Modulatory mechanisms of neuronal gap junctions: the role of intracellular hydrogen and magnesium ions in voltagesensitive gating

Abstract

Electrical synapses formed by connexin-based gap junction (GJ) channels are widely expressed in the mammalian CNS and are essential for the formation of dynamic cellular networks that allow activity coordination for normal brain function. Like other ion channels, GJs are highly regulated according to cellular requirements and respond to various changes in the extracellular and intracellular environments. Voltage-sensitive gates of GJ channels also respond to changes in intracellular pH (pHi) and concentration of free magnesium ([Mg2+]i) ions, which can vary under physiological and pathological conditions. Hence, the effect of pHi and [Mg2+]i on neuronal GJs was studied using dual whole-cell patch clamp and fluorescence microscopy in heterologous expression systems and brain slices. The pHi–dependent modulation of junctional conductance (gj) and its dependence on transjunctional voltage (Vj) was examined in homotypic and heterotypic GJ channels formed by neuronal connexins, Cx45 and Cx57. Stochastic multistate models containing one or two Vj-sensitive gates in each apposed hemichannel (aHC) were used to estimate gating parameters characterizing sensitivity to Vj, open probability (Po) and number of functional channels (NF). First, it is shown that Po and NF of Cx45 and Cx57 GJs, which are expressed in certain neurons of the retina and brain, are strongly affected by changes in pHi through a common mechanism that involves shifting the gj–Vj dependence of aHCs along the Vj axis, without change in the slope of gj–Vj dependence. Cx45 and Cx47 GJs are inhibited at resting pHi and gj was augmented or reduced by increasing or lowering pHi, respectively. The gj–pHi dependence of Cx45 and Cx57 GJs revealed pKas of ~7 and 7.4, respectively. xii The [Mg2+]i–dependent modulation of gj in GJs formed of Cxs 26, 32, 36, 43, 45 and 47 was examined. As opposed to other examined Cxs, GJs formed by neuronal Cx36 are partially inhibited at resting [Mg2+]i and gj was augmented or reduced by lowering or increasing [Mg2+]i, respectively. Similarly, gj was augmented or reduced by using pipette solutions containing K2ATP or MgATP, which decreases or increases [Mg2+]i, respectively. Magnesium ions permeate Cx36 GJ channels and transjunctional asymmetry in [Mg2+]i resulted in asymmetric Vj-gating. Similar to results from pHi studies, changes in [Mg2+]i affect both Po and NF of Cx36 GJ channels through a mechanism that modulates the gj–Vj dependence of aHCs. Cx36-containing electrical synapses between neurons of the trigeminal mesencephalic nucleus and thalamic reticular nucleus in brain slices showed a similar bidirectional Mg2+-dependent modulation of gj. Chimeragenesis and site-directed mutagenesis were used to demonstrate that the first extracellular loop of Cx36 forms a pore-lining Mg2+-sensitive domain, and that residue D47 is critical for determining high Mg2+-sensitivity. In addition, asymmetric transjunctional Mg2+ induced strong instantaneous rectification, providing a novel mechanism for electrical rectification in homotypic Cx36 GJs. In summary, both pHi and [Mg2+]i affect intercellular communication via modulation of Vj-sensitive gating mechanisms of GJ channels. The pHi-dependent modulation of neuronal GJs expressed in the retina, such as Cx45 and Cx57, may explain how changes in pHi during vision modulate electrical transmission. In addition, Mg2+-dependent modulation of Cx36-containing electrical synapses in the CNS could underlie neuronal circuit reorganization via changes in brain metabolism that affects neuronal levels of intracellular ATP and consequently [Mg2+]i.