Interactions of Calmodulin with Two Peptides Derived from the C-terminal Cytoplasmic Domain of the Cav1.2 Ca2+ Channel Provide Evidence for a Molecular Switch Involved in Ca2+-induced Inactivation

When opened by depolarization,l-type calcium channels are rapidly inactivated by the elevation of Ca2+ concentration on the cytoplasmic side. Recent studies have shown that the interaction of calmodulin with the proximal part of the cytoplasmic C-terminal tail of the channel plays a prominent role in this modulation. Two motifs interacting with calmodulin in a Ca2+-dependent manner have been described: the IQ sequence and more recently the neighboring CB sequence. Here, using synthetic peptides and fusion proteins derived from the Cav1.2 channel combined with biochemical techniques, we show that these two peptides are the only motifs of the cytoplasmic tail susceptible to interact with calmodulin. We determined the Kd of the CB interaction with calmodulin to be 12 nm, i.e. below the Kd of IQ-calmodulin, thereby precluding a competitive displacement of CB by IQ in the presence of Ca2+. In place, we demonstrated that a ternary complex is formed at high Ca2+ concentration, provided that calmodulin and the peptides are initially allowed to interact at a low Ca2+ concentration. These results provide evidence that CB and IQ motifs interacting together with calmodulin constitute a minimal molecular switch leading to Ca2+-induced inactivation. In addition, we suggest that they could also be the tethering site of calmodulin.