Mechanism of Inhibition of the Ca2+-ATPase by Melittin

The Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum is inhibited by melittin at pH 7.4. Melittin has no effect on the rate of phosphorylation of the ATPase or on the rate of the Ca2+ transport step, but melittin inhibits dephosphorylation of the phosphorylated ATPase at pH 7.3. At pH 6.0, melittin has no effect on ATPase activity or on the rate of dephosphorylation. At pH 7.4, inhibition of ATPase activity fitted to a Kd of 0.4 micromolar for melittin. Analogues of melittin in which the two Arg residues were replaced by Gln [melittin(RR to QQ)] or the two Lys residues were replaced by Gln [melittin-(KK to QQ)] also inhibited ATPase activity, but with an increased Kd value of 3.4 micromolar. Analogues of melittin containing an extra Lys residue at the C-terminus [melittin(+K)] or in which the Trp residue had been replaced with a Leu residue [melittin(W to L)] had the same effect on activity as melittin. Melittin and all the analogues increased the permeability of the SR membrane to Ca2+ with equal potency at pH 6.4, as shown by a reduction in level of Ca2+ accumulation. Melittin and all the analogues also shifted the E2-E1 equilibrium of the ATPase toward E1 with equal potency at pH 7.2, consistent with stronger binding to the E1 conformation. It is suggested that effects on Ca2+ permeability and on the E2-E1 equilibrium could follow from binding of the N-terminus of melittin at the membrane-water interface, and that effects on ATPase activity could follow from binding of the positively charged C-terminus between the phosphorylation and nucleotide binding domains. Inhibition of ATPase activity by melittin is observed in reconstituted vesicles containing single ATPase molecules. Binding of monoclonal antibodies to the ATPase does not prevent inhibition of ATPase activity by melittin. We conclude that inhibition does not require aggregation of the ATPase molecules