Conduction and blocking properties of a predominantly anion-selective channel from human platelet surface membrane reconstituted into planar phospholipid bilayers

Abstract

We have investigated the basic properties of a predominantly anion-selective channel derived from highly purified human platelet surface membrane. Single channels have been reconstituted into planar phospholipid bilayers by fusion of membrane vesicles and recorded under voltage-clamp conditions. The channel is found to have the following properties: (i) Channel activity occurs in bursts of openings separated by long closed periods. (ii) The current-voltage relationship is nonlinear. Channel current is seen to rectify, with less current flowing at positive than at negative voltages. Rectification may be due to asymmetric block by HEPES/Tris buffers. In 450 mM KCl, 5 mM HEPES/Tris, pH 7.2, the single channel conductance at -40 mV is approximately 160 pS and at +40 mV is approximately 90 pS. (iii) The conductance-concentration relationship follows a simple saturation curve. Half maximal conductance is achieved at a concentration of approximately 1000 mM KCl, and the curve saturates at a conductance of approximately 500 pS. (iv) Reversal potentials interpreted in terms of the Goldman-Hodgkin-Katz equation indicate a Cl: K permeability ratio of 4:1. (v) The channel accepts all of the halides as well as a number of other anions. The following sequence of relative anion permeabilities (in the presence of K+) is obtained: F- less than acetate- less than gluconate- less than Cl- less than Br- less than I- less than NO3- less tha SCN-.(vi) Cations as large as TEA+ are permeant. (vii) Current through the channel is blocked in the presence of DIDS, SITS and ATP, but not by Zn2+.

A chloride channel from lobster walking leg nerves. Characterization of single-channel properties in planar bilayers.

“…In most previous planar bilayer studies of small conductance CI-channels, the observed I-V behavior exhibited a characteristic negative rectification (i.e., trans---, cis CI-flux is less than cis ~ trans CI-flux) (Coronado and Latorre, 1982;Reinhardt et al, 1987;Bridges et al, 1989;Manning and Williams, 1989). In the case of colon epithelia, a similar outward-rectifying CI-channel is known to exist in the intact tissue (Halm et al, 1988).…”

“…Since some Cl-channels exhibit a significant permeability for monovalent cations (Franciolini and Nonner, 1987) including TEA + (Manning and Williams, 1989), we examined the effect of TEA + on the lobster Cl-channel. Fig.…”

“…Some SCI channels exhibit imperfect selectivity for anions with reported PNa,K/Pca ratios in the range of 0.1-0.25 (Blatz and Magleby, 1985;Welsh and Liedtke, 1986;Franciolini and Nonner, 1987;Manning and Williams, 1989). Franciolini and Nonner (1987) have previously proposed that such imperfect selectivity is due to an actual requirement for a monovalent cation such as Na + or K + as a cofactor in anion permeation by a mechanism involving transient ion-pair formation in the channel.…”

Trinitrophenyl-ATP blocks colonic Cl- channels in planar phospholipid bilayers. Evidence for two nucleotide binding sites.

“…Addition of ATP to the extracellular side caused a dosedependent, flickery-type blockade of reconstituted rectifying CI-channels obtained from airway epithelial cells and platelets with an estimated ICs0 of 40 IxM (Manning and Williams, 1989;Alton et al, 1991). Blockade by extracellular ATP is not limited to reconstituted channels since Stutts, Chinet, Mason, Fullton, Clarke, and Boucher (1992) have presented data showing that external ATP increased burst duration of rectifying C1-channels in excised apical membrane patches from airway epithelial cells, which is the result expected if ATP is acting as a fast or flickery-type blocker.…”

Epithelia & Membrane Transport

“…Chloride channels in excised, inside-out membrane patches from human platelets Amanda B. MacKenzie and Martyn P. Mahaut-Smith Physiological Laboratory, Downing Street, Cambridge CB2 3EG Anion-selective channels have been previously reported in human platelet membranes (Manning & Williams, 1989;Mahaut-Smith, 1990), yet their importance in platelet responses remains unknown. To date, only one study has used patch-clamp techniques to record platelet Cl-currents, however, these whole-cell recordings (Mahaut-Smith, 1990) provided only limited information due to the lack of control over the cytosolic environment.…”