Local movement in the S2 region of the voltage-gated potassium channel hKv2.1 studied using cysteine mutagenesis.
The positively charged S4 region of voltage-dependent potassium channels moves outward during depolarization, leading to channel opening, but possible movement of the negatively charged S2 region may be more complex. Here we have studied possible movement of the S2 region of the slowly activating human voltage-dependent potassium channel hKv2.1. For this, cysteine mutants in the S2 region were expressed in Xenopus oocytes by injection of cRNA. Whole-cell currents were measured using the two-electrode voltage-clamp technique, and the effect of the membrane-impermeable cysteine-binding reagent parachloromercuribenzenesulfonate (PCMBS) was studied. For mutant S223C (located just outside the membrane in the S2 region), PCMBS inhibited currents and caused faster deactivation of tail currents. The time course of reactivity of PCMBS on tail current amplitudes was faster at more negative holding potentials. There was no effect of PCMBS on potassium channel currents for mutants D225C, N226C, A230C, and V232C. These data suggest that residue S223 is exposed to the extracellular phase at normal resting potentials, making it accessible to PCMBS, but upon depolarization there is a conformational change, making it less accessible, possibly by a local rather than global movement of S2 residues into the membrane. Voltage-dependent movements of nearby residues could also explain the results.
Milligan
C J
CJ
School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, England.
Wray
D
D
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D051668:Delayed Rectifier Potassium Channels; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014981:Xenopus
D017931:DNA Primers; D051668:Delayed Rectifier Potassium Channels; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D002731:4-Chloromercuribenzenesulfonate; D003545:Cysteine