Influence of GABA‐gated bicarbonate conductance on potential, current and intracellular chloride in crayfish muscle fibres.

Abstract

SUMMARY1. The effects of y-aminobutyric acid (GABA) on membrane potential and conductance as well as on the intracellular Cl-activity (ai4k) and intracellular pH (pHi) were studied in crayfish muscle fibres using a three-microelectrode voltage clamp and ion-selective microelectrodes. In the presence of C02-HC03-, the intracellular HCO3-activity (a' o3) was estimated from pHi.2. In a nominally HCO3 -free solution, a near-saturating concentration of GABA (0-2 mM) produced a marked increase in membrane conductance but little change in potential. In a solution containing 30 mM-HCO3-(equilibrated with 5 % CO2 + 95 % air; pH 7-4), the GABA-induced increase in conductance was associated with a depolarization of about 15 mV, with an increase in ai1 and with a decrease in ah-o.All these effects were blocked by picrotoxin (PTX). The depolarizing action of GABA was augmented following depletion of extracellular and intracellular Cl-.3. The GABA-induced increase in a', which took place in the presence of HCO3 was blocked by clamping the membrane potential at its resting level. This indicates that the increase in a'1 was due to passive redistribution of Cl-. In both the presence and absence of HC03-, the GABA-activated transmembrane flux of Cl-showed reversal at the level of the resting potential, which indicates that under steady-state conditions the Cl-equilibrium potential (Ec1) is identical to the resting potential.4. In a Cl--free, 30 mM-HCO3--containing solution, 0-5 mM-GABA produced a PTX-sensitive increase in conductance which amounted to 15 % of the conductance activated in the presence of Cl-. In the absence of both Cl-and HC03-, the respective figure was 2-8 %. Assuming constant-field conditions, the conductance data yielded a permeability ratio PHCo3/PC1 of 0-42 for the GABA-activated channels.5. In a Cl--containing, HC03--free solution, the reversal potential of the GABAactivated current (EGABA) was, by about 1 mV, less negative than the resting membrane potential (RP). In a solution containing Cl-and 30 mM-HCO3-, EGABA -RP was 12 mV. Simultaneous measurements of EGABA, aci and allCO3 (pHi) gave a PHCO3/PC1 value of 0 33.6. In a Cl--free, HC03--containing solution EGABA was close to the HC03-* To whom correspondence should be addressed. 6 PHY 416K. KAILA AND OTHERS equilibrium potential (EHCO3) and an experimental acidosis which produced a negative shift in EHCO3 was associated with a similar shift in EGABA.7. The present results show that HCO3-permeability of GABA-gated channels can lead to a significant deviation of EGABA from Ecl, and, in particular, to a depolarizing effect of GABA in the absence of a postsynaptic, inwardly directed Clpump. The dependence of EGABA on HCO3-permeability may provide a novel link between postsynaptic regulation of pHi and the efficacy of synaptic inhibition.

Strychnine‐sensitive glycine responses of neonatal rat hippocampal neurones.

“…As proposed for GABA-mediated responses (Ben-Ari et al 1989), this change may be due to modifications in the chloride gradient, resulting from the reversed operation of the chloride membrane pump (Misgeld, Deisz, Dodt & Lux, 1986) during early postnatal days. Alternatively, if glycine-activated channels are also permeable to bicarbonate ions (HC03-), the reversal potential for glycine responses will deviate significantly from the chloride equilibrium potential towards the bicarbonate equilibrium potential (Kaila, Pasternack, Saarikoski & Voipio, 1989). Since the intracellular level of HC03-is determined by the pH regulatory mechanism of the cell (Thomas, 1984), any developmental change in cell metabolism might shift the HC03-equilibrium potential and therefore the glycine equilibrium potential.…”

Inward current caused by sodium‐dependent uptake of GABA in the crayfish stretch receptor neurone.

“…(In a previous paper (Kaila et al 1989), the differential signal was shown with inverted polarity which, of course, does not affect the value of the measured reversal potential.) The Clactivity coefficient was taken as 0 74 (Rauen, 1964).…”

“…The ClV-selective microelectrodes were constructed and calibrated as described in detail previously (Kaila et al 1989). Briefly, dry-bevelled micropipettes pulled from tubing without a fibre (GC150, Clark Electrochemical, Pangbourne, Reading, UK) were silanized and back-filled with 100 mM-NaCl + 20 mM-HEPES (pH 7 6).…”

“…IB of bicarbonate (Kaila et al 1989) since the present experiments were carried out in the nominal absence of CO2-HCO3-. Evidently, the influx of Cl-which leads to the rise in a'l opposes the effect of an as yet unidentified depolarizing mechanism.…”

“…In this work we have examined the effects of GABA on membrane potential, current and on net movements of Cl-in the crayfish stretch receptor neurone (SRN). The experiments were carried out under HCO3--free conditions (see Kaila, Pasternack, Saarikoski & Voipio, 1989;Voipio, Pasternack, Rydqvist & Kaila, 1991) using a two-microelectrode current-voltage clamp and Cl--selective microelectrodes.…”