RESUMEN
Genetic defects leading to epilepsy have been identified in gamma2 GABA(A) receptor subunit. A gamma2(R43Q) substitution is linked to childhood absence epilepsy and febrile seizure, and a gamma2(K289M) mutation is associated with generalized epilepsy with febrile seizures plus. To understand the effect of these mutations, surface targeting of GABA(A) receptors was analyzed by subunit-specific immunofluorescent labeling of living cells. We first transfected hippocampal neurons in culture with recombinant gamma2 constructs and showed that the gamma 2(R43Q) mutation prevented surface expression of the subunit, unlike gamma2(K289M) substitution. Several gamma2-subunit constructs, bearing point mutations within the Arg-43 domain, were expressed in COS-7 cells with alpha3- and beta3-subunits. R43Q and R43A substitutions dramatically reduced surface expression of the gamma2-subunit, whereas R43K, P44A, and D39A substitutions had a lesser, but still significant, impact and K289M substitution had no effect. Whereas the mutant gamma2(R43Q) was retained within intracellular compartments, alphabeta complexes were still targeted at the cell membrane. Coimmunoprecipitation experiments showed that gamma2(R43Q) was able to associate with alpha3- or beta3-subunits, although the stoichiometry of the complex with alpha3 was altered. Our data show that gamma2(R43Q) is not a dominant negative and that the mutation leads to a modification of GABA(A) receptor subunit composition on the cell surface that impairs the synaptic targeting in neurons. This study reveals an involvement of the gamma2-Arg-43 domain in the control of receptor assembly that may be relevant to the effect of the heterozygous gamma2(R43Q) mutation leading to childhood absence epilepsy and febrile seizure.
Asunto(s)
Membrana Celular/metabolismo , Epilepsia Tipo Ausencia/genética , Ligamiento Genético , Mutación Puntual , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Sustitución de Aminoácidos/genética , Animales , Arginina/genética , Células COS , Membrana Celular/genética , Células Cultivadas , Chlorocebus aethiops , Glutamina/genética , Humanos , Neuronas/química , Neuronas/metabolismo , Subunidades de Proteína/biosíntesis , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ratas , Receptores de GABA-A/biosíntesisRESUMEN
Previously, we showed that the peak density of the transient outward K(+) current (I(to)) expressed in GH3 cells was different in the S phase than in other phases of the cell cycle. Using cell synchronization, we show here that I(to) drops precisely at the quiescent (G(0) phase)/proliferating transition. This change is not due to a modification in the voltage dependence of I(to), but rather to a modification in its inactivation kinetics. Molecular determination of K(+) channel subunits showed that I(to) required the expression of Kv1.4, Kv4.1, and Kv4.3. We found that the increase in I(to) density during the quiescent state was accompanied by an increase in Kv1.4 protein expression, whereas Kv4.3 expression remained unchanged. We further demonstrate that the link between I(to) expression and cell proliferation is not mediated by variations in cell excitability. These results provide new evidence for the cell cycle dependence of I(to) expression, which could be relevant in understanding the mechanisms leading to pituitary adenomas.