Cardiac potassium channels are an important component in the excitation- propagation process. Recently, it has been demonstrated that a small protein (dubbed minimal K+, or minK) which is present in a variety of mammalian tissues, leads to the expression of a potassium current (IsK) in Xenopus laevis oocytes; a similar current can also be recorded from human embryonic kidney cells (HEK293) transfected with minK cDNA. The minK protein has been associated with the expression of the delayed rectifier potassium current in guinea pig cardiac ventricular myocytes, and it has been hypothesized that mink may be an essential (if not a constitutive) part of a functional potassium channels in the human heart. Preliminary results from our laboratory show that acidification of both the intra- and extracellular spaces of minK-expressing oocytes lead to IsK closure; the closure occurs at pH values higher than those observed (by other authors) when only the extracellular compartment is acidified; thus, our data suggest that IsK channels may also be sensitive to the proton concentration present in the cytoplasm. Previous studies have also shown that the biophysical properties and regulation by phosphorylation of IsK are determined, at least in part, by the primary sequence of the mink protein. The overall goal of this project is to characterize the molecular bases for acidification-induced closure of the delayed rectifier potassium current IsK that is associated with the expression of the human isoform of the minK protein. We will use a combination of electrophysiological , optical and molecular biological techniques in mink-expressing Xenpous laevis oocytes, as well as in HEK293 cells transfected with mink cDNA, to study the effect of acidification on the biophysical properties of IsK, and to determine a possible structure-function relation between the primary sequence of minK, and the acidification-induced closure of the IsK channels.
The Specific Aims are; 1) To characterize the magnitude, voltage dependence and time course of pH gating of minK-related channels upon acidification of the intr and/or the extracellular space. 2) To determine, by means of non- stationary fluctuation analysis, the effects of intra- and/or extracellular acidification on the unitary conductance and open probability of cell- attached IsK channels expressed in oocytes. 3) To characterize the possible role that soluble components of the cytoplasm may have on pH gating of IsK, and 4) to study the functional expression, and pH sensitivity of mutant forms of minK expressed in oocytes and in HEK292 cells. Successful accomplishment of these experiments should provide a better understanding of the mechanisms by which hydrogen ions inhibit IsK, and may give insight into pH-induced changes on cardiac electrical activity.
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