Regulation of CaV3 channels by NEDD4 Family of Ubiquitin Ligases

Voltage-gated calcium channels (VGCCs) are membrane proteins expressed in electrically excitable cells where they respond to changes in membrane potential to allow for calcium influx. The sub-family of low voltage-activated (LVA; also known as CaV3) calcium channels comprises three distinct genes that encode the pore-forming alpha-1 subunit of the channel. LVA calcium channels open at near-resting potentials, and are physiologically relevant in neuronal firing, cardiac pacemaking, hormone secretion, smooth muscles contraction, and fertilization. To elicit their physiological functions, LVA calcium channels undergo dynamic synthesis, processing, sorting, trafficking, and degradation. The covalent attachment of ubiquitin to proteins is an important regulatory mechanism which has been shown to affect different aspects of protein lifecycle. Ubiquitination occurs via a series of enzymatic reactions in which E3 ligases catalyze the final step involving the covalent transfer of ubiquitin to target substrates. The NEDD4 family of E3 ligases is a well-characterized family of nine E3 ligases each having a catalytic HECT domain for the transfer of ubiquitin to target substrates. Members of this family of E3 ligases have been shown to regulate different ion channels, including LVA channels. However, our understanding of the regulation of LVA channels by NEDD4 family of ubiquitin ligases is incomplete because we do not know the complete list of ligases that regulate the channels, and the effect of ligase regulation on the channels. For this current proposal, we aim to build on exciting preliminary data from HEK293T cells that show that members of the NEDD4 family can differentially modulate the trafficking, stability, and biophysical properties of LVA channels. Specifically, we seek to 1) identify members of the NEDD4 family of E3 ligases that regulated LVA channels , and 2) investigate the molecular determinants governing the ability of distinct NEDD4 ligases to modulate LVA channels . We will use a combination of molecular engineering, patch-clamp electrophysiology, flow-cytometry, and biochemical techniques to definitively answer our questions. (AHA Program: Predoctoral Fellowship)