KATP Channels in the Cardiovascular System
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.
Cardiovascular KATP channels and advanced aging
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.
The cardioprotective role of sirtuins is mediated in part by regulating KATP channel surface expression
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+ (KATP) 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. KATP 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 KATP 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 KATP 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 KATP 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 KATP channel-dependent manner. Overall, our data draw a link between intracellular NAD+, sirtuin activation, KATP channel surface expression, and cardiac protection against ischemic damage.
CL-705G: a novel chemical Kir6.2-specific KATP channel opener
Background: KATP channels have diverse roles, including regulation of insulin secretion and blood flow, and protection against biological stress responses and are excellent therapeutic targets. Different subclasses of KATP channels exist in various tissue types due to the unique assemblies of specific pore-forming (Kir6.x) and accessory (SURx) subunits. The majority of pharmacological openers and blockers act by binding to SURx and are poorly selective against the various KATP channel subclasses. Methods and Results: We used 3D models of the Kir6.2/SUR homotetramers based on existing cryo-EM structures of channels in both the open and closed states to identify a potential agonist binding pocket in a functionally critical area of the channel. Computational docking screens of this pocket with the Chembridge Core chemical library of 492,000 drug-like compounds yielded 15 top-ranked "hits", which were tested for activity against KATP channels using patch clamping and thallium (Tl+) flux assays with a Kir6.2/SUR2A HEK-293 stable cell line. Several of the compounds increased Tl+ fluxes. One of them (CL-705G) opened Kir6.2/SUR2A channels with a similar potency as pinacidil (EC50 of 9 µM and 11 Î¼M, respectively). Remarkably, compound CL-705G had no or minimal effects on other Kir channels, including Kir6.1/SUR2B, Kir2.1, or Kir3.1/Kir3.4 channels, or Na+ currents of TE671 medulloblastoma cells. CL-705G activated Kir6.2Î”36 in the presence of SUR2A, but not when expressed by itself. CL-705G activated Kir6.2/SUR2A channels even after PIP2 depletion. The compound has cardioprotective effects in a cellular model of pharmacological preconditioning. It also partially rescued activity of the gating-defective Kir6.2-R301C mutant that is associated with congenital hyperinsulinism. Conclusion: CL-705G is a new Kir6.2 opener with little cross-reactivity with other channels tested, including the structurally similar Kir6.1. This, to our knowledge, is the first Kir-specific channel opener.
CL-705G: a novel chemical Kir6.2-specific KATP channel opener
Rab35 GTPase positively regulates endocytic recycling of cardiac KATP channels
ATP-sensitive K+ (KATP) channel couples membrane excitability to intracellular energy metabolism. Maintaining KATP channel surface expression is key to normal insulin secretion, blood pressure and cardioprotection. However, the molecular mechanisms regulating KATP channel internalization and endocytic recycling, which directly affect the surface expression of KATP channels, are poorly understood. Here we used the cardiac KATP channel subtype, Kir6.2/SUR2A, and characterized Rab35 GTPase as a key regulator of KATP channel endocytic recycling. Electrophysiological recordings and surface biotinylation assays showed decreased KATP 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 KATP channels. Rab35 also regulated KATP 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 KATP channel trafficking defects.
KATP channel trafficking
Sarcolemmal/plasmalemmal ATP-sensitive K+ (KATP) channels have key roles in many cell types and tissues. Hundreds of studies have described how the KATP channel activity and ATP sensitivity can be regulated by changes in the cellular metabolic state, by receptor signaling pathways and by pharmacological interventions. These alterations in channel activity directly translate to alterations in cell or tissue function, that can range from modulating secretory responses, such as insulin release from pancreatic Î²-cells or neurotransmitters from neurons, to modulating contractile behavior of smooth muscle or cardiac cells to elicit alterations in blood flow or cardiac contractility. It is increasingly becoming apparent, however, that KATP channels are regulated beyond changes in their activity. Recent studies have highlighted that KATP channel surface expression is a tightly regulated process with similar implications in health and disease. The surface expression of KATP channels is finely balanced by several trafficking steps including synthesis, assembly, anterograde trafï¬cking, membrane anchoring, endocytosis, endocytic recycling and degradation. This review aims to summarize the physiological and pathophysiological implications of KATP channel trafficking and mechanisms that regulate KATP channel trafficking. A better understanding of this topic has potential to identify new approaches to develop therapeutically useful drugs to treat KATP channel-related diseases.
The volume-regulated anion channel LRRC8C suppresses T cell function by regulating cyclic dinucleotide transport and STING-p53 signaling
Targeting Piezo1 unleashes innate immunity against cancer and infectious disease
Piezo1 is a mechanosensitive ion channel that has gained recognition for its role in regulating diverse physiological processes. However, the influence of Piezo1 in inflammatory disease, including infection and tumor immunity, is not well studied. We postulated that Piezo1 links physical forces to immune regulation in myeloid cells. We found signal transduction via Piezo1 in myeloid cells and established this channel as the primary sensor of mechanical stress in these cells. Global inhibition of Piezo1 with a peptide inhibitor was protective against both cancer and septic shock and resulted in a diminution in suppressive myeloid cells. Moreover, deletion of Piezo1 in myeloid cells protected against cancer and increased survival in polymicrobial sepsis. Mechanistically, we show that mechanical stimulation promotes Piezo1-dependent myeloid cell expansion by suppressing the retinoblastoma gene Rb1 We further show that Piezo1-mediated silencing of Rb1 is regulated via up-regulation of histone deacetylase 2. Collectively, our work uncovers Piezo1 as a targetable immune checkpoint that drives immunosuppressive myelopoiesis in cancer and infectious disease.
Palmitoylation of the KATP channel Kir6.2 subunit promotes channel opening by regulating PIP2 sensitivity
A physiological role for long-chain acyl-CoA esters to activate ATP-sensitive K+ (KATP) channels is well established. Circulating palmitate is transported into cells and converted to palmitoyl-CoA, which is a substrate for palmitoylation. We found that palmitoyl-CoA, but not palmitic acid, activated the channel when applied acutely. We have altered the palmitoylation state by preincubating cells with micromolar concentrations of palmitic acid or by inhibiting protein thioesterases. With acyl-biotin exchange assays we found that Kir6.2, but not sulfonylurea receptor (SUR)1 or SUR2, was palmitoylated. These interventions increased the KATP channel mean patch current, increased the open time, and decreased the apparent sensitivity to ATP without affecting surface expression. Similar data were obtained in transfected cells, rat insulin-secreting INS-1 cells, and isolated cardiac myocytes. Kir6.2Î”C36, expressed without SUR, was also positively regulated by palmitoylation. Mutagenesis of Kir6.2 Cys166 prevented these effects. Clinical variants in KCNJ11 that affect Cys166 had a similar gain-of-function phenotype, but was more pronounced. Molecular modeling studies suggested that palmitoyl-C166 and selected large hydrophobic mutations make direct hydrophobic contact with Kir6.2-bound PIP2 Patch-clamp studies confirmed that palmitoylation of Kir6.2 at Cys166 enhanced the PIP2 sensitivity of the channel. Physiological relevance is suggested since palmitoylation blunted the regulation of KATP channels by Î±1-adrenoreceptor stimulation. The Cys166 residue is conserved in some other Kir family members (Kir6.1 and Kir3, but not Kir2), which are also subject to regulated palmitoylation, suggesting a general mechanism to control the open state of certain Kir channels.