Abstract
Sodium-calcium exchange current was isolated in inside-out patches
excised from guinea pig ventricular cells using the giant patch method.
The outward exchange current decayed exponentially upon activation
by cytoplasmic sodium (sodium-dependent inactivation). The kinetics
and mechanism of the inactivation were studied. (a) The rate of inactivation
and the peak current amplitude were both strongly temperature dependent
(Q10 = 2.2). (b) An increase in cytoplasmic pH from 6.8 to 7.8 attenuated
the current decay and shifted the apparent dissociation constant
(Kd) of cytoplasmic calcium for secondary activation of the exchange
current from 9.6 microM to < 0.3 microM. (c) The amplitude of exchange
current decreased synchronously over the membrane potential range
from -120 to 60 mV during the inactivation, indicating that voltage
dependence of the exchanger did not change during the inactivation
process. The voltage dependence of exchange current also did not
change during secondary modulation by cytoplasmic calcium and activation
by chymotrypsin. (d) In the presence of 150 mM extracellular sodium
and 2 mM extracellular calcium, outward exchange current decayed
similarly upon application of cytoplasmic sodium. Upon removal of
cytoplasmic sodium in the presence of 2-5 microM cytoplasmic free
calcium, the inward exchange current developed in two phases, a fast
phase within the time course of solution changes, and a slow phase
(tau approximately 4 s) indicative of recovery from sodium-dependent
inactivation. (e) Under zero-trans conditions, the inward current
was fully activated within solution switch times upon application
of cytoplasmic calcium and did not decay. (f) The slow recovery phase
of inward current upon removal of cytoplasmic sodium was also present
under the zero-trans condition. (g) Sodium-dependent inactivation
shows little or no dependence on membrane potential in guinea pig
myocyte sarcolemma. (h) Sodium-dependent inactivation of outward
current is attenuated in rate and extent as extracellular calcium
is decreased. (i) Kinetics of the sodium-dependent inactivation and
its dependence on major experimental variables are well described
by a simple two-state inactivation model assuming one fully active
and one fully inactive exchanger state, whereby the transition to
the inactive state takes place from a fully sodium-loaded exchanger
conformation with cytoplasmic orientation of binding sites (E1.3Ni).
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