Abstract 17301: Functional Characterization of KCNK3 Mutants Associated With Pulmonary Arterial Hypertension Under Physiologically Relevant Heterozygous Conditions

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Michael S Bohnen ◽  
Danilo Roman-Campos ◽  
Cecile Terrenoire ◽  
Jack Jnani ◽  
Lei Chen ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Ph Dependence ◽  
Functional Characterization ◽  
Resting Membrane Potential ◽  
Channel Activity ◽  
Loss Of Function ◽  
Pulmonary Arterial ◽  
Specific Impact

KCNK3 encodes a two-pore domain K+ channel, TASK-1, which is inhibited by extracellular acidity and hypoxia. Expressed in a variety of tissues, including human pulmonary artery smooth muscle cells (hPASMCs), the central nervous system, pancreas, and adrenal glands, TASK-1 contributes to the resting membrane potential of cells in which it is expressed. Recently, our group reported mutations in KCNK3 underlying idiopathic pulmonary arterial hypertension (PAH), resulting from loss of TASK-1 function, partially pharmacologically rescuable with ONO-RS-082. TASK-1 dimerizes in vivo, forming functional channels with another TASK-1 subunit or with the related TASK-3 channel. TASK-1 and TASK-3 often are expressed in the same cells, although it has been reported that TASK-1 alone is expressed in the lung. Our initial study examined mutant and wildtype (WT) homodimeric TASK-1 channels expressed heterologously in COS-7 cells. Here we further characterize PAH-linked TASK-1 mutations in physiologically relevant heterozygous conditions in COS-7 and hPASMC cell lines. We engineered heterodimeric channels consisting of one mutant and one WT subunit; compared this with co-expression of mutant and WT channels; and measured channel activity with whole cell patch clamp procedures. We found a mutation specific impact of heterozygosity on channel activity. One mutation, V221L, produces a shift in pH dependence accounting for loss of function at physiological pH 7.4, partially rescued by dimerization with a WT subunit, while another, G203D, produces near complete loss of function as a homo- or hetero-dimer. The presence of TASK-3 results in greater rescue of V221L TASK-1 activity at pH 7.4 than does WT TASK-1. Additionally, under current clamp we found that ONO-RS-082 hyperpolarizes the membrane potential in hPASMCs expressing WT or V221L TASK-1, reversible by selective block of TASK-1 with ML365. Together, our results suggest (a) TASK-1 mutant heterodimers exhibit loss of function with mutation specific severity; (b) TASK-3 may rescue mutant TASK-1 and underlie a tissue specific impact of the TASK-1 mutations observed clinically; and (c) PAH TASK-1 mutants can be pharmacologically modulated in hPASMCs and alter the critically important resting membrane potential.

Functional characterization ofBMPR2gene in pulmonary arterial hypertension

2015 ◽  
Author(s):  
Guillermo Pousada ◽  
Vincenzo Lupo ◽  
María Álvarez-Satta ◽  
Adolfo Baloira ◽  
Carmen Espinós ◽  
...  
Keyword(s):  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Functional Characterization ◽  
Pulmonary Arterial

scholarly journals Loss-of-function thrombospondin-1 mutations in familial pulmonary hypertension

2012 ◽  
Vol 302 (6) ◽  
pp. L541-L554 ◽  
Author(s):  
James P. Maloney ◽  
Robert S. Stearman ◽  
Todd M. Bull ◽  
David W. Calabrese ◽  
Megan L. Tripp-Addison ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Pulmonary Arterial Hypertension ◽  
Cell Growth ◽  
Arterial Hypertension ◽  
Rna Splicing ◽  
Type I ◽  
Loss Of Function ◽  
Intronic Mutation ◽  
Thrombospondin 1 ◽  
Pulmonary Arterial

Most patients with familial pulmonary arterial hypertension (FPAH) carry mutations in the bone morphogenic protein receptor 2 gene ( BMPR2). Yet carriers have only a 20% risk of disease, suggesting that other factors influence penetrance. Thrombospondin-1 (TSP1) regulates activation of TGF-β and inhibits endothelial and smooth muscle cell proliferation, pathways coincidentally altered in pulmonary arterial hypertension (PAH). To determine whether a subset of FPAH patients also have mutations in the TSP1 gene ( THBS1) we resequenced the type I repeats of THBS1 encoding the TGF-β regulation and cell growth inhibition domains in 60 FPAH probands, 70 nonfamilial PAH subjects, and in large control groups. We identified THBS1 mutations in three families: a novel missense mutation in two (Asp362Asn), and an intronic mutation in a third (IVS8+255 G/A). Neither mutation was detected in population controls. Mutant 362Asn TSP1 had less than half of the ability of wild-type TSP1 to activate TGF-β. Mutant 362Asn TSP1 also lost the ability to inhibit growth of pulmonary arterial smooth muscle cells and was over threefold less effective at inhibiting endothelial cell growth. The IVS8+255 G/A mutation decreased and/or eliminated local binding of the transcription factors SP1 and MAZ but did not affect RNA splicing. These novel mutations implicate THBS1 as a modifier gene in FPAH. These THBS1 mutations have implications in the genetic evaluation of FPAH patients. However, since FPAH is rare, these data are most relevant as evidence for the importance of TSP1 in pulmonary vascular homeostasis. Further examination of THBS1 in the pathogenesis of PAH is warranted.

scholarly journals Molecular and functional characterization of the BMPR2 gene in Pulmonary Arterial Hypertension

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Guillermo Pousada ◽  
Vincenzo Lupo ◽  
Sheila Cástro-Sánchez ◽  
María Álvarez-Satta ◽  
Ana Sánchez-Monteagudo ◽  
...  
Keyword(s):  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Functional Characterization ◽  
Pulmonary Arterial

scholarly journals Implication of Potassium Channels in the Pathophysiology of Pulmonary Arterial Hypertension

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1261
Author(s):  
Hélène Le Ribeuz ◽  
Véronique Capuano ◽  
Barbara Girerd ◽  
Marc Humbert ◽  
David Montani ◽  
...  
Keyword(s):  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Therapeutic Potential ◽  
Pulmonary Vasculature ◽  
K Channel ◽  
Loss Of Function ◽  
Epigenetic Factors ◽  
Regulatory Subunits ◽  
Pulmonary Arterial ◽  
Or Task

Pulmonary arterial hypertension (PAH) is a rare and severe cardiopulmonary disease without curative treatments. PAH is a multifactorial disease that involves genetic predisposition, epigenetic factors, and environmental factors (drugs, toxins, viruses, hypoxia, and inflammation), which contribute to the initiation or development of irreversible remodeling of the pulmonary vessels. The recent identification of loss-of-function mutations in KCNK3 (KCNK3 or TASK-1) and ABCC8 (SUR1), or gain-of-function mutations in ABCC9 (SUR2), as well as polymorphisms in KCNA5 (Kv1.5), which encode two potassium (K+) channels and two K+ channel regulatory subunits, has revived the interest of ion channels in PAH. This review focuses on KCNK3, SUR1, SUR2, and Kv1.5 channels in pulmonary vasculature and discusses their pathophysiological contribution to and therapeutic potential in PAH.

scholarly journals SOX17 Loss-of-Function Mutation Underlying Familial Pulmonary Arterial Hypertension

2021 ◽  
Author(s):  
Tian-Ming Wang ◽  
Shan-Shan Wang ◽  
Ying-Jia Xu ◽  
Cui-Mei Zhao ◽  
Xiao-Hui Qiao ◽  
...  
Keyword(s):  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Loss Of Function ◽  
Pulmonary Arterial

Abstract 15092: The Yin-yang of Bmpr2 and Ces1 in the Pulmonary Endothelium Aad Its Role in Pulmonary Arterial Hypertension

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Stuti Agarwal ◽  
Ananya Chakraborty ◽  
David Condon ◽  
Salvador Tello ◽  
Karthik Suresh ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Redox Balance ◽  
Tube Formation ◽  
Vascular Lesions ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Pulmonary Endothelium ◽  
Pulmonary Arterial

Background/Hypothesis: Pulmonary Arterial Hypertension (PAH) is a life-threatening disease characterized by loss of pulmonary microvessels and vascular remodeling. Loss of function BMPR2 mutations contribute to pulmonary endothelial cell (EC) apoptosis and oxidative stress, but their reduced penetrance suggests need for additional modifiers. Carboxylesterase1 (CES1) is endoplasmic reticulum (ER) enzyme responsible for detoxification, proteostasis, and redox balance. Similar to BMPR2 mutations, we found that loss of CES1 is associated with oxidative stress and apoptosis. In this study, we explore a plausible link between BMPR2 pathway and CES1 regulation in promoting EC survival and angiogenesis. Methods: PECs & lung tissue from healthy donors and PAH patients were obtained from PHBI. To induce oxidative stress, we used H 2 O 2 (100 μM) & methamphetamine HCl (METH, 0.5-2mM). Both siRNA and pharmacological approaches were used to inhibit BMPR2, Nrf2, and CES1 expression. Caspase and Matrigel assays were used to assess PEC survival and tube formation, respectively. Results: RNAseq of BMPR2-mutant PECs showed significantly less CES1 expression, which correlated with reduced protein expression in PEC lysates and within lung vascular lesions. In healthy PECs, BMP9 stimulation led to increase in CES1 expression that was absent post BMPR2 knockdown. CES1 gene transcription was by BMPR2-dependent activation of Nrf2, a transcription factor responsible for antioxidant gene expression and mitochondrial biogenesis. Inhibition of Nrf2 activation by ML385 (5μM) abrogates BMP9 induced CES1 mRNA levels similar to BMPR2 knockdown. The connection between BMPR2 and CES1 was further strengthened by CES1 knockdown studies in PECs that demonstrated reduction in BMPR2 protein synthesis associated with ER stress and reduced autophagy. Finally, lung examination in CRISPR generated CES1 +/- mice demonstrated increased microvascular muscularization at normoxia compared to wild type mice. Conclusion: BMPR2 and CES1 are part of common signaling pathway that protects PECs against oxidative stress and mitochondrial damage through a positive feedback loop. Interventions that restore CES1 activity could rescue BMPR2 signaling and serve as novel PAH therapeutics.

scholarly journals Structural consequences of BMPR2 kinase domain mutations causing pulmonary arterial hypertension

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Apirat Chaikuad ◽  
Chancievan Thangaratnarajah ◽  
Frank von Delft ◽  
Alex N. Bullock
Keyword(s):  
Protein Structure ◽  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Transforming Growth Factor ◽  
Catalytic Domain ◽  
Clinical Investigation ◽  
Kinase Domain ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Pulmonary Arterial

AbstractBone morphogenetic proteins (BMPs) are secreted ligands of the transforming growth factor-β (TGF-β) family that control embryonic patterning, as well as tissue development and homeostasis. Loss of function mutations in the type II BMP receptor BMPR2 are the leading cause of pulmonary arterial hypertension (PAH), a rare disease of vascular occlusion that leads to high blood pressure in the pulmonary arteries. To understand the structural consequences of these mutations, we determined the crystal structure of the human wild-type BMPR2 kinase domain at 2.35 Å resolution. The structure revealed an active conformation of the catalytic domain that formed canonical interactions with the bound ligand Mg-ADP. Disease-associated missense mutations were mapped throughout the protein structure, but clustered predominantly in the larger kinase C-lobe. Modelling revealed that the mutations will destabilize the protein structure by varying extents consistent with their previously reported functional heterogeneity. The most severe mutations introduced steric clashes in the hydrophobic protein core, whereas those found on the protein surface were less destabilizing and potentially most favorable for therapeutic rescue strategies currently under clinical investigation.

scholarly journals Stimulants and Pulmonary Arterial Hypertension: An Update

2018 ◽  
Vol 17 (2) ◽  
pp. 49-54
Author(s):  
Ramon L. Ramirez ◽  
Vinicio De Jesus Perez ◽  
Roham T. Zamanian
Keyword(s):  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Science Research ◽  
New Drugs ◽  
Loss Of Function ◽  
Ongoing Research ◽  
Appetite Suppressants ◽  
Genomic Studies ◽  
Pulmonary Arterial ◽  
The 1960S

The connection between stimulants and pulmonary arterial hypertension (PAH) was first made apparent in the 1960s during an outbreak associated with anorexigen (amphetamine-like appetite suppressants) use. Since then, a total of 16 drugs and toxins have been linked to PAH (ie, drug and toxin-associated PAH [DT-APAH]), including illicit stimulants like methamphetamine. Recently, basic science research and novel genomic studies have started to shed light on possible pathologic and genetic mechanisms implicated in disease development, namely loss of function variants in genes involved in drug detoxification. This review will discuss the history and current state of knowledge regarding stimulants and their association with PAH. It will also discuss clinical management of patients with DT-APAH. Lastly, it will highlight the importance of ongoing research efforts to identify susceptibility factors implicated in DT-APAH and the need for increased pharmacovigilance and awareness to identify new drugs that may be risk factors for PAH. Ultimately, this may be our best strategy to improve clinical outcomes and prevent deadly future outbreaks of DT-APAH.

Sign in / Sign up

Export Citation Format

Share Document