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

Abstract

A novel, small conductance of Cl-channel was characterized by incorporation into planar bilayers from a plasma membrane preparation of lobster walking leg nerves. Under conditions of symmetrical 100 mM NaC1, 10 mM Tris-HC1, pH 7.4, single C1-channels exhibit rectifying current-voltage (I-V) behavior with a conductance of 19.2 -+ 0.8 pS at positive voltages and 15.1 _+ 1.6 pS in the voltage range of -40 to 0 mV. The channel exhibits a negligible permeability for Na + compared with Cl-and displays the following sequence of anion permeability relative to C1-as measured under near bi-ionic conditions: I-(2.7) > NO~-(1.8) > Br-(1.5) > CI-(1.0) > CHACO ~ (0.18) > HCO~-(0.10) > gluconate (0.06) > F-(0.05). The unitary conductance saturates with increasing CI-concentration in a Michaelis-Menten fashion with a K m of 100 mM and "gm~ ----33 pS at positive voltage. The I-Vcurve is similar in 10 mM Tris or 10 mM HEPES buffer, but substitution of 100 mM NaC1 with 100 mM tetraethylammonium chloride on the c/s side results in increased rectification with a 40% reduction in current at negative voltages. The gating of the channel is weakly voltage dependent with an open-state probability of 0.23 at -75 mV and 0.64 at + 75 mV. Channel gating is sensitive to c/s pH with an increased opening probability observed for a pH change of 7.4 to 11 and nearly complete inhibition for a pH change of 7.4 to 6.0. The lobster CI-channel is reversibly blocked by the anion transport inhibitors, SITS (4-acetamido, 4'-isothiocyanostilbene-2,2'-disufonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid). Many of these characteristics are similar to those previously described for small conductance C1-channels in various vertebrate cells, including epithelia. These functional comparisons suggest that this invertebrate C1-channel is an evolutionary prototype of a widely distributed class of small conductance anion channels.Address reprint requests to Dr.

Block of neuronal fast chloride channels by internal tetraethylammonium ions.

“…TEA block of the lobster CI channel appears quite similar to that of neuronal fast CI channels in that the major effect is a reduction of single-channel current amplitude, presumably because of time averaging of rapid on and off kinetics. Other than the reduction of conductance, Luk~cs and Moczydlowski (1990) observed no alterations of single-channel kinetic behavior. The increase in mean open lifetime observed in our experiments would probably not have been resolved in the experiments on the lobster channel because of the necessary bandwidth limitations of bilayer recording.…”

“…Other CI channels are also blocked by millimolar concentrations of TEA. Luk~cs and Moczydlowski (1990) described a CI channel isolated from lobster nerve and inserted into planar lipid bilayers. This channel was blocked by a factor of 40-60% in the presence of 100 mM TEA.…”

Rectifying conductance substates in a large conductance Ca(2+)-activated K+ channel: evidence for a fluctuating barrier mechanism.

“…One application concerns the mechanistic interpretation of unitary LVrelations that rectify under conditions of symmetrical permeant ion concentration. Specifically, single-channel LVcurves of various C1 channels exhibit outward rectification in the presence of symmetrical C1-solutions (Welsh and Liedtke, 1986;Chen et al, 1989;Lukacs and Moczydlowski, 1990). Similarly, unitary I-V relations of a number of voltage-sensitive K-channels also exhibit moderate inward rectification in the presence of symmetrical K + (Isacoff et al, 1991;Wonderlin and French, 1991).…”

Modulation of calcium-activated chloride current via pH-induced changes of calcium channel properties in cone photoreceptors

“…Other Cl channels, insensitive to calcium, exhibit pronounced sensitivity to pH. For example, the chloride conductance of frog skeletal muscle is a steep, sigmoidal function of external pH (Hutter and Warner, 1967) and the weakly voltage-dependent Cl channels of lobster nerve are irreversibly suppressed by acidification of the cytoplasmic solution to pH 6.0 (Lukacs and Moczydlowski, 1990).…”