TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac.
Cantú syndrome (CS), first described in 1982, is caused by pathogenic variants in ABCC9 and KCNJ8, which encode the regulatory and pore forming subunits of ATP‐sensitive potassium (KATP) channels, respectively. Multiple case reports of affected individuals have described the various clinical features of CS, but systematic studies are lacking. To define the effects of genetic variants on CS phenotypes and clinical outcomes, we have developed a standardized REDCap‐based registry for CS. We report phenotypic features and associated genotypes on 74 CS subjects, with confirmed ABCC9 variants in 72 of the individuals. Hypertrichosis and a characteristic facial appearance are present in all individuals. Polyhydramnios during fetal life, hyperflexibility, edema, patent ductus arteriosus (PDA), cardiomegaly, dilated aortic root, vascular tortuosity of cerebral arteries, and migraine headaches are common features, although even with this large group of subjects, there is incomplete penetrance of CS‐associated features, without clear correlation to genotype.
SummaryThe mechanosensitive two-pore domain (K2P) K+ channels (TREK-1, TREK-2, and TRAAK) are important for mechanical and thermal nociception. However, the mechanisms underlying their gating by membrane stretch remain controversial. Here we use molecular dynamics simulations to examine their behavior in a lipid bilayer. We show that TREK-2 moves from the “down” to “up” conformation in direct response to membrane stretch, and examine the role of the transmembrane pressure profile in this process. Furthermore, we show how state-dependent interactions with lipids affect the movement of TREK-2, and how stretch influences both the inner pore and selectivity filter. Finally, we present functional studies that demonstrate why direct pore block by lipid tails does not represent the principal mechanism of mechanogating. Overall, this study provides a dynamic structural insight into K2P channel mechanosensitivity and illustrates how the structure of a eukaryotic mechanosensitive ion channel responds to changes in forces within the bilayer.
Cantu syndrome (CS) is characterized by multiple vascular and cardiac abnormalities including vascular dilation and tortuosity, systemic hypotension, and cardiomegaly. The disorder is caused by gain-of-function (GOF) mutations in genes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) ATP-sensitive potassium (KATP) channel subunits. However, there is little understanding of the link between molecular dysfunction and the complex pathophysiology observed, and there is no known treatment, in large part due to the lack of appropriate preclinical disease models in which to test therapies. Notably, expression of Kir6.1 and SUR2 does not fully overlap, and the relative contribution of KATP GOF in various cardiovascular tissues remains to be elucidated. To investigate pathophysiologic mechanisms in CS we have used CRISPR/Cas9 engineering to introduce CS-associated SUR2[A478V] and Kir6.1[V65M] mutations to the equivalent endogenous loci in mice. Mirroring human CS, both of these animals exhibit low systemic blood pressure and dilated, compliant blood vessels, as well dramatic cardiac enlargement, the effects being more severe in V65M animals than in A478V animals. In both animals, whole-cell patch-clamp recordings reveal enhanced basal KATP conductance in vascular smooth muscle, explaining vasodilation and lower blood pressure, and demonstrating a cardinal role for smooth muscle KATP dysfunction in CS etiology. Echocardiography confirms in situ cardiac enlargement and increased cardiac output in both animals. Patch-clamp recordings reveal reduced ATP sensitivity of ventricular myocyte KATP channels in A478V, but normal ATP sensitivity in V65M, suggesting that cardiac remodeling occurs secondary to KATP overactivity outside of the heart. These SUR2[A478V] and Kir6.1[V65M] animals thus reiterate the key cardiovascular features seen in human CS. They establish the molecular basis of the pathophysiological consequences of reduced smooth muscle excitability resulting from SUR2/Kir6.1-dependent KATP GOF, and provide a validated animal model in which to examine potential therapeutic approaches to treating CS.
Background: In pulmonary arterial hypertension (PAH), pathological changes in pulmonary arterioles progressively raise pulmonary artery pressure and increase pulmonary vascular resistance, leading to right heart failure and high mortality rates. Recently, the first potassium channelopathy in PAH, due to mutations in KCNK3, was identified as a genetic cause and pharmacological target. Methods: Exome sequencing was performed to identify novel genes in a cohort of 99 pediatric and 134 adult onset group I pulmonary arterial hypertension patients. Novel rare variants in the gene identified were independently identified in a cohort of 680 adult onset patients. Variants were expressed in COS cells and function assessed by patch-clamp and rubidium flux analysis. Results: We identified a de novo novel heterozygous predicted deleterious missense variant c.G2873A (p.R958H) in ABCC8 (ATP-binding cassette, subfamily C, member 8) in a child with idiopathic PAH. We then evaluated all individuals in the original and a second cohort for rare or novel variants in ABCC8 and identified 11 additional heterozygous predicted damaging ABCC8 variants. ABCC8 encodes sulfonylurea receptor 1 (SUR1), a regulatory subunit of the ATP-sensitive potassium channel (KATP). We observed loss of KATP function for all ABCC8 variants evaluated, and pharmacological rescue of all channel currents in vitro by the SUR1 activator, diazoxide. Conclusions: Novel and rare missense variants in ABCC8 are associated with pulmonary arterial hypertension. Identified ABCC8 mutations decreased KATP channel function, which was pharmacologically recovered.
TREK channels, which are gated open by a wide range of stimuli, exist in at least two conformations known as “up” and “down.” McClenaghan et al. show that the channel can be open in both of these conformations and that gating is primarily achieved by the channel’s selectivity filter.
Coronavirus (CoV) outbreaks have recently emerged as a global public health threat due to their exceptional zoonotic potential — a feature arising from their ability to infect a diverse range of potential hosts combined with their high capacity for mutation and recombination. After Severe Acute Respiratory Syndrome (SARS) CoV-1 in 2003 and Middle East Respiratory Syndrome (MERS) CoV in 2012, with the current SARS-CoV-2 pandemic we are now in the midst of the third deadly international CoV outbreak in less than 20 years. Coronavirus outbreaks present a critical threat to global public health and an urgent necessity for therapeutic options. Here, we critically examine the current evidence for ion channel activity in CoV proteins and the potential for modulation as a therapeutic approach.
sensitivity of specific CS mutations, require further study, and ideal therapy for CS may ultimately require an agent with much improved selectivity or potency for VSM Kir6.1/SUR2B channels. However, there is immediate need for a targeted therapy for CS, and the present findings clearly demonstrate the in vivo potential of glibenclamide for correcting CS cardiovascular abnormalities. Moreover, they suggest that the undesired glucoselowering effects in nondiabetic animals are temporary, and may not therefore be prohibitive for the use of glibenclamide as a therapy in CS. Methods Mouse models. CRISPR/Cas9 genome-edited SUR2 wt/AV and Kir6.1 wt/VM Cantu mice were previously reported (19) (see also Supplemental Methods; supplemental material available online with this article;
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