A Novel Gene, hKCa4, Encodes the Calcium-activated Potassium Channel in Human T Lymphocytes

Abstract

We have isolated a novel gene, hKCa4, encoding an intermediate conductance, calcium-activated potassium channel from a human lymph node library. The translated protein comprises 427 amino acids, has six transmembrane segments, S1-S6, and a pore motif between S5 and S6. hKCa4 shares 41-42% similarity at the amino acid level with three small conductance calcium-activated potassium channels cloned from brain. Northern blot analysis of primary human T lymphocytes reveals a 2.2-kilobase transcript 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 hematopoietic origin. Patch-clamp recordings of hKCa4-transfected HEK 293 cells reveal a large voltage-independent, inwardly rectifying potassium 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, kaliotoxin, scyllatoxin (K d > 1 M), and margatoxin (K d > 100 nM). Single hKCa4 channels have a conductance of 33 ؎ 2 picosiemens in symmetrical potassium solutions. The channel is activated by intracellular calcium (K d ‫؍‬ 270 ؎ 8 nM) with a highly cooperative interaction of approximately three calcium ions per channel. These properties of the cloned channel are very similar to those reported for the native KCa channel in activated human T lymphocytes, indicating that hKCa4 encodes this channel type.Potassium channels play a critical role in modulating calcium signaling of lymphocytes (1). Human T lymphocytes express at least two types of potassium channels (2) (8), and B lymphocytes (9), as well as in other peripheral tissues. However, the molecular identity of this channel type was hitherto unknown. We report the cloning and characterization of an intermediate conductance, CTX-sensitive KCa channel, which we call hKCa4, from a human lymph node cDNA library. We present convergent molecular, biophysical, and pharmacological evidence that hKCa4 encodes the predominant KCa channel in human T cells. EXPERIMENTAL PROCEDURESData Base Search-We performed a BLAST search of a proprietary EST data base (licensed from Incyte Pharmaceuticals, Palo Alto, CA) for unannotated potassium channel sequences using the pore sequence of hKv2.1, a Shab-related K ϩ channel (PASFWWATITMTTVGYGDIYP; Ref. 10). Two overlapping clones of interest were identified, and their sequences were determined (Applied Biosystems PRISM™377 automated sequencer). Both of these clones were from a cDNA library of adherent mononuclear cells, which came from a pool of male and female donors.Library Screening and Computer Analysis-A 32 P-labeled DNA fragment from one of the above clones, corresponding to nucleotides 262-1265 in Fig. 1A, was used as a probe to screen ϳ600,000 recombinant plaques from a human lymph node gt10 cDNA library (CLONTECH). Hybridizations w...

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