Voltage-gated L-type calcium (Ca²⁺) channels (Ca_v1.2/1.3) are essential to neuronal and cardiac physiology. They convey extracellular Ca²⁺ after membrane depolarization, a crucial event in muscle contraction, cardiac adrenergic response, neurotransmission, memory, and learning. CaV1.2/1.3 are fine-tuned by auxiliary proteins that orchestrate Ca²⁺ influx into cells, and human variants of these proteins can disrupt channel function leading to disease. The present work probes in depth molecular mechanisms of Ca_v1.2/1.3 regulation by small cytosolic proteins calmodulin (CaM) and Leucine-rich repeat containing protein 10 (Lrrc10), as well as their relevance in physiology and pathophysiology. Chapter 1 introduces basic concepts of ion channel function, classification of voltage-gated Ca²⁺ channels, molecular components of Ca_v1.2/1.3 channel complexes, and participation of Ca_v1.2/1.3 in cardiac and neuronal physiology and disease. Chapter 2 dissects the potential contribution of a selectivity-filter gate on both VDI and CDI of Ca_v1.3 through extensive biophysical characterization, revealing asymmetric participation of conformational changes in the domain IV selectivity filter. The uncovered inactivation mechanism may be of relevance for reversing the molecular phenotype observed in Timothy syndrome, an arrhythmogenic disorder that partially stems from reduced Ca_v1.2 inactivation. Chapter 3 considers Lrrc10 as a regulatory subunit of CaV channels, uncovers molecular mechanisms, including binding interfaces that support Ca_v1.2 upregulation, and evaluates the functional consequences of human variants in Lrrc10. As Lrr proteins can interact with a wide range of targets, Chapter 4 probes the promiscuity of Lrrc10 as an ion channel modulator. Using FRET analysis, I find that Lrrc10 can, in fact, associate with various ion channels. Further analysis revealed that Lrrc10 interaction with one of its potential targets, the cardiac NaV1.5 channel, alters channel function. More broadly, these studies establish a framework to systematically screen cross talk between ion channel subunits. Finally, in Chapter 5, I leverage insights obtained from in-depth characterization of Lrrc10 modulation to engineer a genetically encoded actuator that upregulates Ca_v1.2/1.3 currents in distinct physiological settings. Altogether, this work contributes to our molecular understanding of Ca_v1.2/1.3 regulation by small cytosolic proteins, and establishes new strategies to probe and manipulate a Ca²⁺ channel function that may ultimately aid in discovering potential new targets and tools for research and therapeutics.

Vitamin A is an essential nutrient; it is not synthesized by mammals and therefore must be obtained through the diet. During times of fasting or dietary vitamin A insufficiency, retinol, the alcohol form of the vitamin is released from the liver, its main storage tissue, for circulation in complex with retinol-binding protein 4 (RBP) to provide an adequate supply to peripheral tissues. Stimulated by Retinoic Acid 6 (STRA6), the transmembrane RBP receptor, mediates retinol uptake across blood-tissue barriers such as the retinal pigment epithelium of the eye, the placenta and the choroid plexus of the brain. Our understanding as to how this protein functions has been greatly enhanced by the high-resolution 3D structure of zebrafish STRA6 in complex with calmodulin (CaM) solved by single-particle cryogenic-electron microscopy. However, the nature of the interaction of STRA6 with retinol remains unclear. Here, I present the high-resolution structures of zebrafish and sheep STRA6 reconstituted in nanodisc lipid bilayers in the presence and absence of retinol. The nanodisc reconstitution system has allowed us to study this protein in a close to physiological environment and examine its interaction with the cell membrane and relationship with its ligand, retinol. We also present the structure of sheep STRA6 in complex with human RBP. The structure of the STRA6-RBP complex confirms predictions in the literature as to the nature of the protein-protein interaction needed for retinol transport. Calcium-bound CaM is bound to STRA6 in the RBP-STRA6 structure, consistent with a regulatory role of this calcium binding protein in STRA6-RBP interaction. The analysis of the three states of STRA6 – pre, post and during interaction with retinol – provide unique insights into the mechanism of STRA6-mediated cellular retinol uptake.

In the heart, sodium (Na+) influx via NaV1.5 channels initiates the action potential, and calcium (Ca2+) influx via CaV1.2 channels has a key role in excitation-contraction coupling and determining the plateau phase of the action potential. Mutations in the genes that encode these ion channels or in proteins that modulate them are linked to arrhythmias and cardiomyopathy, underscoring the need for characterizing mechanisms of regulation. The work presented in this thesis is subdivided into three different chapters, each with a distinct focus on ion channel modulation. The first chapter details our investigation of the functional PKA phosphorylation target for β-adrenergic regulation of CaV1.2. Physiologic β-adrenergic activation of PKA during the sympathetic “fight or flight” response increases Ca2+ influx through CaV1.2 in cardiomyocytes, leading to increased cardiac contractility. The molecular mechanisms of β-adrenergic regulation of CaV1.2 in cardiomyocytes are incompletely known, but activation of PKA is required for this process. Recent data suggest that β-adrenergic regulation of CaV1.2 does not require any combination of PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α1C subunits. To test if any non-conserved sites are required for regulation, we generated mice with inducible cardiac-specific expression of α1C with mutations at both conserved and non- conserved predicted PKA phosphorylation sites (35-mutant α1C). Additionally, we createdanother mouse with inducible cardiac-specific expression of β2 with mutations at predicted PKA phosphorylation sites (28-mutant β2B). In each of these mice, β-adrenergic stimulation of Ca²⁺ current was unperturbed. Finally, to test the hypothesis that redundant functional PKA phosphorylation sites exist on the α1C subunit and β2 subunit or that several sites confer incremental regulation, we crossed the 35-mutant α1C mice with the 28-mutant β2B mice to generate offspring expressing both mutant subunits. In these offspring, intact regulation was observed. These results provide the definitive answer that phosphorylation of the α1C subunit or β2 subunit is not required for β-adrenergic regulation of CaV1.2 in the heart. In the second chapter, we study the influence of calmodulin and fibroblast growth homologous factor (FHF) FGF13 on late Na+ current. Studies in heterologous expression systems show that the Ca²⁺-binding protein calmodulin plays a key role in decreasing late Na⁺ current. The effect of loss of calmodulin binding to NaV1.5 on late Na+ current has yet to be resolved in native cardiomyocytes. We created transgenic mice with cardiac-specific expression of human NaV1.5 channels with alanine substitutions for the IQ motif (IQ/AA), disrupting calmodulin binding to the C-terminus. Surprisingly, we found that the IQ/AA mutation did not cause an increase late Na⁺ current in cardiomyocytes. These findings suggest the existence of endogenous protective mechanisms that counteract the increase in late Na+ current that occurs with loss of calmodulin binding. We reasoned that FGF13, a known modulator of late Na+ current that is endogenously expressed in cardiomyocytes but not HEK cells, might play a protective role in limiting late Na+ current. Finally, we coexpressed the IQ/AA mutant NaV1.5 channel in HEK293 cells with FGF13 and found that FGF13 diminished the late Na⁺ currentcompared to cells without FGF13, suggesting that endogenous FHFs may serve to prevent late Na⁺ current in mouse cardiomyocytes. The third chapter of this thesis focuses on the use of proximity labeling and multiplexed quantitative proteomics to define changes in the NaV1.5 macromolecular complex in Duchenne muscular dystrophy (DMD), in which the absence of dystrophin predisposes affected individuals to arrhythmias and cardiac dysfunction.. Standard methods to characterize macromolecular complexes have relied on candidate immunoprecipitation or immunocytochemistry techniques that fall short of providing a comprehensive view of the numbers and types of interactors, as well as the potential dynamic nature of the interactions that may be perturbed by disease states. To provide an inclusive understanding of NaV1.5 macromolecular complexes, we utilize live-cell APEX2 proximity labeling in cardiomyocytes. We identify several proximal changes that align with the electrophysiological NaV1.5 phenotype of young dystrophin-deficient mice, including a decrease in Ptpn3 and Gdp1l and an increase in proteasomal machinery. Whole-cell protein expression fold-change results were used to reveal the altered global expression profile and to place context behind NaV1.5-proximal changes. Finally, we leveraged the neighborhood- specificity of proteins at the lateral membrane, intercalated disc, and transverse tubules of cardiomyocytes to demonstrate that NaV1.5 channels can traffic to all three membrane compartments even in the absence of dystrophin. Thus, the approach of proximity labeling in cardiomyocytes from an animal model of human disease offers new insights into molecular mechanisms of NaV1.5 dysfunction in DMD and provides a template for similar investigations in other cardiac diseases.