| dc.description | 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. | |
