R-isovaline: a subtype-specific agonist at GABAB-receptors?

The R-enantiomer of isovaline, an analgesic amino acid, has a chemical structure similar to glycine and GABA. Although its actions on thalamic neurons are strychnine-resistant and independent of the Cl− gradient, R-isovaline increases membrane conductance for K+. The purpose of this study was to determine if R-isovaline activated metabotropic GABAB receptors. We used whole-cell voltage-clamp recordings to characterize the effects of R-isovaline applied by bath perfusion and local ejection from a micropipette to thalamic neurons in 250 μm thick slices of rat brain. The immunocytochemical methods that we employed to visualize GABAB1 and GABAB2 receptor subunits showed extensive staining for both subunits in ventrobasal nuclei, which were the recording sites. Bath or local application of R-isovaline caused a slowly developing increase in conductance and outward rectification in 70% (54/77) of neurons, both effects reversing near the K+ Nernst potential. As with the GABAB agonist baclofen, G proteins likely mediated the R-isovaline effects because they were susceptible to blockade by non-hydrolyzable substrates of guanosine triphosphate. The GABAB antagonists CGP35348 and CGP52432 prevented the conductance increase induced by R-isovaline, applied by bath or local ejection. The GABAB allosteric modulator CGP7930 enhanced the R-isovaline induced increase in conductance. At high doses, antagonists of GABAA, GABAC, glycineA, μ-opioid, and nicotinic receptors did not block R-isovaline responses. The observations establish that R-isovaline increases the conductance of K+ channels coupled to metabotropic GABAB receptors. Remarkably, not all neurons that were responsive to baclofen responded to R-isovaline. The R-isovaline–induced currents outlasted the fast baclofen responses and persisted for a 1–2-h period. Despite some similar actions, R-isovaline and baclofen do not act at identical GABAB receptor sites. The binding of R-isovaline and baclofen to the GABAB receptor may not induce the same conformational changes in receptor proteins or components of the intracellular signaling pathways.

▶R-isovaline activated metabotropic GABAB receptors and K current in thalamic neurons. ▶GABAB receptor antagonists or non-hydrolyzable GTP substrates blocked these effects. ▶The responses were enhanced by a positive allosteric modulator of GABAB receptors. ▶Responses were slow, poorly reversible and not seen in all baclofen-responsive cells. ▶R-isovaline and baclofen do not act at the same GABAB receptor/signaling sites.