Apocalmodulin and Ca2+ calmodulin-binding sites on the CaV1.2 channel

Area of Science:

• Molecular and Cellular Biology
• Cardiovascular Physiology
• Biochemistry

Background:

• The cardiac L-type voltage-dependent calcium channel initiates excitation-contraction coupling.
• Specific alpha(1) subunit sequences (A, C, IQ) mediate calmodulin (CaM) binding and Ca(2+)-dependent inactivation.
• Understanding these interactions is key to cardiac function.

Purpose of the Study:

• To investigate the binding interactions between CaM and specific peptide sequences of the cardiac calcium channel.
• To elucidate the role of these sequences in Ca(2+)-dependent inactivation.
• To model the mechanism of apoCaM binding and subsequent CaM activation.

Main Methods:

• Peptide synthesis corresponding to A, C, and IQ sequences and their combinations (A-C, C-IQ).
• Binding assays using Ca(2+)-bound CaM (Ca(2+)CaM) and apocalmodulin (ApoCaM).
• Analysis of Ca(2+) affinity changes in CaM lobes upon peptide binding.
• Utilizing mutant CaM unable to bind Ca(2+) at specific sites.

Main Results:

• Peptides A, C, and IQ all bind Ca(2+)CaM; longer peptides (A-C, C-IQ) bind only one Ca(2+)CaM molecule.
• ApoCaM shows differential affinity, binding C-IQ more strongly than IQ.
• Binding to IQ and C peptides enhances Ca(2+) affinity of CaM's C-lobe; IQ peptide uniquely affects the N-lobe.
• Mutations in the IQ motif disrupt inactivation and apoCaM interaction.
• Mutant CaM lacking Ca(2+) binding at sites 3 and 4 binds IQ but not C or A peptides.

Conclusions:

• ApoCaM binding to the IQ motif region is essential for rapid Ca(2+) binding to CaM's C-lobe.
• Ca(2+) binding to the C-lobe likely facilitates interaction with the A-C region.
• These findings provide a mechanistic model for Ca(2+)-dependent inactivation of the cardiac calcium channel.