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.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/ymre-jc92 |
| Date | January 2024 |
| Creators | del Rivero Morfin, Pedro Javier |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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