Interactions of Calmodulin With the Multiple Binding Sites of Cav1.2 Ca2+ Channels

Abstract

Abstract. Although calmodulin binding to various sites of the Cav1.2 Ca 2+ channel has been reported, the mechanism of the interaction is not fully understood. In this study we examined calmodulin binding to fragment channel peptides using a semi-quantitative pull-down assay. Calmodulin bound to the peptides with decreasing affinity order: IQ > preIQ > I-II loop > N-terminal peptide. A peptide containing both preIQ and IQ regions (Leu 1599 -Leu 1668 ) bound with approximately 2 mol of calmodulin per peptide. These results support the hypothesis that two molecules of calmodulin can simultaneously bind to the C-terminus of the Cav1.2 channel and modulate its facilitatory and inhibitory activities.

The Ca2+‐dependent interaction of calpastatin domain L with the C‐terminal tail of the Cav1.2 channel

“…Thus, it is proposed that CS L may compete with CaM for binding with Cav1.2 as a partial agonist [8], and this kind of competitive binding of CaM and CS L was eventually confirmed on the IQ motif [8]. , in IQ motif, significantly affect the CaM binding to the preIQ and IQ peptides, respectively [12]. Similarly, in present study, we found that the amounts of CS L binding with the mutants of above a.a. residues were dramatically decreased, suggesting that those amino acids are also important for the CS L binding.…”

“…A previous study [12] examined the effect of the I/E mutation on the IQ region binding to CaM. Those results indicated that the mutation nearly completely abolished CaM binding and confirmed that I1653 in the IQ region interacted with CaM, and that multiple mutations (E/K-I/D-LL/RR) decreased the affinity of the CaM-PreIQ interaction, supporting the hypothesis that these a.a. residues are important for the binding [12].…”

“…The purification of the GST-fusion proteins was as described in the previous reports [8,12,13] As for CS L fusion protein, the GST portion of it was removed by using the PreScission Protease (GE Healthcare). All steps were performed on ice or at 4°C.…”

“…Those results indicated that the mutation nearly completely abolished CaM binding and confirmed that I1653 in the IQ region interacted with CaM, and that multiple mutations (E/K-I/D-LL/RR) decreased the affinity of the CaM-PreIQ interaction, supporting the hypothesis that these a.a. residues are important for the binding [12]. To demonstrate the binding of CS L with IQ and PreIQ, we generated mutated IQ and mutated PreIQ GST-fusion peptides (Fig.…”

Post-transcriptional modifications and “Calmodulation” of voltage-gated calcium channel function: Reflections by two collaborators of David T Yue

“…38 It was the words of David Yue: 'one bad apple spoils the pie'. Thereafter, crystallographic and biochemical studies revealed that CaM could at least bind at 2 other sites including the C-terminal domain of Cav1.2 channels 39,40 and the N-terminal region called NSCaTE. 41 Thus, the gap of knowledge between functional counting of CaM and these latter results is still unresolved.…”