Y. Saimi et Ky. Ling, PARAMECIUM NA-CALMODULIN - CALMODULIN IS THE CA2+ SENSOR IN THE CHANNEL GATING MECHANISM( CHANNELS ACTIVATED BY CA2+), The Journal of membrane biology, 144(3), 1995, pp. 257-265
Paramecium Na+ channels, which were Ca2+-calmodulin activated, were st
udied in the inside-out mode of patch clamp. After excision of the mem
brane patch, they were active in the presence of 10(-5) to 10(-3) M Ca
2+ in the bath. They became much less active in the presence of 10(-6)
M Ca2+, and their activity subsided completely at 10(-8) M Ca2+. A Hi
ll plot showed a dissociation constant of 6 mu M for Ca2+ binding. Thi
s dissociation constant shifted to a submicromolar range in the presen
ce of 1 mM Mg2+. The channels also exhibited a mild voltage dependence
. When exposed to 10(-8) M Ca2+ for an extended period of 2-4 min, cha
nnels were further inactivated even after bath Ca2+ was restored to 10
(-4) M. Whereas neither high voltage (+100 mV) nor high Ca2+ (10(-3) M
) was effective in reactivation of the inactive channels, addition of
Paramecium wild-type calmodulin together with high Ca2+ to the bath re
stored channel activity without a requirement of additional Mg2+ and m
etabolites such as ATP. The channels reactivated by calmodulin had the
same ion conductance, ion selectivity and Ca2+ sensitivity as those p
rior to inactivation. These inactivation and reactivation of the chann
els could be repeated, indicating that the direct calmodulin effect on
the Na+ channel was reversible. Thus, calmodulin is a physiological f
actor critically required for Na+ channel activation, and is the Ca2sensor of the Na+-channel gating machinery.