Permeability and single channel conductance of human homomeric rho1 GABAC receptors

Abstract

1. Homomeric human rho1 GABAC receptors were expressed in Xenopus oocytes and in human embryonic kidney cells (HEK293) in order to examine their conductance and permeability. 2. Reversal potentials of currents elicited by gamma-aminobutyric acid (GABA) were measured in extracellular solutions of various ionic composition to determine relative permeability of homomeric rho1 receptors. The rank order of anionic permeability was: SCN- > I- > NO3- > Br- > Cl- > formate (For-) > HCO3- > acetate (Ac-) approximately proprionate (Prop-) approximately isethionate (Ise-) approximately F- approximately PO4-. 3. In the oocyte expression system, relative permeabilities to SCN-, I-, NO3-, Br- and HCO3- were higher for rho1 GABAC receptors than alpha1beta2gamma2L GABAA receptors. 4. Expression of rho1 GABAC receptors in Xenopus oocytes and in HEK293 cells gave similar relative permeabilities for selected anions, suggesting that the expression system does not significantly alter permeation properties. 5. The pore diameter of the homomeric rho1 GABAC receptor expressed in oocytes was estimated to be 0.61 nm, which is somewhat larger than the 0.56 nm pore diameter estimated for alpha1beta2gamma2L GABAA receptors. 6. Homomeric rho1 GABA receptors expressed in oocytes had a single channel chord conductance of 0.65 +/- 0.04 pS (mean +/- s.e.m.) when the internal chloride concentration ([Cl-]i) was 20 mM. With a [Cl-]i of 100 mM, the single channel chord conductance was 1.59 +/- 0.24 pS. 7. The mean open time directly measured from 43 GABA-induced channel openings in six patches was 3. 2 +/- 0.8 s. The mean open time in the presence of 100 microM picrotoxin was 0.07 +/- 0.01 s (77 openings from 3 patches). 8. The differences observed in ionic permeabilities, pore size, single channel conductance and mean open time suggest that the rho1 homomeric receptor may not be the native retinal GABAC receptor reported previously.

Figure 1. A ramp protocol was used to determine reversal potentials of GABA-induced currents

The holding potential was ramped from -70 mV to +10 mV over a period of 1 s and repeated five times in rapid succession. Each trace shown is the average of these five. a and c, control ramps before GABA application and after recovery, respectively, are superimposed. b, ramp during steady-state application of 0.5 μm GABA. d, GABA ramp after subtraction of the average of a and c (control ramps). Note that the point at which the GABA ramp and the control ramp cross is the reversal potential of the GABA-induced current and is the same as where the subtracted ramp crosses the x-axis. Inset, current trace of two-electrode voltage clamp ramp experiment in an oocyte, showing the sets of five ramps before, during, and after recovery from GABA application, which were averaged to obtain the ramps labelled a, b and c in the main figure.

Figure 2. Reversal potentials are shifted by anion substitutions but not by cation substitutions

A, 50 mm NaCl in the extracellular solution was replaced by 50 mm sodium salt of the anion indicated by each trace. The holding potential was ramped from -70 mV to +10 mV over a period of 1 s, except in the case of SCN⁻, when a ramp from -85 mV to -25 mV was used. B, a similar experiment, varying the cation composition of the extracellular solutions.

Figure 3. Comparison of the relative permeabilities to anions of ρ1 receptors and GABA_A receptors

For most of the anions tested, relative permeabilities of ρ1 receptors were higher than GABA_A receptors in our oocyte expression system. Permeabilities of α1β2γ2L expressed in oocytes to SCN⁻, I⁻, NO₃⁻, Br⁻ and HCO₃⁻ were significantly different from the ρ1 permeabilities. The corresponding permeabilities reported by Bormann (1987) from spinal cord neurons are provided for comparison to our results. α1β2γ2L expressed in oocytes has essentially the same permeability profile as native spinal neurons.

Figure 4. The ρ1 GABA-induced single channel conductance is small

A, GABA (20 μm) induced a single channel current in an excised outside-out patch from an oocyte expressing homomeric ρ1 GABA receptors. The membrane voltage of the patch was clamped to -200 mV in order to maximize the amplitude of channel openings. Even at this extreme holding potential, the single channel current was only ≈0.1 pA. Note that the channel remains open after the end of the GABA application. B, the all-points amplitude histogram of the current in A shows two peaks. The continuous smooth line is the least squares fit of a double Gaussian distribution to the histogram. The difference between the means of the two peaks is the single channel current. The single channel conductance was calculated according to eqn (4) (0.65 ± 0.04 pS, n = 5).

Figure 5. The single channel chord conductance is dependent upon [Cl⁻]_i

A, examples of current traces in different solutions show that the current amplitude was higher when [Cl⁻]_i was increased. The upper trace shows a GABA-activated current in a multichannel patch held at -200 mV with a [Cl⁻]_i of 20 mm. The all-points amplitude histogram of this trace shows a single channel current of 0.10 pA under these conditions. In the lower trace, another multichannel patch shows a single channel chord conductance of 1.59 ± 0.24 pS (n = 3) when [Cl⁻]_i was increased to 100 mm. The all-points amplitude histograms, B and C, were derived from the portions of trace indicated by arrows in A.

Figure 6. Ensemble average of single channel currents has a time course similar to the macroscopic current

A, representative current traces of 20 μm GABA-activated single channel activity from a single patch are shown; the bottom trace is the average of 12. The membrane voltage of the patch was clamped to -200 mV. GABA (20 μm) was applied through a computer-controlled piezo fast-switching system for 2 s. B, comparison of the ensemble average with the GABA response in a macropatch. The upper trace is the same ensemble average from A, on a different scale. The lower trace is the GABA-induced current in a macropatch from an oocyte expressing ρ1 GABA receptors.

Figure 7. GABA-induced single channel current can be blocked by picrotoxin

Application of 20 μm GABA to an outside-out patch excised from an oocyte expressing ρ1 GABA receptors induced an inward current, which was blocked when perfusion was briefly switched to GABA (20 μm) plus picrotoxin (100 μm). The holding potential was -200 mV with a [Cl⁻]_i of 20 mm. Note that although the current returns to baseline during the picrotoxin application, some brief channel openings were evident. After removal of the antagonist, the GABA-induced current recovered.

Figure 8. The ρ1 GABA_C pore diameter is larger than that of the α1β2γ2L GABA_A receptor

The anionic permeabilities relative to chloride from Table 1 were plotted against the Stokes diameters of the ions (determined from the limiting conductances, Robinson & Stokes, 1959). A fit of eqn (3) using the permeabilities of the larger anions (chloride, formate, bicarbonate, and acetate) gave a pore size of 0.61 nm. This is larger than the estimated α1β2γ2L GABA_A receptor pore size of 0.56 nm.