This thesis represents the culmination of the main project I have undertaken during my master's program. It is important to note that additional data collection and analysis were conducted by intern students under my supervision, which will be integrated into a forthcoming manuscript where I will be credited as the first co-author. Due to space and focus limitations of this thesis, these additional findings have not been included here. / La famille des canaux potassiques à rectification entrante (Kir), exprimée de manière ubiquitaire, repolarise et maintient le gradient de tension à travers les membranes des cellules excitables et non-excitables. Les canaux Kir sont fortement régulés par divers lipides membranaires, tels que les phosphoinositides, les phospholipides anioniques secondaires, le cholestérol, le Coenzyme A (CoA) à longue chaîne et l'acide arachidonique. Kir2.1 est fortement exprimé dans le tissu musculaire strié des cellules cardiaques auriculaires et ventriculaires. Il joue un rôle essentiel dans la régulation du potentiel de membrane au repos et de la contraction des cellules musculaires cardiaques et lisses en générant le courant K+ à rectification entrante (IK1). (IK1). Les mutations de Kir2.1 avec perte de fonction sont à l'origine du syndrome d'Andersen-Tawil (ATS). Par conséquent, l'altération de la fonction de Kir2.1 est un déterminant essentiel au bon fonctionnement du cœur. Les endocannabinoïdes sont une classe spéciale de lipides naturellement exprimés dans une variété de cellules, y compris les cellules cardiaques, neuronales et immunitaires. Le système endocannabinoïde, y compris les récepteurs cannabinoïdes (CBR), agit comme un système de réponse au stress qui s'active. Des études menées chez l'animal et chez l'homme suggèrent que la modulation pharmacologique de ce système pourrait représenter une nouvelle approche thérapeutique. Cependant, ces dernières années, il est devenu clair que si les endocannabinoïdes peuvent déclencher des changements de signalisation en aval par l'intermédiaire des CBR, ils peuvent également interagir directement avec les canaux ioniques indépendamment des CBR pour moduler la fonction cellulaire. Dans cette étude, nous avons utilisé la technique de double électrode en voltage imposé pour examiner les effets d'un panel d'endocannabinoïdes sur la fonction de Kir2.1. Nous avons montré qu'un sous-ensemble d'endocannabinoïdes, mais pas tous, peut réguler la fonction de Kir2.1 à des degrés divers, indépendamment des CBR. Nous avons également démontré que les endocannabinoïdes peuvent également réguler les protéines mutées menant à l'ATS (G144S et V302M). Nous avons également observé que l'effet des endocannabinoïdes n'est pas conservé parmi les membres de la famille Kir, avec des différences observées entre les canaux Kir2.1, Kir4.1 et Kir7.1. Ces résultats pourraient avoir des implications plus larges pour les fonctions des cellules cardiaques, neuronales et immunitaires. Mots clés : Kir2.1, Endocannabinoïdes, LQT7, Rectification entrante, G144S, Kir7.1, Kir4.1 / The ubiquitously expressed family of inward rectifier potassium (Kir) channels repolarizes and maintains the voltage gradient across excitable and non-excitable cell membranes. Kir channels are highly regulated by various membrane lipids, such as phosphoinositides, secondary anionic phospholipids, cholesterol, long chain acyl- Coenzyme A (CoA), and arachidonic acid. Kir2.1 is highly expressed in striated muscle tissue of atrial and ventricular heart cells. It is critically involved in regulating the resting membrane potential and contraction of cardiac and smooth muscle cells through the generation of the current IK1. Loss-of-function mutations in Kir2.1 cause Andersen-Tawil syndrome (ATS). Therefore, altered Kir2.1 function is a critical determinant of proper heart function. Endocannabinoids are a special class of lipids that are naturally expressed in a variety of cells, including cardiac, neuronal, and immune cells. The endocannabinoid system, including cannabinoid receptors (CBRs), acts as a stress response system that is activated. Studies in both animals and humans suggest that pharmacological modulation of this system might represent a novel approach to treatment. However, in recent years, it is becoming clear that while endocannabinoids can trigger downstream signaling changes through CBRs, they can also directly interact with ion channels independently of CBRs to modulate cellular function. In this study, we used the electrophysiology technique called two-electrode-voltage-clamp (TEVC) in combination with mutagenesis studies to examine the effects of a panel of endocannabinoids on the function of Kir2.1. We showed that a subset of endocannabinoids, but not all, can regulate the Kir2.1 function to varying degrees, independent of CBRs. We also demonstrated that endocannabinoids can also regulate mutants linked with ATS (G144S and V302M). We also observed that the effect of endocannabinoids is not conserved among Kir family members, with differences observed between Kir2.1, Kir4.1 and Kir7.1 channels. These findings could have broader implications for cardiac, neuronal, and immune cell functions. Key words: Kir2.1, Endocannabinoids, LQT7, Inward rectification, G144S, Kir7.1, Kir4.1

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The process of respiration is a pattern of spontaneity and automatic motor control that originate in the brainstem. The mechanism by which the brainstem detects CO2 is termed central CO2 chemoreception (CCR). Since the early 1960’s there have been tremendous efforts placed on identification of central CO2 chemoreceptors (molecules that detect CO2). Even with these efforts, what a central CO2 chemoreceptor looks like remain unknown. To test the hypothesis that inward rectifier K+ (Kir) channels are CO2 sensing molecules in CCR, a series of experiments were carried out. 1) The first question asked was whether the Kir4.1-Kir5.1 channel is expressed in brainstem chemosensitive nuclei. Immunocytochemistry was performed on transverse medullary and pontine sections using antibodies raised against Kir4.1 and Kir5.1. Positive immunoassays for both Kir4.1 and Kir5.1 subunits were found in CO2 chemosensitive neurons. In the LC the Kir4.1 and Kir5.1 were co-expressed with the neurokinin-1 receptor that is the natural receptor for substance P. 2) The second question asked was whether the Kir4.1-Kir5.1 channel is subject to modulation by neurotransmitters critical for respiratory control. My studies demonstrated that indeed the Kir4.1-Kir5.1 channel is subject to modulation by substance P, serotonin and thyrotropin releasing hormone. 3) I performed studies to demonstrate the intracellular signaling system underlying the Kir4.1-Kir5.1 channel modulation by these neurotransmitters. The modulation by all three neurotransmitters was dependent upon the activation of protein kinase C (PKC). The Kir4.1-Kir5.1 but not the Kir4.1 channel was modulated by PKC. Both the Kir4.1 and Kir5.1 subunits can be phosphorylated by PKC in vitro. However, systematic mutational analysis failed to reveal the phosphorylation site. 4) The fourth question asked was whether Kir channels share a common pH gating mechanism that can be identified. Experiments were performed to understand the gating of the Kir6.2+SUR1 channel as specific sites for ligand binding and gating have been demonstrated. I identified a functional gate that was shared by multiple ligands that is Phe168 in the Kir6.2. Other Kir channels appear to share a similar gating mechanism. Taken together, these studies demonstrate the modulation of Kir channels in central CO2 chemoreception.