Title: The Regulation and Function of RGK Proteins on Voltage-Gated Calcium Channel Physiology
Description: Rad/Rem/Rem2/Gem/Kir (RGK) proteins are Ras-like GTPases with diverse (and expanding) functions including: regulating cytoskeleton dynamics, cell proliferation, synaptogenesis, and inhibition of high voltage-dependent calcium (CaV) channels. Furthermore, they have tissue-specific distribution with Rem and Rad most highly expressed in the heart. Indeed, the importance of Rem and Rad in the cardiovascular system is underscored by a number of studies linking them to disease states including cardiac hypertrophy, cardiac fibrosis, and inflammation. A hallmark feature of RGK proteins is their ability to inhibit current through CaV channels (ICa) and in fact, they are recognized as the most potent endogenous inhibitors of ICa. However, how RGK proteins are regulated and what their physiological role is are unknown. Understanding these points is critical for defining the patho-physiological roles of RGK proteins. My thesis work contributes towards the RGK field on two fronts: First, we demonstrate that RGK proteins are non-canonical G-proteins in the context of their ability to undergo nucleotide regulation and second, we reveal a novel paradigm of RGK-mediated inhibition on CaV channels.

In Chapters 2 and 3, we show that Rem and Rad are are non-canonical G-proteins with respect to the regulatory role of their guanine nucleotide binding pocket (GNBP). Canonical Raslike G-proteins contain a conserved G-domain that encompass a GNBP and is important for guanine nucleotide binding and hydrolysis. Since RGK proteins also possess a G-domain and GNBP as well as demonstrate bona fide nucleotide binding, it was initially thought that they were regulated in a manner similar to other Ras proteins. However, subsequent studies suggested that RGK proteins may not obey such a classical model and as a result, the regulatory role of their GNBP in the G-domain was unclear. By using a wide range of functional measurements (CaV1.2 currents, Ca2+ transients, β-subunit binding), we demonstrate that RGK proteins Rem and Rad are non-canonical G-proteins. Utilizing point mutants that abolish GTPbinding and prevent GTPase activity (RemT94N and RadS105N), we show that only some cellular functions are dependent on an operational nucleotide binding pocket while others are unperturbed.

Specifically, Rem- and Rad-mediated inhibition of ICa is independent of guanine nucleotide regulation whereas protein interactions with the b-subunit of CaV channels (CaVβ) and protein stability are sensitive to nucleotide regulation. We also discover skeletal and cardiac actin to be novel binding partners of Rem. And lastly, we observe differences between the effects of Rem and Rad on their degree of ICa inhibition in cardiac myocytes. Thus, Rem and Rad are non-canonical G-proteins with respect to the regulatory role of their GNBP. In collaboration with a close colleague, Akil Puckerin, Chapter 4 reveals a novel mechanism behind RGK-mediated inhibition of ICa. Together, we show RGK proteins display different modes of inhibition against specific CaV channels and that we can utilize this property to design calcium channel blockers which inhibit CaV channels in an isoform specific manner. We demonstrate this by designing Rem and Rad mutants which have diminished CaVβ capacity, termed Rem-βNULL and Rad-βNULL, respectively.

Characterization of these mutants using wholecell patch clamp experiments revealed that Rem-βNULL inhibits only CaV1.2 whereas Rad-βNULL inhibits only CaV1.2 and CaV2.2. Thus, our results describe the first genetically encoded calcium channel blocker that can selectively distinguish amongst L-type channels. Altogether, this thesis work contributes towards our understanding of RGK protein regulation function and the underlying mechanisms by which they inhibit ICa. These findings advance the field both from a mechanistic and physiological standpoint, and will be of great importance towards investigating the patho-physiological role of RGK proteins.