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UBC Theses and Dissertations

Structural insights into calmodulin regulation and dysregulation of the L-type voltage-gated calcium channel Wang, Kaiqian


Calmodulinopathies are life-threatening arrhythmia syndromes that arise from mutations in calmodulin (CaM), a calcium-sensing protein whose sequence is completely conserved across vertebrates. Although these mutations have been shown to interfere with the function of cardiac ion channels including the voltage-gated calcium channel CaV1.2, in a mutation-specific manner, direct structural insights into any CaM disease variant have been lacking. Here, we utilize X-ray crystallography, NMR, and binding assays to probe the interaction of CaM disease variants with the IQ domain of CaV1.2 from a structural and biophysical standpoint. We present crystal structures of several C-lobe mutants and an N-lobe mutant in complex with the IQ domain. Surprisingly, two variants (D95V and N97I) cause major distortion of the C-lobe, resulting in a new pathological conformation not reported before. These structural changes result in altered interactions with IQ domain. Ca²⁺ binding to EF-hands normally proceeds with high cooperativity, but we find N97S CaM can adopt different conformations with either one or two Ca²⁺ ions bound to the C-lobe, possibly disrupting cooperativity. Another mutation (D129G) results in complete separation of EF-hands within the C-lobe and loss of Ca²⁺-binding in EF-hand 4. Q135P CaM has severely reduced affinity for the IQ domain, and shows changes in the CD spectra under Ca²⁺-saturating conditions when unbound to IQ domain. F141L CaM exhibits structural changes in the Ca²⁺-free state that increase affinity for IQ domain. An N-lobe variant (N53I) does not display major changes in complex with the IQ domain, providing a structural basis for why this mutant does not affect function of CaV1.2. These findings demonstrate that different CaM mutants have distinct effects on both CaM structure and interactions with protein targets, and act via distinct pathological mechanisms to cause disease. We also investigate a binding site for CaM in the intracellular linker between domains I and II of CaV1.1 and CaV1.2. We describe crystal structures of Ca²⁺/CaM in complex with I-II loops and examine binding thermodynamics via isothermal titration calorimetry, revealing a high affinity CaM-binding site in the cytoplasmic I-II loop of L-type calcium channels which may contribute to the complex CaM-mediated regulation of the channel.

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