0000000000416651
AUTHOR
Riikka Mäkelä
Cerebellar granule-cell-specific GABAAreceptors attenuate benzodiazepine-induced ataxia: evidence from α6-subunit-deficient mice
Benzodiazepine- and alcohol-induced ataxias in rodents have been proposed to be affected by the gamma-aminobutyric acid type A (GABAA) receptor alpha 6 subunit, which contributes to receptors specifically expressed in cerebellar granule cells. We have studied an alpha 6 -/- mouse line for motor performance and drug sensitivity. These mice, as a result of a specific genetic lesion, carry a precise impairment at their Golgi-granule cell synapses. On motor performance tests (rotarod, horizontal wire, pole descending, staircase and swimming tests) there were no robust baseline differences in motor function or motor learning between alpha 6 -/- and alpha 6 +/+ mice. On the rotarod test, however,…
Blunted furosemide action on cerebellar GABAA receptors in ANT rats selectively bred for high alcohol sensitivity.
Furosemide specifically reverses the inhibition by gamma-aminobutyric acid (GABA) of t-[35S]-butylbicyclophosphorothionate ([35S]TBPS) binding and increases the basal [35S]TBPS binding to the cerebellar granule cell layer GABAA receptors. For the selectivity of furosemide, an interplay between GABAA receptor alpha 6 and beta 2 or beta 3 subunits is needed. We have now investigated the furosemide sensitivity of cerebellar [35S]TBPS binding in the alcohol-sensitive (ANT) rat line that harbors a pharmacologically critical point mutation in the alpha 6 subunit [alpha 6 (Q1000)], increasing benzodiazepine affinity of the normally insensitive alpha 6-containing receptors. ANT receptors were less …
The main determinant of furosemide inhibition on GABA(A) receptors is located close to the first transmembrane domain.
Inhibitory GABA(A) receptors are regulated by numerous allosteric modulators, the most receptor-subtype specific of which is furosemide. It recognises receptors of the subunit composition alpha6beta2/3gamma2, restricted to cerebellar granule cells. To locate furosemide's site of action we constructed chimeras of the furosemide-sensitive alpha6 and the furosemide-insensitive alpha1 subunit, and expressed and studied them together with the beta3 and gamma2 subunits in Xenopus oocytes by the two-electrode voltage clamp technique. The inhibition of GABA-induced currents by furosemide mainly depended on a short domain proximal to the first transmembrane region of the alpha6 subunit.
Quantification of GABA(A) receptor subunit mRNAs by non-radioisotopic competitive RT-PCR utilizing plate-based EIA methodology.
We developed a non-radioisotopic quantitative competitive RT-PCR method for the measurement of gamma-aminobutyric acid (GABA) type A receptor subunit mRNA levels. The specificity of the method was optimized by the use of four subunit-specific oligonucleotides in the sequential steps: reverse transcription, polymerase chain reaction (PCR), and detection. The biotinylated PCR products were bound on streptavidin-coated microtiter plates allowing detection of the products using dinitrophenyl (DNP)-labeled probes and anti-DNP alkaline phosphatase conjugate. The method was set up for the six major cerebellar GABA(A) receptor subunits: alpha1; alpha6; beta2; beta3; gamma2 and delta. The method is …
Furosemide action on cerebellar GABA(A) receptors in alcohol-sensitive ANT rats.
Furosemide increases the basal tert-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding and reverses the inhibition of the binding by gamma-aminobutyric acid (GABA) in the cerebellar GABA(A) receptors containing the alpha6 and beta2/beta3 subunits. These effects are less pronounced in the alcohol-sensitive (ANT) than in the alcohol-insensitive (AT) rat line. The difference between the rat lines in the increase of basal [35S]TBPS binding was removed after a longer preincubation with ethylendiaminetetraacetic acid (EDTA) containing buffer, but long preincubation did not reduce the GABA content of the incubation fluid or remove the difference in GABA antagonism by furosemide. The GABA sensi…