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...

3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors, or statins, reduce the incidence of strokes and reduce infarct volume after cerebral ischemia in mice. Excitoxicity caused by overstimulation of glutamate receptors is a major cause of neuronal death after an ischemic brain insult. Experiments presented here explored whether statins protect cultured neurons from excitotoxic death caused by the glutamate receptor agonist NMDA. Treatment with statins preserved NMDA receptor-expressing cortical neurons and potently and substantially reduced lactate dehydrogenase release caused by exposure of embryonic mouse neocortical cultures to NMDA. The rank order of neuroprotective potency was rosuvastatin = simvastatin > atorvastatin = mevastatin > pravastatin, which is similar to the known rank order of potency for inhibition of the HMG-CoA reductase enzyme. Resistance of cultures to NMDA excitotoxicity developed after several days of statin exposure. Neuroprotection by rosuvastatin was coincident with a decrease in cell sterols and occurred with a similar potency as inhibition of cholesterol biosynthesis. Neuroprotection was substantially attenuated by cotreatment with either mevalonate or cholesterol and was mimicked by acute treatment with the cholesterol-extracting agent beta-cyclodextrin, suggesting that neuroprotection was mediated by depletion of a cellular pool of cholesterol because of the inhibition of cholesterol biosynthesis. These results suggest the possibility that, in addition to effects on cerebrovascular function, statins have the potential to render cortical neurons more resistant to NMDA-induced excitotoxic death as a result of changes in cell cholesterol homeostasis.

We have obtained evidence that the Ca(2+)-selective current activated by Ca2+ store depletion (Ca2+ release-activated Ca2+ current; Icrac) in Jurkat T lymphocytes is augmented in a time-dependent manner by Ca2+ itself. Whole cell patch clamp experiments employed high cytosolic Ca(2+)-buffering conditions to passively deplete Ca2+ stores. Rapidly switching to nominally Ca(2+)-free extracellular buffer instantaneously reduced Icrac measured at -100 mV to leak current level. Unexpectedly, readmission of 2 mM Ca2+ instantaneously restored only 38 +/- 5% (mean +/- SEM, n = 9) of the full Icrac amplitude. The remainder reappeared in a monotonic time-dependent manner over 10 to 20 sec. Rapid vs. slow intracellular Ca2+ chelators did not alter this process, and inorganic Icrac blockers did not regenerate it, arguing against an intracellular site of action. The effect was specific to Ca2+: introduction of the permeant ions, Ba2+ or Sr2+, failed to invoke time-dependent Icrac reappearance. Moreover, equimolar substitution of Ba2+ for Ca2+ initially produced Ba2+ current of similar magnitude to the full Ca2+ current, but the Ba2+ current decayed monotonically to < 50% of its initial amplitude in < 20 sec. Conversely, return to Ca2+ produced a time-dependent increase in Icrac to its larger Ca2+ permeation level. Thus Ca2+ appears to selectively promote a reversible transition of Icrac that results in larger current flux, and at least partially explains the selectivity of this current for Ca2+ over other divalent ions.

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1. Intracellular recordings were made from C-fiber neurons identified by antidromic conduction velocity in intact guinea pig nodose ganglia maintained in vitro, and whole-cell patch clamp recordings were made from dissociated guinea pig nodose neurons to investigate the contribution of various K+ conductances to action-potential repolarization. 2. The repolarizing phase of the intracellularly recorded action potential was prolonged in a concentration-dependent manner by charybdotoxin (Chtx; EC50 = 39 nM) or iberiatoxin (Ibtx; EC50 = 48 nM) in a subpopulation of 16/36 C-fiber neurons. In a subset of these experiments, removal of extracellular Ca2+ reversibly prolonged action-potential duration (APD) in the same 4/9 intracellularly recorded C-fiber neurons affected by Chtx (> or = 100 nM). These convergent results support that a Ca(2+)-activated K+ current (IC) contributes to action-potential repolarization in a restricted subpopulation of C-fiber neurons. 3. Tetraethylammonium (TEA; 1-10 mM) increased APD considerably further in the presence of 100-250 nM Chtx or Ibtx, or in nominally Ca(2+)-free superfusate in 14/14 intracellularly recorded C-fiber neurons. TEA affected APD similarly in subpopulations of neurons with and without IC, suggesting that a voltage-dependent K+ current (IK) contributes significantly to action-potential repolarization in most nodose C-fiber neurons. 4. Substitution of Mn2+ for Ca2+ reduced outward whole-cell currents elicited by voltage command steps positive to -30 mV (2-25 ms) in a subpopulation of 21/36 dissociated nodose neurons, supporting the heterogeneous expression of IC. The kinetics of outward tail current relaxations (tau s of 1.5-2 ms) measured at the return of 2-3 ms depolarizing steps to -40 mV were indistinguishable in neurons with and without IC, precluding a separation of the nodose IC and IK by a difference in deactivation rates. 5. Chtx (10-250 nM) reduced in a subpopulation of 3/8 C-fiber neurons the total outward current elicited by voltage steps depolarized to -30 mV in single microelectrode voltage-clamp recordings. TEA (5-10 mM) further reduced outward current in the presence of 100-250 nM Chtx in all eight experiments. The Chtx-sensitive current was taken to represent IC, and the TEA-sensitive current, the IK component contributing to action-potential repolarization. 6. Rapidly inactivating current (IA) was implicated in action-potential repolarization in a subpopulation of intracellularly recorded C-fiber neurons. In 4/7 neurons, incremented hyperpolarizing prepulses negative to -50 mV progressively shortened APD.(ABSTRACT TRUNCATED AT 400 WORDS)

D-Arg[Hyp3-Thi5-D-Tic7-Tic8]-bradykinin (NPC 16731) inhibited bradykinin (BK) binding and BKinduced contraction in guinea-pig ileum, being markedly more potent than D-Phe7-BK analogues as a BK2 receptor antagonist. In isolated trachea NPC 16731, unlike other BK2 antagonists, inhibited BK binding and BK-induced contraction, and 45Ca2+ efflux in tracheal smooth muscle cells. That NPC 16731 potently inhibits BK effects in trachea provides further evidence for the existence of the airway BK3 receptor.

1 Standard whole cell patch clamp recording techniques were used to study the pharmacological characteristics and site of econazole-mediated inhibition of calcium release-activated calcium current (Ic,_,)