Intracellular magnesium-dependent modulation of gap junction channels formed by neuronal connexin36

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 ([Mg²⁺]_i). We examined junctional conductance (g_j) and its dependence on transjunctional voltage (V_j) at different [Mg²⁺]_i in cultures of HeLa or N2A cells expressing Cx36. We found that Cx36 GJs are partially inhibited at resting [Mg²⁺]_i. Thus, g_j can be augmented or reduced by lowering or increasing [Mg²⁺]_i, respectively. Similar changes in g_j and V_j-gating were observed using MgATP or K₂ATP in pipette solutions, which increases or decreases [Mg²⁺]_i, respectively. Changes in phosphorylation of Cx36 or in intracellular free calcium concentration were not involved in the observed Mg²⁺-dependent modulation of g_j. Magnesium ions permeate the channel and transjunctional asymmetry in [Mg²⁺]_i resulted in asymmetric V_j-gating. The gj of GJs formed of Cx26, Cx32, Cx43, Cx45, and Cx47 was also reduced by increasing [Mg²⁺]_i, but was not increased by lowering [Mg²⁺]_i; single-channel conductance did not change. We showed that [Mg²⁺]_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 [Mg²⁺]_i. Thus, this novel modulatory mechanism could underlie changes in neuronal synchronization under conditions in which ATP levels, and consequently [Mg²⁺]_i, are modified.