Phosphorylation of Maurocalcine Strongly Modifies its Effect on Type 1 Ryanodine Receptor

Abstract

Maurocalcine (MCa) is a 33-amino acid peptide isolated from the venom of Scorpio maurus palmatus, a Tunisian scorpion. It possesses efficient cell penetration properties and has been shown to strongly modify ryanodine receptor (RyR1) channel behavior by increasing open probability and inducing a long-lasting subconductance state. The amino acid residue threonine at position 26 belongs to a putative phosphorylation site within MCa sequence. We investigated the effect of a) T26 phosphorylation (MCa T26Phospho) by protein kinase A and b) replacement of T26 by glutamic acid residue to mimic phosphorylation on the effect of MCa on RyR1 properties. Using [ 3 H]ryanodine ([ 3 H]Ry) binding and single channel voltage-clamp measurements, we show that both MCa T26Phospho and MCa T26E analogs almost completely lose the ability to induce RyR1 activation. Neither MCa T26Phospho nor MCa T26E (up to 2mM) increase [ 3 H]rya binding. Small increase (~1.65-fold activation; p<0.01) was observed with high concentrations (10mM) of either MCa T26Phospho or MCa T26E. Single channel measurements revealed that neither MCa T26E nor MCa T26Phospho (up to 2mM) were able to induce the characteristic long-lasting subconductance state of RyR1 observed in presence of wild-type MCa. MCa T26Phospho altered RyR1 channel gating in a timedependent biphasic manner with initial activation of Po followed by almost complete inactivation. These effects of MCa T26Phospho could be reversed by 200nM wt MCa. At concentrations R2mM, MCa T26E enhances Po~3 fold by shortening t c without altering t o. These results show that single phosphorylation of MCa modifies RyR1 activity.1P01 AR52354.