for proper folding.…”

Disulfide mapping the voltage-sensing mechanism of a voltage-dependent potassium channel

“…The modification can be detected as a mobility shift in the SDS-PAGE analysis2627. In order to estimate the intramolecular SS-bond formation in the VSD double Cys mutants by detecting free thiol groups, VSD double Cys mutants, which were pre-denatured in SDS and urea, were treated with mal-PEG with a molecular weight of ca.…”