RESUMEN
Neuronal G-protein-gated potassium (GIRK) channels mediate the inhibitory effects of many neurotransmitters. Although the overlapping distribution of GIRK subunits suggests that channel composition varies in the CNS, little direct evidence supports the existence of structural or functional diversity in the neuronal GIRK channel repertoire. Here we show that the GIRK channels linked to GABAB receptors differed in two neuron populations. In the substantia nigra, GIRK2 was the principal subunit, and it was found primarily in dendrites of neurons in the substantia nigra pars compacta (SNc). Baclofen evoked prominent barium-sensitive outward current in dopamine neurons of the SNc from wild-type mice, but this current was completely absent in neurons from GIRK2 knock-out mice. In the hippocampus, all three neuronal GIRK subunits were detected. The loss of GIRK1 or GIRK2 was correlated with equivalent, dramatic reductions in baclofen-evoked current in CA1 neurons. Virtually all of the barium-sensitive component of the baclofen-evoked current was eliminated with the ablation of both GIRK2 and GIRK3, indicating that channels containing GIRK3 contribute to the postsynaptic inhibitory effect of GABAB receptor activation. The impact of GIRK subunit ablation on baclofen-evoked current was consistent with observations that GIRK1, GIRK2, and GABAB receptors were enriched in lipid rafts isolated from mouse brain, whereas GIRK3 was found primarily in higher-density membrane fractions. Altogether, our data show that different GIRK channel subtypes can couple to GABAB receptors in vivo. Furthermore, subunit composition appears to specify interactions between GIRK channels and organizational elements involved in channel distribution and efficient receptor coupling.
Asunto(s)
Encéfalo/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/biosíntesis , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Neuronas/metabolismo , Animales , Baclofeno/farmacología , Encéfalo/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Masculino , Ratones , Ratones Noqueados , Neuronas/efectos de los fármacos , Neuronas/ultraestructuraRESUMEN
Sphingosine-1-phosphate (S1P) is an endogenous agonist for a family of five G protein-coupled receptors (S1P(1-5)) involved in cell proliferation, cardiovascular development and lymphocyte trafficking. The sphingolipid drug FTY720 displays structural similarity to S1P and efficacy as an immunosuppressant in models of autoimmune disease and in solid organ transplantation. While FTY720 is well-tolerated in humans, it produces a transient reduction of heart rate (HR). As S1P activates the cardiac G protein-gated potassium channel I(KACh), we speculated that the FTY720-induced HR reduction reflects I(KACh) activation. We examined FTY720 effects on atrial myocytes from wild-type and I(KACh)-deficient mice. In wild-type myocytes, the active phosphate metabolite of FTY720 (FTY720-P) induced single channel activity with conductance, open time, GTP sensitivity and rectification identical to that of I(KACh). In whole-cell recordings, FTY720-P evoked an inwardly rectifying potassium current in approximately 90% of myocytes responding to acetylcholine. Comparable channel activity was never observed in myocytes from I(KACh)-deficient mice. In wild-type mice, acute FTY720 administration produced a dose-dependent, robust HR reduction. In contrast, the HR reduction induced by FTY720 in I(KACh)-deficient mice was blunted. We conclude that the effect of acute FTY720 administration on HR is mediated primarily by I(KACh) activation.