A human intermediate conductance calcium-activated potassium channel

T Ishii

C Silvia

B Hirschberg

C Bond

J Adelman

J Maylie

^{Vollum Institute and}^{Department of Obstetrics and Gynecology, Oregon Health Sciences University, Portland, OR 97201; and}^{ICAgen Corporation, Research Triangle Park, NC 27703}

^{To whom reprint requests should be addressed at: Department of Obstetrics and Gynecology, Oregon Health Sciences University, L-458, 3181 SW Sam Jackson Park Road, Portland, OR 97201. e-mail:}ude.usho@jeilyam.

Edited by Lily Yeh Jan, University of California, San Francisco, San Francisco, CA, and approved August 7, 1997

^{Vollum Institute and}^{Department of Obstetrics and Gynecology, Oregon Health Sciences University, Portland, OR 97201; and}^{ICAgen Corporation, Research Triangle Park, NC 27703}

Edited by Lily Yeh Jan, University of California, San Francisco, San Francisco, CA, and approved August 7, 1997

Received 1997 Jun 26

Abstract

An intermediate conductance calcium-activated potassium channel, hIK1, was cloned from human pancreas. The predicted amino acid sequence is related to, but distinct from, the small conductance calcium-activated potassium channel subfamily, which is ≈50% conserved. hIK1 mRNA was detected in peripheral tissues but not in brain. Expression of hIK1 in Xenopus oocytes gave rise to inwardly rectifying potassium currents, which were activated by submicromolar concentrations of intracellular calcium (K_0.5 = 0.3 μM). Although the K_0.5 for calcium was similar to that of small conductance calcium-activated potassium channels, the slope factor derived from the Hill equation was significantly reduced (1.7 vs. 3.5). Single-channel current amplitudes reflected the macroscopic inward rectification and revealed a conductance level of 39 pS in the inward direction. hIK1 currents were reversibly blocked by charybdotoxin (K_i = 2.5 nM) and clotrimazole (K_i = 24.8 nM) but were minimally affected by apamin (100 nM), iberiotoxin (50 nM), or ketoconazole (10 μM). These biophysical and pharmacological properties are consistent with native intermediate conductance calcium-activated potassium channels, including the erythrocyte Gardos channel.

Abstract

Three distinct classes of calcium-activated K^{channels (K}_Ca channels) have been described. Large conductance calcium-activated K^{(BK) channels are gated by the concerted actions of internal calcium ions and membrane potential and have a unit conductance of between 100 and 220 pS. Small (SK) and intermediate (IK) conductance calcium-activated K}^{channels are gated solely by internal calcium ions, with a unit conductance of 2–20 and 20–85 pS, respectively, and are more sensitive to calcium than are BK channels (for review, see ref.}1). In addition, each type of K_Ca channel shows a distinct pharmacology, and the activity of each hyperpolarizes the membrane potential. Members of the BK (2–4) and SK (5) subfamilies have been cloned and expressed in heterologous cell types, where they recapitulate the fundamental properties of their native counterparts.

The first demonstration that internal calcium ions regulate potassium flux was provided by Gardos from red blood cells (6). A rise in intracellular Ca^{in red blood cells opens the Gardos channel, causing potassium loss and cell dehydration, a condition that is exacerbated in sickle cell anemia. Promising therapeutic approaches for sickle cell anemia involve specifically blocking the Gardos channel (}7, 8). Like IK channels, the Gardos channel is opened by submicromolar concentrations of internal calcium and has a rectifying unit conductance, ranging from 50 pS at −120 mV to 13 pS at 120 mV (symmetrical 130 mM K^{; ref.}9). It is blocked by charybdotoxin (CTX) but not the structurally related peptide iberiotoxin (IBX), both of which block BK channels (10). Apamin, a potent blocker of certain native (11, 12) and cloned (5) SK channels, does not block IK channels (13). The Gardos channel is also blocked by some imidazole compounds, such as clotrimazole, but not ketoconazole (ref. 7; S. Alper, personal communication). The electrophysiological and pharmacological properties of the Gardos channel show that it belongs to the IK subfamily.

IK channels have been described in a variety of other cell types. IK channels have also been implicated in the microvasculature of the kidney, where they may be responsible for the vasodilatory effects of bradykinin (14). In brain capillary endothelial cells, IK channels, activated by endothelin that is produced by neurons and glia, shunt excess K^{into the blood (}15). Neutrophil granulocytes, mobile phagocytic cells that defend against microbial invaders, undergo a large depolarization subsequent to agonist stimulation, and IK channels have been implicated in repolarizing the stimulated granulocyte (16).

IK channels are distinguished from other K_Ca channel types by their biophysical and pharmacological profiles. Here, we describe the isolation and heterologous expression of the first member of this class of calcium-activated potassium channels. hIK1 is structurally related to the SK subfamily, and expression in Xenopus oocytes results in intermediate conductance calcium-activated K^{channels with a pharmacology that is consistent with the Gardos channel from red blood cells and IK channels from other tissues.}

Acknowledgments

We thank Dr. Martin Köhler for some initial database searches and Dr. Kay Wagner for patience and support. T.M.I. is supported by grants from the Uehara Memorial Foundation and the Naito Foundation. This work was supported by National Institutes of Health Grants (J.P.A. and J.M.).

Acknowledgments

Footnotes

This paper was submitted directly (Track II) to the Proceedings Office.

Abbreviations: BK, SK, and IK, large, small, and intermediate conductance calcium-activated K^{channels, respectively; CTX, charybdotoxin; IBX, iberiotoxin; RACE, rapid amplification of cDNA ends.}

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. {"type":"entrez-nucleotide","attrs":{"text":"AF022150","term_id":"2655058"}}AF022150).

Footnotes

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