RESUMEN
Gap junction (GJ) channels composed of Connexin36 (Cx36) are widely expressed in the mammalian CNS and form electrical synapses between neurons. Here we describe a novel modulatory mechanism of Cx36 GJ channels dependent on intracellular free magnesium ([Mg(2+)]i). We examined junctional conductance (gj) and its dependence on transjunctional voltage (Vj) at different [Mg(2+)]i in cultures of HeLa or N2A cells expressing Cx36. We found that Cx36 GJs are partially inhibited at resting [Mg(2+)]i. Thus, gj can be augmented or reduced by lowering or increasing [Mg(2+)]i, respectively. Similar changes in gj and Vj-gating were observed using MgATP or K2ATP in pipette solutions, which increases or decreases [Mg(2+)]i, respectively. Changes in phosphorylation of Cx36 or in intracellular free calcium concentration were not involved in the observed Mg(2+)-dependent modulation of gj. Magnesium ions permeate the channel and transjunctional asymmetry in [Mg(2+)]i resulted in asymmetric Vj-gating. The gj of GJs formed of Cx26, Cx32, Cx43, Cx45, and Cx47 was also reduced by increasing [Mg(2+)]i, but was not increased by lowering [Mg(2+)]i; single-channel conductance did not change. We showed that [Mg(2+)]i affects both open probability and the number of functional channels, likely through binding in the channel lumen. Finally, we showed that Cx36-containing electrical synapses between neurons of the trigeminal mesencephalic nucleus in rat brain slices are similarly affected by changes in [Mg(2+)]i. Thus, this novel modulatory mechanism could underlie changes in neuronal synchronization under conditions in which ATP levels, and consequently [Mg(2+)]i, are modified.
Asunto(s)
Conexinas/fisiología , Uniones Comunicantes/fisiología , Líquido Intracelular/metabolismo , Activación del Canal Iónico/fisiología , Magnesio/metabolismo , Neuronas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos , Fenómenos Biofísicos/efectos de los fármacos , Fenómenos Biofísicos/fisiología , Cationes Bivalentes/metabolismo , Línea Celular Tumoral , Quelantes/farmacología , Conexina 26 , Conexinas/genética , Relación Dosis-Respuesta a Droga , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Femenino , Uniones Comunicantes/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Humanos , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Magnesio/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Neuronas/citología , Técnicas de Placa-Clamp , Fosforilación , Ratas , Ratas Sprague-Dawley , Tegmento Mesencefálico/citología , Transfección , Proteína delta-6 de Union ComunicanteRESUMEN
Neuronal gap junction (GJ) channels composed of connexin36 (Cx36) play an important role in neuronal synchronization and network dynamics. Here we show that Cx36-containing electrical synapses between inhibitory neurons of the thalamic reticular nucleus are bidirectionally modulated by changes in intracellular free magnesium concentration ([Mg(2+)]i). Chimeragenesis demonstrates that the first extracellular loop of Cx36 contains a Mg(2+)-sensitive domain, and site-directed mutagenesis shows that the pore-lining residue D47 is critical in determining high Mg(2+)-sensitivity. Single-channel analysis of Mg(2+)-sensitive chimeras and mutants reveals that [Mg(2+)]i controls the strength of electrical coupling mostly via gating mechanisms. In addition, asymmetric transjunctional [Mg(2+)]i induces strong instantaneous rectification, providing a novel mechanism for electrical rectification in homotypic Cx36 GJs. We suggest that Mg(2+)-dependent synaptic plasticity of Cx36-containing electrical synapses could underlie neuronal circuit reconfiguration via changes in brain energy metabolism that affects neuronal levels of intracellular ATP and [Mg(2+)]i.