C terminus L-type Ca²⁺ channel calmodulin-binding domains are 'auto-agonist' ligands in rabbit ventricular myocytes

L-type Ca²⁺ channel C terminus calmodulin (CaM)-binding domains are molecular determinants for Ca²⁺-CaM-dependent increases in L-type Ca²⁺ current (I_Ca), and a CaM-binding IQ domain mimetic peptide (IQmp) increases L-type Ca²⁺ channel current by promoting a gating mode with prolonged openings (mode 2), suggesting the intriguing possibility that CaM-binding domains are 'auto-agonist' signalling molecules. In order to test the breadth of this concept, we studied the effect of a second C terminus CaM-binding domain (CB) mp (CBmp), in conjunction with IQmp, on single L-type Ca²⁺ channel currents in excised cell membrane patches from rabbit ventricular myocytes. Here we show that both CBmp and IQmp are agonist ligands that non-additively increase L-type Ca²⁺ channel opening probability (P_o) by inducing mode 2 gating. CBmp and IQmp agonist effects were lost under conditions favouring calcification of CaM (Ca²⁺-CaM, 150 nM free Ca²⁺ and 10-20 μM CaM), but persisted in the presence of CaM (0-20 μM) under conditions adverse to Ca²⁺-CaM (20 mM BAPTA), indicating that CaM-binding domains increase L-type Ca²⁺ channel P_o by a low Ca²⁺-CaM activity mechanism. Increasing Ca²⁺-CaM in the bath (cytosol) reduced the efficacy of CBmp and IQmp signals with Ba²⁺ as charge carrier, suggesting that CaM binding motifs target a site outside of the pore region. We measured the combined effects of CBmp and Ca²⁺-CaM-dependent protein kinase II (CaMKII) on L-type Ca²⁺ channels by using an engineered Ca²⁺-CaM-independent form of CaMKII that remains active under low Ca²⁺-CaM conditions, permissive for CBmp signalling. CBmp and CaMKII increased L-type Ca²⁺ channel P_o in a non-additive manner, suggesting that low and high Ca²⁺-CaM-dependent L-type Ca²⁺ channel facilitation pathways converge upon a common signalling mechanism.