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Gap junction channels (GJC)
provide an intercellular pathway for the exchange of small molecules
and ions. Each channel consists of two hemichannels composed of six
transmembrane proteins (connexins). Different types of cells express
different kinds of connexins (Cx). Cx45 plays an important role in the
propagation of the action potential in the heart. It has also been found
in kidney, lung and smooth muscle.
So far, electrical properties of GJC have been studied in pairs of isolated
cells or transfected cells in culture. To gain further insight into
the properties of GJC, we have used an alternative approach. It involves
examination of gap junction hemichannels (hc) with a standard voltage-clamp
method in the whole-cell configuration. Hc exist in the cell membrane
of single cells and are thought to be closed under normal conditions.
However, they can be opened by lowering [Ca2+]o. In our study, we have
used human HeLa cells transfected with cDNA of mouse Cx45. Interference
from K+ channels and volume-regulated Cl- channels was minimized by
adding Ba2+, Cs+, TEA+ and Mibefradil, respectively. Lowering the [Ca2+]o
revealed an extra current component. Its sensitivity to extracellular
Ca2+, impairment by gap junction channel blockers (18-b-glycyrrhetinic
acid, palmitoleic acid, heptanol) and its absence in wild-type HeLa
cells suggest that it is carried by Cx45 hc. Using a double-step pulse
protocol, we have examined the instantaneous and steady-state currents
[I(hc,inst) and I(hc,ss)] and conductances [g(hc,inst) and g(hc,ss)].
I(hc) was sensitive to the membrane potential V(m) and inactivated with
time. It was maximal at positive V(m) and minimal, but different from
zero, at negative V(m). The plot of normalized g(hc,inst) versus V(m)
was constant, the plot of g(hc,ss) versus V(m) was sigmoidal (Boltzmann
parameters: V(m,0) = -2.6 mV, z = 4.1). The inactivation of I(hc) grew
faster with negative V(m). The analysis showed that I(hc) inactivates
as a single exponential. The plot of the time constant of inactivation
tau(i) versus V(m) followed an exponential with a minimum at negative
V(m). The recovery of I(hc) from inactivation was fastest at negative
V(m). In essence, it followed a single exponential.
We conclude that Cx45 expressed in human HeLa cells forms voltage-sensitive
hc which are gated by negative voltage. These channels are fully open
at positive V(m) and partially closed at negative V(m), properties reminiscent
of GJC. They are completely closed in the presence of normal [Ca2+]o.
It will be interesting to see if hc contribute to the membrane conductance
under physiological conditions.
Supported by the
SNSF (31-67230.01).
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