The minK potassium channel exists in functional and nonfunctional forms when expressed in the plasma membrane of Xenopus oocytes.
The minK protein induces a slowly activating voltage-dependent potassium current when expressed in Xenopus oocytes. In order to measure the levels of minK protein in the plasma membrane, we have modified the minK gene by inserting a 9 amino acid epitope into the N-terminal domain of the protein sequence. When intact live oocytes are injected with the modified minK RNA and subsequently incubated with an antibody to this epitope, specific binding is detected, indicating that the N-terminal domain is extracellular. We found that when oocytes are injected with amounts of minK mRNA up to 50 ng, the levels of protein at the surface are proportional to the amount of injected mRNA. In contrast, the amplitude of the minK current recorded in the oocytes saturates at 1 ng of injected mRNA. Although the amplitude of the currents is not altered by increasing mRNA levels above 1 ng, the kinetics of activation of the current differ in oocytes with high or low levels of minK RNA. In particular, activation is slower with higher levels of minK protein in the plasma membrane. Finally, we find that increasing intracellular cAMP levels, which increases the amplitude of minK currents, does not alter surface expression of the minK protein but produces a small increase in the rate of activation of the current. Our results support a model in which minK protein forms functional potassium channels by association with a factor endogenous to the oocyte.
Blumenthal
E M
EM
Interdepartmental Neuroscience Program, Yale University School of Medicine, New Haven, Connecticut 06510.
Kaczmarek
L K
LK
D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D004594:Electrophysiology; D000939:Epitopes; D007267:Injections; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D009865:Oocytes / Q000378:metabolism*; D015221:Potassium Channels / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D012333:RNA, Messenger; D014981:Xenopus / Q000378:metabolism*
D000939:Epitopes; D015345:Oligonucleotide Probes; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D012333:RNA, Messenger; C059983:potassium channel protein I(sk); D000242:Cyclic AMP