A novel gene, hKCa4, encodes the calcium-activated potassium channel in human T lymphocytes.

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 (Kd = 30 +/- 7 mM), charybdotoxin (Kd = 10 +/- 1 nM), and clotrimazole (Kd = 387 +/- 34 nM), but is resistant to apamin, iberiotoxin, kaliotoxin, scyllatoxin (Kd > 1 microM), and margatoxin (Kd > 100 nM). Single hKCa4 channels have a conductance of 33 +/- 2 picosiemens in symmetrical potassium solutions. The channel is activated by intracellular calcium (Kd = 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.

Extracellular site for econazole-mediated block of Ca2+ release-activated Ca2+ current (Icrac) in T lymphocytes.

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 (Icrac) in the human leukaemic T cell line, Jurkat. 2. Extracellularly applied econazole blocked Icrac in a concentration-dependent manner (IC50 approximately 14 microM). Block developed over a relatively slow timecourse of 30-60 s (10 microM), and only partially reversed over minutes. 3. Econazole dialysed from the pipette into the cytosol at concentrations ranging from 0.1 to 30 microM did not reduce Icrac, or quantitatively affect Icrac block by extracellularly applied econazole. 4. A less lipophilic quaternary iodide derivative of econazole was synthesized to retard absorption through the cell membrane. When applied extracellularly, this compound blocked Icrac in a concentration-dependent manner with onset kinetics comparable to econazole. 5. Results with intracellularly dialysed econazole and the quaternary econazole derivative provide convergent evidence that econazole blocks Icrac via an extracellular interaction. 6. The inability of intracellularly applied econazole to inhibit Icrac argues against the notion that econazole inhibits capacitative Ca2+ entry pathways secondary to its known inhibitory effects on cytochrome P-450.

Calcium-dependent enhancement of depletion-activated calcium current in Jurkat T lymphocytes.

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.

Excitable properties and underlying Na+ and K+ currents in neurons from the guinea-pig jugular ganglion.

Neurons in the superior vagal (jugular) ganglion relay afferent information from thoracic visceral organs and may be important in inflammatory processes due to the peripheral release of bioactive neuropeptides such as substance P. We characterized the excitable properties and underlying voltage-gated Na+ (INa) and K+ (IKv) currents in acutely dissociated guinea pig jugular ganglion neurons with microelectrode and whole-cell patch-clamp recording techniques. Current clamp recordings revealed a resting potential of approx. -55 mV and input resistance of approx. 100 M ohms. Brief depolarizing steps evoked an overshooting action potential (approx. 2 ms duration), fast (< 20 ms duration) afterhyperpolarization (AHPF) sequence in all neurons, followed by a slow (> 1 s) Cd(2+)-sensitive afterhyperpolarization (AHPS) in 45% of the neurons. The AHPS was implicated in limiting repetitive action potential firing during maintained depolarizing steps. The action potential in 15/17 neurons, and a major component of the whole cell INa in 13/13 neurons were insensitive to TTX (1-10 microM), indicating that jugular neurons express predominantly a TTX-resistant type of INa. Cd2+ (200 microM) did not affect action potential repolarization, while tetraethylammonium (TEA; 10 mM) in the presence of Cd2+ markedly prolonged action potential repolarization, and blocked the AHPF in 11/11 neurons. This suggested that the action potential repolarization and the AHPF are mediated by IKv, with little contribution by Ca(2+)-dependent IK (IK(Ca)). Whole cell IKv activated rapidly (tau < 1.5 ms), and inactivated variably over a time period of seconds. IKv activation and inactivation voltage dependencies and TEA sensitivity were compatible with its availability during the action potential and AHPF. Only 1/26 neurons exhibited current with the rapid inactivation kinetics and voltage-dependencies characteristic of classic IA-type current. These results highlight differences in the properties of jugular neurons (e.g., deficiency of rapid IA, and lack of a TTX-sensitive subpopulation), relative to those known for other visceral and somatic afferents, and thus provide a basis for further functional studies.

Calcium current characterization in dissociated adult guinea-pig jugular ganglion neurons.

Voltage-dependent calcium (Ca2+) channel currents in freshly dissociated adult guinea-pig jugular ganglion neurons were examined and characterized using the whole-cell patch-clamp technique. Electrophysiological analysis demonstrated a high-threshold current, but no low-threshold or T-type current. A fraction of the total Ca2+ current was inhibited by omega (omega)-conotoxin GVIA (Cg (inTX; 10 microM); the dihydropyridine antagonist nifedipine (NIF; 10 microM), inhibited a large fraction of the CgTX-insensitive current. The remaining CgTX/NIF-insensitive current was completely inhibited by omega-agatoxin IVA (AgIVA; 100 nM). These results demonstrated that the whole-cell Ca2+ channel current consisted only of N-, L- P-type components. Of these currents, only the L-type was partially inhibited by both histamine and carbachol (0.01-100 microM).

A retrograde labeling technique for the functional study of airway-specific visceral afferent neurons.

The development of a method is described whereby primary afferent neurons that specifically innervate the airways in the guinea pig can be retrogradely labeled, acutely dissociated and studied functionally with electrophysiological techniques. Following administration of either dextran-tetramethylrhodamine, Fast Blue, or Fluorogold dye into the tracheal lumen, dye-labeled neurons can be visualized in 100 microns serial nodose ganglion sections. Control experiments show that labeling does not result from the undesirable spread of the dyes to target innervation fields in the gastrointestinal (GI) or cardiovascular (CV) systems. Neuronal somata retain dye label when acutely dissociated. Microelectrode studies provide evidence that the presence of the Rhodamine dye label and its fluorescent excitation neither alter basic electrophysiological membrane parameters nor the chemoreceptive properties of isolated neurons. Thus this new method will allow the isolation of individual airway-specific primary visceral afferent neurons for functional studies with multidisciplinary techniques.