CELLULAR MECHANISMS INVOLVED IN VOLTAGE-GATED POTASSIUM CHANNEL REGULATION: FOCUS ON SIGMA-1R DRIVEN PATHWAYS

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Understanding the cellular and molecular mechanisms modulating potassium voltage-gated channel (Kv) activity and cell surface expression is of the utmost importance. Encoding 40 a-subunits divided in twelve subfamilies, the Kv channels represent the most diverse family of ion channels expressed in the nervous system. This diversity allows Kv channels to uniquely influence several aspects of electrical information processing. These channels are functionnal when bound to auxiliary subunits, which modulate the Kv plasma membrane (PM) targeting, intracellular trafficking, conductance, and gating (open or closed state). Past studies have identified SIGMA-1R (S1R), an endoplasmic reticulum (ER) chaperone, as an atypical auxiliary subunit for several Kv a-subunits. However, the molecular mechanisms involved in S1R regulation of Kv channels is still poorly understood. Therefore, the long term objective of my Discovery grant program will focus on identifying the pathways and key components engaged in S1R-mediated Kv channel trafficking in cells and to define the S1R-Kv complexes' role in neuronal excitability, a fundamental mechanism for proper brain functions. For the next five years, my team will focus on studying the role of S1R in Kv1.2 channel trafficking. Using homologous expression system or S1R-depleted cells combined with ELISA-cell based assays, confocal microscopy and GST-Pull down analyses, the following specific objectives will be investigated: Axis 1:  Impact of S1R on Kv1.2 channel stability: role in autophagy and the ubiquitin-proteasome mechanisms. My most recent work exposed that Kv.12 basal expression is reduced when co-expressed with S1R. Given that S1R is involved in ERAD, UPR, and autophagy, we will investigate if S1R enhances Kv1.2 degradation along the proteasomal and autophagy pathways. Axis 2: S1R regulation of Kv1.2 channel Cell Surface Expression. S1R has been shown to have ligand-dependent and independent effects on several Kv channels. However, it remains unclear how these distinctive drug-inducible or drug-independent events are orchestrated within cells and how it is guiding S1R-mediated channels trafficking. Hence, I plan to monitor Kv1.2 PM expression and trafficking upon S1R activation or inactivation. Axis 3: Characterization of S1R-Kv1.2 channel interaction: ligand-regulated association and mapping of the binding domain.  Herein, I suggest examining S1R association with Kv1.2 in an agonist and antagonist-specific manner using overexpression assays in immortalized cell lines and in vitro GST-Pull down. Axis 4: Develop induced Neurons (iNS) as a new working model. We will develop a human primary neuronal model to study S1R regulation of Kv1.2 channel trafficking. Significance and expected contributions to NSE research: This research program will provide answers to major pending questions related to S1R fundamental biology and S1R-Kv channels trafficking that enable neurons to tightly regulate their excitability.