PARAMECIUM NA-CALMODULIN - CALMODULIN IS THE CA2+ SENSOR IN THE CHANNEL GATING MECHANISM( CHANNELS ACTIVATED BY CA2+)

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.