ISSN:
1432-1424
Schlagwort(e):
Whole-cell recording
;
Anion selectivity
;
Cl-channel blockers
Quelle:
Springer Online Journal Archives 1860-2000
Thema:
Biologie
,
Chemie und Pharmazie
Notizen:
Abstract During whole-cell patch-clamp recording from normal (nontransformed) human T lymphocytes a chloride current spontaneously activated in 〉98% of cells (n 〉 200) in the absence of applied osmotic or pressure gradients. However, some volume sensitivity was observed, as negative pressure pulses reduced the current. With iso-osmotic bath and pipette solutions the peak amplitude built up (time constant ≈23 sec at room temperature), a variable-duration plateau phase followed, then the current ran down spontaneously (time constant ≈280 sec). The anion permeability sequence, calculated from reversal potentials was I−, Br− 〉 NO 3 − , Cl− 〉 CH3SO 3 − , HCO 3 − 〉 CH3COO− 〉 F− 〉 aspartate, gluconate, SO 4 2− and there was no measurable monovalent cation permeability. The Cl− current was independent of time during long voltage steps and there was no evidence of voltage-dependent gating; however, the current showed intrinsic outward rectification in symmetrical Cl− solutions. The conductance of the channels underlying the whole-cell current was calculated from fluctuation analysis, using power-spectral density and variance-vs.-mean analysis. Both methods yielded a single channel conductance of about 0.6 pS at −70 mV (close to the normal resting potential of T lymphocytes). The power spectral density function was best fit by the sum of two Lorentzian functions, with corner frequencies of 30 and 295 Hz, corresponding to mean open times of 0.54 and 5.13 msec. The pharmacological profile included rapid block by external application of flufenamic acid (50 μm), 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 100 μm, [6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5-y1) oxy] acetic acid (IAA-94, 250 μm) or 100 μm 1,9-dideoxyforskolin. The stilbene derivatives DIDS (4,4′-diisothiocyano-2,2′ di-sulphonic acid stilbene, 500 μm) and SITS (4-acetamido-4′-isothiocyano-2, 2′-disulphonic acid stilbene, 500 μm) prevented buildup of Cl− current after a 30-min preincubation at 500 μm. When tested in a mitogenic assay, DIDS, flufenamic acid, NPPB and IAA-94 all inhibited T-cell proliferation, suggesting a physiological function in addition to the observed volume sensitivity.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1007/BF00232714
