Magnesium-dependent modulation of junctional conductance and gating properties of Connexin36 Gap junction channels

Abstract

Gap junction (GJ) channels formed by connexin36 (Cx36) play an important role in neuronal synchronization, and calcium oscillations in insulinsecreting beta cells. Here we describe a new form of plasticity of Cx36 GJ channels dependent on intracellular free magnesium ([Mg2þ ]i). We examined junctional conductance (gj) and its dependence on transjunctional voltage (Vj) in HeLa and neuroblastoma N2A cells expressing Cx36 at different [Mg2þ ]i. A remarkable ~3.5-fold increase in gj was observed when [Mg2þ ]i was reduced to 0.01 mM, and a reduction to ~1/5th of initial values when [Mg2þ ]i was augmented to 5 mM; for [Mg2þ ]i action EC50= ~0.45 mM. By using a stochastic 16-state model of voltage gating, we demonstrate that lowered [Mg2þ ]i increases open channel probability while enhanced [Mg2þ ]i reduces it. Similar changes in conductance and Vj-gating are observed with MgATP or K2ATP, which increases or decreases [Mg2þ ]i, respectively. Changes in phosphorylation of Cx36 or [Ca2þ ]i are not involved in the observed Mg-dependent modulation of gj. Magnesium ions permeate the channel and transjunctional asymmetry in [Mg2þ ]i results in asymmetric Vj-gating. We propose that the lumen of Cx36 GJ channels contains binding site(s) for Mg2þ , and that Mg2þ stabilizes a closed channel conformation. Conductance of GJs formed by Cx26, 32, 43, 45 and 47 expressed in HeLa cells are also reduced by increasing [Mg2þ ]i above resting levels. However, none of these Cxs show increase in gj upon reduction in [Mg2þ ]I; thus, Cx36 is the only tested Cx sensitive to lowering of physiological levels of free Mg2þ . This novel Mg2þ -dependent modulation of Cx36 GJ channels can be important for changes in neuronal synchronization and insulin secretion under physiological and pathological conditions when ATP levels, and consequently [Mg2þ ]i, are modified.