cDNA Cloning and Functional Characterization of the Mouse Ca2+-gated K+ Channel, mIK1

Abstract

We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca 2؉ -gated K ؉ (K Ca ) 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 K Ca channel activity expressed in Xenopus oocytes closely resembled the K ca of red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation, inward rectification, and Ca 2؉ 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, Ca 2؉ -dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes increased outward currents that were Ca 2؉ -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 K Ca mRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood stem cells in liquid culture.Terminal differentiation of erythroid precursor cells is marked by enucleation and reduction in cell volume. A major component of cell volume reduction is achieved by reduction of cell K ϩ content. Mature, circulating erythrocytes retain two major ion transport pathways mediating K ϩ efflux (1). These are: 1) electroneutral K-Cl cotransport and 2) a voltage-insensitive, Ca 2ϩ -activated potassium (K ϩ ) channel of intermediate conductance (2-4), also known as the Gardos channel (5). The Gardos channel is thought to play a major role in volume regulation in normal (6) and sickle (SS) 1 human erythrocytes (7,8).Especially in the chronically hypoxic environment of adherent or trapped sickle cells, the Gardos channel appears to mediate the major component of K ϩ loss from the erythrocyte (9), leading to an increased concentration of intracellular hemoglobin S, and exponentially decreasing the lag time for accelerated hemoglobin S polymerization (10). The Gardos channel's biophysical and pharmacological properties have been characterized in excised inside-out human red cell membrane patches, in which Ca 2ϩ -activated K (K Ca ) currents show inwardly rectifying properties with a unitary slope conductance ranging from 15 to 40 picosiemens, depending on the ionic conditions used (11-13). The channel is sensitive to block by charybdotoxin (14 -16), but insensitive to the SK channel blocker, apamin, and to the K ATP channel blockers, the antihypoglycemic drugs (17). 2Sickle...

The Cytoplasmic C-terminal Fragment of Polycystin-1 Regulates a Ca2+-permeable Cation Channel

“…Xenopus oocyte isolation, culture, and microinjection were performed using standard techniques (28,29). Oocytes injected with 12-25 ng of cRNA in a volume of 50 nl maintained integrity up to 18 days after injection.…”

“…Relative change in intracellular calcium concentration ([Ca 2ϩ ] i ) was determined by exciting the fluorophore at 490 nm and imaging at 530 nm as described (29). Additional oocytes were loaded for 45 min with 8 M Fura2-AM, then subjected to bath [Ca 2ϩ ] changes from 1.8 mM to nominal 0 mM to 10 mM.…”

“…Patch Clamp Recording of Cation Currents-Patch pipettes were pulled to a resistance of 5-8 megohms, and oocytes were devitellinized as described previously (29). Gigaseals were first attained in the cellattached mode.…”

“…In these oocytes [Ca 2ϩ ] i was determined by alternately exciting the fluorophore at 340 and 380 nm. 510-nm emission images were acquired and analyzed as described (29), using IMAGE1 and IMAGE1-FL software (Universal Imaging). …”

Delineation of the Clotrimazole/TRAM-34 Binding Site on the Intermediate Conductance Calcium-activated Potassium Channel, IKCa1

“…The intermediate conductance calcium-activated potassium channel IKCa1 1 (also known as IK1, KCa4, SK4, KCNN4, and the "Gardos channel") plays an important role in the physiology of lymphocytes, erythrocytes, and intestinal and airway epithelial cells (1)(2)(3)(4). IKCa1 channels are voltage-independent, and their activation is steeply sensitive to rises in intracellular Ca 2ϩ , calcium sensitivity being conferred by calmodulin tightly complexed to the C terminus of the channel (5).…”