Bicarbonate and thiocyanate ions affect the gating of g-aminobutyric acid A receptors in cultured rat cortical cells Ga´ bor Maksay a, * , La´ szlo´ Fodor b , Norbert Bielik b , Istva´ n Tarnawa b a Chemical Research Center ofthe Hungarian Academy of Sciences, Department of Molecular Pharmacology, P.O. Box17, H-1525 Budapest, Hungary b Gedeon Richter Limited, Department of Electrophysiology and Cell Biology, P.O. Box100, Budapest, Hungary Received 29 June 2001; received in revised form 27 July 2001; accepted 30 July 2001 Abstract The ionophore function of g-aminobutyric acid A (GABA A ) receptors was studied by whole-cell patch clamp electro- physiology in primary cultures of rat cerebral cortex. Chloride ions were replaced for SCN 2 (thiocyanate) and HCO 3 2 (bicarbonate) ions. The EC 50 values of the GABA A agonist muscimol (HCO 3 2 . Cl 2 . SCN 2 ) varied parallel with the free energies of dehydration of the anions, while the inhibition constants of the GABA A antagonist bicuculline methiodide were not affected. These findings might be relevant in the interpretation of the contribution of HCO 3 2 versus Cl 2 currents to the pharmacological differences between depolarizing and hyperpolarizing GABA responses. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: g-aminobutyric acid (GABA) A receptor-ionophore function; Bicarbonate; Thiocyanate; Muscimol; Bicuculline; Dehydration of anions; GABAergic depolarization g-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. GABA A receptors function as ionophores permeable for monovalent anions. The permeabilities of these anions have been corre- lated with their Stokes diameters [3] and their free energies of dehydration [8]. Chloride is the most important perme- able anion physiologically but the contribution of bicarbo- nate ions has been recently established for the depolarizing effects of GABA A receptors [12,14,23]. Pharmacological studies revealed that the sensitivities of the hyperpolarizing and depolarizing GABA responses are different for antago- nists [2], neurosteroids [4], ethanol [21] and barbiturates [18,21]. This led to different conclusions about the hetero- geneity of the participating GABA A receptors [18–20]. We examined the potencies of muscimol, a selective GABA A receptor agonist and the antagonist bicuculline methiodide in the presence of Cl 2 and HCO 3 2 ions to reveal the conse- quences of the full replacement of the permeable anions on the pharmacology of GABA A receptors. The effects of the substitution of Cl 2 for SCN 2 ions were also investigated for the following reasons: (1) SCN 2 has the highest permeabil- ity through GABA A ionophores [3,8]; (2) it has anomalous properties of binding to the anion binding sites within the ionophore [3]; (3) SCN 2 potentiates binding of bicuculline to GABA A receptors via entropy-driven hydrophobic inter- actions [17]; and (4) SCN 2 is a chaotropic agent [11]. Primary cultures of cortical neurons were prepared from 17-day-old Wistar rat embryos. Cortical tissue was removed and washed with Ca 21 - and Mg 21 -free Hank’s balanced salt solution (HBSS) and incubated in HBSS containing 0.2% trypsin (type XI, Sigma) for 4 min at 378C. After five washes with HBSS the tissue was triturated using a Pasteur pipette and placed into Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco), and antibiotics (penicillin, 100 Unit/ml, streptomycin, 0.1 mg/ ml, amphotericin, 0.25 mg/ml, Sigma). The suspension was centrifuged twice at 125 £ g for 5 min, the pellet was resus- pended in 10% serum supplemented medium. Cells were plated at a density of 10 5 cells/cm 2 on sterilized glass cover- slips previously coated with poly-d-lysine (Sigma). Cultures were kept at 378C in 5% CO 2 , half of the medium was changed to fresh serum-free medium twice a week. Conventional whole-cell patch clamp [10] recordings were made from cultured cortical cells 6–11 days after plat- ing. Cultures were transferred to a recording chamber and constantly superfused with the extracellular solution (e.s.) at Neuroscience Letters 311 (2001) 169–172 0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(01)02163-2 www.elsevier.com/locate/neulet * Corresponding author. Tel.: 1361-438-0411 ext. 282; fax: 1361-325-7554. E-mail address: maksay@chemres.hu (G. Maksay).