A NOVEL GENE, HKCA4, ENCODES THE CALCIUM-ACTIVATED POTASSIUM CHANNEL IN HUMAN T-LYMPHOCYTES

We have isolated a novel gene, hKCa4, encoding an intermediate conduct ance, calcium-activated potassium channel from a human lymph node libr ary, The translated protein comprises 427 amino acids, has six transme mbrane segments, S1-S6, and a pore motif between S5 and S6. hKCa4 shar es 41-42% similarity at the amino acid level with three small conducta nce calcium-activated potassium channels cloned from brain, Northern b lot analysis of primary human T lymphocytes reveals a 2.2-kilobase tra nscript that is highly up-regulated in activated compared with resting cells, concomitant with an increase in KCa current, hKCa4 transcript is also detected by Northern blots or by polymerase chain reaction in placenta, prostate, thymus, spleen, colon, and many cell lines of hema topoietic origin. Patch-clamp recordings of hKCa4-transfected HEK 293 cells reveal a large voltage-independent, inwardly rectifying potassiu m current that is blocked by externally applied tetraethylammonium (K- d = 30 +/- 7 mM), charybdotoxin (K-d = 10 +/- 1 nM and clotrimazole (K -d = 387 +/- 34 nM), but is resistant to apamin, iberiotoxin, kaliotox in, scyllatoxin (K-d > 1 mu M), and margatoxin (K-d > 100 nM). Single hKCa4 channels have a conductance of 33 +/- 2 picosiemens in symmetric al potassium solutions. The channel is activated by intracellular calc ium (K-d = 270 +/- 8 nM) with a highly cooperative interaction of appr oximately three calcium ions per channel, These properties of the clon ed channel are very similar to those reported for the native KCa chann el in activated human T lymphocytes, indicating that hKCa4 encodes thi s channel type.