Depolarization induces intersubunit cross-linking in a S4 cysteine mutant of the Shaker potassium channel.
Voltage-gated potassium (K(v)) channels are integral membrane proteins, composed of four subunits, each comprising six (S1-S6) transmembrane segments. S1-S4 comprise the voltage-sensing domain, and S5-S6 with the linker P-loop forms the ion conducting pore domain. During activation, S4 undergoes structural rearrangements that lead to the opening of the channel pore and ion conduction. To obtain details of these structural changes we have used the engineered disulfide bridge approach. For this we have introduced the L361C mutation at the extracellular end of S4 of the Shaker K channel and expressed the mutant channel in Xenopus oocytes. When exposed to mild oxidizing conditions (ambient oxygen or copper phenanthroline), Cys-361 formed an intersubunit disulfide bridge as revealed by the appearance of a dimeric band on Western blotting. As a consequence, the mutant channel suffered a significant loss in conductance (measured by two-electrode voltage clamp). Removal of native cysteines failed to prevent the disulfide formation, indicating that Cys-361 forms a disulfide with its counterpart in the neighboring subunit. The effect was voltage-dependent and occurred during channel activation after Cys-361 has been exposed to the extracellular phase. Although the disulfide bridge reduced the maximal conductance, it caused a hyperpolarizing shift in the conductance-voltage relationship and reduced the deactivation kinetics of the channel. The latter two effects suggest stabilization of the open state of the channel. In conclusion, we report that during activation the intersubunit distance between the N-terminal ends of the S4 segments of the L361C mutant Shaker K channel is reduced.
School of Biomedical Sciences, Leeds University, Leeds LS2 9JT, United Kingdom.
Qadeer H
QH
Aziz
Christopher J
CJ
Partridge
Tim S
TS
Munsey
Asipu
A
Sivaprasadarao
D019943:Amino Acid Substitution; D000818:Animals; D003432:Cross-Linking Reagents; D003545:Cysteine; D004220:Disulfides / Q000378:metabolism; D005260:Female; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000494:pharmacology; D015221:Potassium Channels / Q000737:chemistry* / Q000187:drug effects / Q000502:physiology*; D021122:Protein Subunits / Q000737:chemistry / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D051637:Shaker Superfamily of Potassium Channels; D014981:Xenopus
D003432:Cross-Linking Reagents; D004220:Disulfides; D010618:Phenanthrolines; D015221:Potassium Channels; D021122:Protein Subunits; D011994:Recombinant Proteins; D051637:Shaker Superfamily of Potassium Channels; D003545:Cysteine; C025205:1,10-phenanthroline