Faculty Bibliography

KATP channels are integral to the functions of many cells and tissues. The use of electrophysiological methods has allowed for a detailed characterization of KATP channels in terms of their biophysical properties, nucleotide sensitivities, and modification by pharmacological compounds. However, even though they were first described almost 25 years ago (Noma 1983, Trube and Hescheler 1984), the physiological and pathophysiological roles of these channels, and their regulation by complex biological systems, are only now emerging for many tissues. Even in tissues where their roles have been best defined, there are still many unanswered questions. This review aims to summarize the properties, molecular composition, and pharmacology of KATP channels in various cardiovascular components (atria, specialized conduction system, ventricles, smooth muscle, endothelium, and mitochondria). We will summarize the lessons learned from available genetic mouse models and address the known roles of KATP channels in cardiovascular pathologies and how genetic variation in KATP channel genes contribute to human disease.

With advanced aging, there is a decline in innate cardiovascular function. This decline is not general in nature. Instead, specific changes occur that impact the basic cardiovascular function, which include alterations in biochemical pathways and ion channel function. This review focuses on a particular ion channel that couple the latter two processes, namely the KATP channel, which opening is promoted by alterations in intracellular energy metabolism. We show that the intrinsic properties of the KATP channel changes with advanced aging and argue that the channel can be further modulated by biochemical changes. The importance is widespread, given the ubiquitous nature of the KATP channel in the cardiovascular system where it can regulate processes as diverse as cardiac function, blood flow and protection mechanisms against superimposed stress, such as cardiac ischemia. We highlight questions that remain to be answered before the KATP channel can be considered as a viable target for therapeutic intervention.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive cancer that frequently presents with disseminated disease. The PDA metastatic microenvironment imposes distinct metabolic stressors, potentially generating context-dependent vulnerabilities. Therefore, we employed CRISPR-based genetic screening in a model of PDA liver metastasis to identify novel and possibly targetable liabilities. Remarkably, ferritin heavy chain (FTH1) emerged as the most prominent liver-specific dependency - loss of FTH1 suppressed tumor growth specifically in the liver microenvironment. FTH1 deletion and subsequent disruption of iron handling triggers mitochondrial dysfunction and ionic imbalance, including cytosolic calcium overload. These perturbations result in the activation of a transcriptional program that triggers anti-tumor immunity mediated by immunostimulatory cytokine IL36G. Mechanistically, FTH1 deletion and subsequent ionic imbalance causes decreased protein levels of the tumor suppressor Stk11 (LKB1) which we propose to be mediated by an RNA G-quadruplex located in the 5'-UTR of LKB1. The loss of LKB1 protein levels alters signaling cascades resulting in reduced SIK signaling and inhibition of nonsense mediated decay, ultimately leading to Il36g mRNA stabilization. Taken together, this work elucidates novel ionic disruptions that regulate the translation of LKB1 through a previously undescribed quadruplex in the 5'UTR, altering signaling axes that can be targeted to generate an anti-tumor immune response in PDA.

Ameloblasts are specialized epithelial cells that form enamel during the secretory and maturation stages, the latter involving an increase in Ca²⁺ transport to mineralize the enamel crystals. During enamel formation, ameloblasts travel several microns while secreting a matrix and are surrounded by several cell layers in the confined space of the enamel organ. Presumably, ameloblasts are subjected to mechanical stimuli e.g. pressure, stretch. Mechanosensitive (MS) or stretch-gated channels are expressed in the membranes of many cells including mineralizing cells. The opening of MS channels occurs in response to physical stimuli and results in the influx of ions. Piezo1 is a non-selective class of MS channel permeable to Ca²⁺ and hence it may contribute to Ca²⁺ homeostasis in ameloblasts. Here we show that secretory and maturation stage ameloblasts express similar protein levels of Piezo1. Cultured rat primary secretory and maturation stage ameloblasts showed stretch-activated currents by patch-clamp. Ameloblasts loaded with the cytosolic Ca²⁺ indicator Fura-2 were also stimulated with the Piezo1-selective activator Yoda1. We show that ameloblasts are sensitive to Piezo1 stimulation which evoked an increase in cytosolic Ca²⁺. This effect was inhibited by Piezo1 blockers. Mechanical analysis of the incisors of Piezo1 cKO mice showed no alterations in hardness or elastic modulus relative to littermate control mice. Our work provides the first evidence that Piezo1 channels are functional in both ameloblast stages and their activation leads to an elevation in cytosolic Ca²⁺, however, Piezo1 does not appear to be essential for enamel mineralization.

Ion channels are the second most common clinical drug target besides G protein-coupled receptors. Aneurysmal diseases pose a significant threat to human life. Novel drug targets for its treatment remain to be explored. We investigated the role of an ion channel, calcium homeostasis modulators 5 (CALHM5), on the development of aortic aneurysms. We characterized CALHM5 as a plasma membrane ion channel abundant in smooth muscle cells of both humans and mice, playing a pivotal role in regulating calcium homeostasis. Notably, CALHM5 deficiency suppressed the transcription of the L-type calcium channel (LTCC) pore-forming subunit by downregulating cAMP-response element binding proteins. This in turn diminished blood vessel contractility and decreased blood flow. Intriguingly, CALHM5 expression is downregulated in smooth muscle tissues of aortic aneurysm patients. Furthermore, CALHM5 deficiency was observed to ameliorate the development of abdominal aortic aneurysms in mice, partly by stimulating smooth muscle cell proliferation. CALHM5 emerges as an ion channel prominently expressed in arterial smooth muscles, serving as a physiological regulator of smooth muscle contraction and presenting itself as a promising therapeutic target for aortic aneurysms.

Ion channels, exchangers and pumps are expressed ubiquitously in cells from all phyla of life. In mammals, their role is best described in excitable cells, where they regulate the initiation and propagation of action potentials. There are over 70 different types of K⁺ channels subunits that contribute to these processes. In non-excitable cells, K⁺ channels set the resting membrane potential, which in turn drives the activity of other translocators. K⁺ channels also help maintain cell volume, influence cell proliferation and apoptosis and regulate Ca²⁺ signaling, which in turn is crucial for many cellular processes, including metabolism, secretion, and gene expression. K⁺ channels play crucial roles in the activation, proliferation and a variety of other functions in cells of the innate and adaptive immune system. The ATP-sensitive K⁺ (K_ATP) channel has an established role in diverse cells, but its presence and function in immunity is scantly described. Public gene expression databases show that K_ATP channel subunits are highly expressed in NKT and NK cells, and that they are significantly upregulated after infection in CD8+ T cells and macrophages. We discuss these findings in the light of the available literature and propose a role for K_ATP channels in cytotoxicity of cells that are primed for a rapid immune response. Possible underlying molecular mechanisms are discussed.

INTRODUCTION/UNASSIGNED:Involved in immunity and reproduction, natural killer (NK) cells offer opportunities to develop new immunotherapies to treat infections and cancer or to alleviate pregnancy complications. Most current strategies use cytokines or antibodies to enhance NK-cell function, but none use ion channel modulators, which are widely used in clinical practice to treat hypertension, diabetes, epilepsy, and other conditions. Little is known about ion channels in NK cells. RESULTS/UNASSIGNED:NK cells in the bone barrow and spleen. DISCUSSION/UNASSIGNED:subunit Kir6.1 has a key role in NK-cell development.

Sirtuins are NAD⁺-dependent deacetylases with beneficial roles in conditions relevant to human health, including metabolic disease, type II diabetes, obesity, cancer, aging, neurodegenerative diseases, and cardiac ischemia. Since ATP-sensitive K⁺ (K_ATP) channels have cardioprotective roles, we investigated whether they are regulated by sirtuins. Nicotinamide mononucleotide (NMN) was used to increase cytosolic NAD⁺ levels and to activate sirtuins in cell lines, isolated rat and mouse cardiomyocytes or insulin-secreting INS-1 cells. K_ATP channels were studied with patch clamping, biochemistry techniques, and antibody uptake experiments. NMN led to an increase in intracellular NAD⁺ levels and an increase in the K_ATP channel current, without significant changes in the unitary current amplitude or open probability. An increased surface expression was confirmed using surface biotinylation approaches. The rate of K_ATP channel internalization was diminished by NMN, which may be a partial explanation for the increased surface expression. We show that NMN acts via sirtuins since the increased K_ATP channel surface expression was prevented by blockers of SIRT1 and SIRT2 (Ex527 and AGK2) and mimicked by SIRT1 activation (SRT1720). The pathophysiological relevance of this finding was studied using a cardioprotection assay with isolated ventricular myocytes, in which NMN protected against simulated ischemia or hypoxia in a K_ATP channel-dependent manner. Overall, our data draw a link between intracellular NAD⁺, sirtuin activation, K_ATP channel surface expression, and cardiac protection against ischemic damage.

ATP-sensitive K⁺ (K_ATP) channel couples membrane excitability to intracellular energy metabolism. Maintaining K_ATP channel surface expression is key to normal insulin secretion, blood pressure and cardioprotection. However, the molecular mechanisms regulating K_ATP channel internalization and endocytic recycling, which directly affect the surface expression of K_ATP channels, are poorly understood. Here we used the cardiac K_ATP channel subtype, Kir6.2/SUR2A, and characterized Rab35 GTPase as a key regulator of K_ATP channel endocytic recycling. Electrophysiological recordings and surface biotinylation assays showed decreased K_ATP channel surface density with co-expression of a dominant negative Rab35 mutant (Rab35-DN), but not other recycling-related Rab GTPases, including Rab4, Rab11a and Rab11b. Immunofluorescence images revealed strong colocalization of Rab35-DN with recycling Kir6.2. Rab35-DN minimized the recycling rate of K_ATP channels. Rab35 also regulated K_ATP channel current amplitude in isolated adult cardiomyocytes by affecting its surface expression but not channel properties, which validated its physiologic relevance and the potential of pharmacologic target for treating the diseases with K_ATP channel trafficking defects.