GABA and glycine channels in isolated ganglion cells from the goldfish retina.
1. Adult goldfish retinas were enzymatically dissociated and ganglion cells were maintained in culture for periods of 1-5 days. Ganglion cells could be identified by their morphology, and this identification was confirmed by retrograde transport of the fluorescent dye Fast Blue injected into the optic nerve stub. 2. All the ganglion cells tested responded to 30 microM-GABA or 100 microM-glycine between 2 and 30 h after enzymatic dissociation of the retina. 3. Whole-cell responses to 30 microM-GABA or glycine declined over a period of seconds during sustained applications of the agonists, probably as a result of desensitization. There was an irreversible decline in the peak whole-cell response to repeated applications of 30 microM-GABA unless the pipette-filling solution contained 2 mM-ATP, 4 mM-Mg2+, 10 mM-EGTA and no added Ca2+. Both GABA and glycine responses also showed an irreversible decline in outside-out patches but, in this case, Mg2+, ATP, and very low Ca2+ failed to stabilize the response. 4. Whole-cell currents activated by both GABA and glycine were demonstrated to be chloride-selective by investigating the dependence of reversal potential (Vr) on internal chloride concentration ([Cl-]i). For GABA responses, the dependence of Vr on [Cl-]i could not be distinguished from that predicted by the Nernst relation. For glycine, deviations from Nernstian dependence were observed, but the permeability to Cl- was at least 20 times greater than to isethionate, SO4(2-), or monovalent cations (Na+ and Cs+). 5. Bicuculline methochloride (10 microM) selectively blocked responses to 3-30 microM-GABA without affecting responses to 30 microM-glycine. Bicuculline itself was not as selective. At agonist concentrations of 30 microM, 3 microM-bicuculline partially blocked the response to GABA but not that to glycine, but bicuculline at 10 microM blocked responses to both GABA and glycine. Strychnine (0.3-1 microM) blocked responses to 30 microM-glycine but also competitively antagonized GABA responses. Picrotoxinin (10 microM) blocked responses to 3 microM-GABA in some cells but also partially antagonized responses to 30 microM-glycine. 6. GABA channels had at least two conductance states at 10-12 degrees C in nearly symmetrical (141 mM in, 142 mM out) chloride. The slope conductance of the most frequently observed (main) state was 16 +/- 2 pS. The reversal potential for the main state was not significantly different from the chloride equilibrium potential (0 mV).(ABSTRACT TRUNCATED AT 400 WORDS)
Cohen
B N
BN
Department of Ophthalmology, Jules Stein Eye Institute, University of California School of Medicine, Los Angeles 90024.
Fain
G L
GL
Fain
M J
MJ
D000818:Animals; D001640:Bicuculline / Q000494:pharmacology; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism; D005071:Evoked Potentials / Q000187:drug effects; D005998:Glycine / Q000378:metabolism* / Q000494:pharmacology; D006054:Goldfish; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism* / Q000494:pharmacology
D018118:Chloride Channels; D002712:Chlorides; D008565:Membrane Proteins; D005680:gamma-Aminobutyric Acid; D005998:Glycine; D001640:Bicuculline