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cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles in regulatory volume
D H Vandorpe1, B E Shmukler, L Jiang
1Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center Boston, Massachusetts 02215, USA.
Abstract:
We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca2+-gated K+ (KCa) channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci.(U. S. A. 94, 11651-11656). mIK1 mRNA was detected at varied levels in many tissue types. mIK1 KCa channel activity expressed in Xenopus oocytes closely resembled the Kca of red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation, inward rectification, and Ca2+ concentration dependence. mIK1 also resembled the erythroid channel in its pharmacological properties, mediating whole cell and unitary currents sensitive to low nM concentrations of both clotrimazole (CLT) and its des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to low nM concentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression conferred on oocytes a novel, Ca2+-dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes increased outward currents that were Ca2+-dependent, CLT-sensitive, and reversed near the K+ equilibrium potential. mIK1 mRNA levels in ES cells increased steadily during erythroid differentiation in culture, in contrast to other KCa mRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood stem cells in liquid culture.
Insights
We identified the mIK1 calcium-gated potassium channel in mouse cells. Its expression in oocytes enabled a novel volume decrease response, and clotrimazole inhibited stem cell proliferation.
Area of Science:
- Molecular Biology
- Cell Physiology
- Ion Channel Research
Background:
- The Ca2+-gated K+ (KCa) channel, hIK1, is crucial in various cell types.
- Understanding mammalian KCa channels is vital for cellular function and disease research.
Purpose of the Study:
- To clone and characterize the mouse homolog of hIK1, named mIK1.
- To investigate the functional properties and physiological roles of the mIK1 channel.
Main Methods:
- Cloning of mIK1 cDNA from murine erythroleukemia (MEL) cells, thymus, and stomach.
- Expression of mIK1 in Xenopus oocytes for electrophysiological analysis.
- Assessing mIK1 channel activity, Ca2+ dependence, and pharmacological properties.
- Investigating the role of mIK1 in oocyte regulatory volume decrease and stem cell differentiation.
Main Results:
- mIK1 mRNA is expressed in multiple mouse tissues.
- Expressed mIK1 channels exhibit properties similar to red blood cell (Gardos) and MEL cell KCa channels.
- mIK1 expression in oocytes confers a Ca2+-dependent, clotrimazole-sensitive regulatory volume decrease.
- Low nanomolar clotrimazole concentrations inhibit stem cell proliferation and erythroid differentiation.
Conclusions:
- mIK1 is a functional Ca2+-gated K+ channel in mice with characteristics similar to known erythroid KCa channels.
- mIK1 plays a role in cellular volume regulation and is a potential target for modulating stem cell proliferation and differentiation.