Contribution of the NH2 terminus of Kv2.1 to channel activation.
Opening and closing of voltage-operated channels requires the interaction of diverse structural elements. One approach to the identification of channel domains that participate in gating is to locate the sites of action of modifiers. Covalent reaction of Kv2.1 channels with the neutral, sulfhydryl-specific methylmethanethiosulfonate (MMTS) caused a slowing of channel gating with a predominant effect on the kinetics of activation. These effects were also obtained after intracellular, but not extracellular, application of a charged MMTS analog. Single channel analysis revealed that MMTS acted primarily by prolonging the latency to first opening without substantially affecting gating transitions after the channel first opens and until it inactivates. To localize the channel cysteine(s) with which MMTS reacts, we generated NH2- and COOH-terminal deletion mutants and a construct in which all three cysteines in transmembrane regions were substituted. Only the NH2-terminal deletion construct gave rise to currents that activated slowly and displayed MMTS-insensitive kinetics. These results show that the NH2-terminal tail of Kv2.1 participates in transitions leading to activation through interactions involving reduced cysteine(s) that can be modulated from the cytoplasmic phase.
Pascual
J M
JM
Center for Molecular Recognition, Columbia University, New York, New York 10032, USA.
Shieh
C C
CC
Kirsch
G E
GE
Brown
A M
AM
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D051668:Delayed Rectifier Potassium Channels; D004229:Dithiothreitol / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000502:physiology; D015221:Potassium Channels / Q000096:biosynthesis / Q000737:chemistry / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017384:Sequence Deletion; D051669:Shab Potassium Channels
D051668:Delayed Rectifier Potassium Channels; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011994:Recombinant Proteins; D051669:Shab Potassium Channels; C014674:methyl methanethiosulfonate; D008741:Methyl Methanesulfonate; D004229:Dithiothreitol