| Summary: | Divalent cations are known to exert a charge screening effect on voltage-gated ion
channels either through non-specific interactions with fixed negative charges on the cell
membrane or via binding to negatively charged sites on or electrically close to the channel
forming protein. In some instances, divalent cations bind directly to the gating apparatus
of voltage-sensitive sodium and potassium channels thereby stabilizing the channels in a
closed conformation.
Most of the investigations into the effects of divalent cations on voltage-gated ion
channels have concentrated on the Na + and delayed rectifier type K + channels. Although,
there has been a recent explosion of information regarding the molecular structure of the
transient outward potassium channel, few investigators have examined the actions of divalent
cations on the behaviour of the transient outward potassium current (TOC).
A transient outward potassium current, (IK(f)), has been characterized in
melanotrophs, the major cell type found in the pars intermedia of the pituitary gland in rats
(Kehl, 1989). I K(f) activates and inactivates rapidly. Cd2+ (5 mM) reduced the peak
amplitude of IK(f) and increased the 50% rise time of this current (Kehl, 1989). The present
study elaborated on these observations and examined the effects of varying the extracellular
concentrations of Cd 2+ , Zn2+ , Ca2+ and Mg2+ on the behaviour of IK(f).
Acutely dissociated melanotrophs were obtained from male Wistar rats and wholecell
currents were recorded, using conventional patch clamp techniques, from cells
maintained in culture for 1-14 hrs.
Divalent cations shift the activation and inactivation curves and the gating kinetics
of IK(f) right-ward along the voltage axis. The cations tested varied in their ability to shift
the potential-sensitive parameters of IK(f) and ranked in the following order: Zn2+ ≥ Cd2+
> > Ca2+ > Mg2+ , in good agreement with previous observations of their effect on sodium
channels.
The mean control half-activation potential was -13.6 mV with a slope-factor of + 12.8
mV (n = 55) and the mean control half-inactivation potential was -54.7 mV with a slopefactor
of -4.4 mV (n =50). The relationships between the shift of the half-activation
potential and the divalent cation concentration indicated that the K m's for the half-maximal
shift of the activation curve were 221 μM (Cd2+), 92 μM (Zn2 +) and 3.4 mM (Ca2+) and the
maximal shifts of the activation curve were, respectively, +28 mV, +34 mV and + 15.6 mV.
Mg2+ was far less potent than any of the other divalent cations examined. Shifts of the
inactivation curve were equal to the shifts of the activation curve at each divalent cation
concentration tested. The slope-factors of the activation and inactivation curves were not
altered by the application of divalent cations. Removal of Ca2+ from the external media
significantly increased the slope-factor for the activation curve. That is, zero Ca2+ resulted
in a decrease in the equivalent charge transferred during the activation gating process.
The prediction central to non-specific charge screening is that all divalent cations will
be equally effective. The results reported here show that this is not the case for IK(f). It is
proposed that a specific binding site for divalent cations exists on or electrically close to the
channel protein. Divalent cations also slowed the rise time of I K(f) suggesting that they
might stabilize the channel conducting this current in the closed conformation. === Medicine, Faculty of === Cellular and Physiological Sciences, Department of === Graduate
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