Hypertension resistance polymorphisms in ROMK (Kir1.1) alter channel function by different mechanisms

The renal outer medullary K+ (ROMK) channel plays a critical role in renal sodium handling. Recent genome sequencing efforts in the Framingham Heart Study offspring cohort (Ji W, Foo JN, O'Roak BJ, Zhao H, Larson MG, Simon DB, Newton-Cheh C, State MW, Levy D, and Lifton RP. Nat Genet 40: 592–599, 2008) recently revealed an association between suspected loss-of-function polymorphisms in the ROMK channel and resistance to hypertension, suggesting that ROMK activity may also be a determinant of blood pressure control in the general population. Here we examine whether these sequence variants do, in fact, alter ROMK channel function and explore the mechanisms. As assessed by two-microelectrode voltage clamp in Xenopus oocytes, 3/5 of the variants (R193P, H251Y, and T313FS) displayed an almost complete attenuation of whole cell ROMK channel activity. Surface antibody binding measurements of external epitope-tagged channels and analysis of glycosylation-state maturation revealed that these variants prevent channel expression at the plasmalemma, likely as a consequence of retention in the endoplasmic reticulum. The other variants (P166S, R169H) had no obvious effects on the basal channel activity or surface expression but, instead, conferred a gain in regulated-inhibitory gating. As assessed in giant excised patch-clamp studies, apparent phosphotidylinositol 4,5-bisphosphate (PIP2) binding affinity of the variants was reduced, causing channels to be more susceptible to inhibition upon PIP2 depletion. Unlike the protein product of the major ROMK allele, these two variants are sensitive to the inhibitory affects of a G protein-coupled receptor, which stimulates PIP2 hydrolysis. In summary, we have found that hypertension resistance sequence variants inhibit ROMK channel function by different mechanisms, providing new insights into the role of the channel in the maintenance of blood pressure.

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Human epithelial Na+ channel missense variants identified in the GenSalt study alter channel activity

AJP Renal Physiology ◽

10.1152/ajprenal.00426.2016 ◽

2016 ◽

Vol 311 (5) ◽

pp. F908-F914 ◽

Cited By ~ 7

Author(s):

Evan C. Ray ◽

Jingxin Chen ◽

Tanika N. Kelly ◽

Jiang He ◽

L. Lee Hamm ◽

...

Keyword(s):

Blood Pressure ◽

Cell Surface ◽

Surface Expression ◽

Cell Surface Expression ◽

Channel Activity ◽

Dietary Salt ◽

Α Subunit ◽

Γ Subunit ◽

Genes Encoding ◽

Altered Sensitivity

Mutations in genes encoding subunits of the epithelial Na+ channel (ENaC) can cause early onset familial hypertension, demonstrating the importance of this channel in modulating blood pressure. It remains unclear whether other genetic variants resulting in subtler alterations of channel function result in hypertension or altered sensitivity of blood pressure to dietary salt. This study sought to identify functional human ENaC variants to examine how these variants alter channel activity and to explore whether these variants are associated with altered sensitivity of blood pressure to dietary salt. Six-hundred participants of the Genetic Epidemiology Network of Salt Sensitivity (GenSalt) study with salt-sensitive or salt-resistant blood pressure underwent sequencing of the genes encoding ENaC subunits. Functional effects of identified variants were examined in a Xenopus oocyte expression system. Variants that increased channel activity included three in the gene encoding the α-subunit (αS115N, αR476W, and αV481M), one in the β-subunit (βS635N), and one in the γ-subunit (γL438Q). One α-subunit variant (αA334T) and one γ-subunit variant (βD31N) decreased channel activity. Several α-subunit extracellular domain variants altered channel inhibition by extracellular Na+ (Na+ self-inhibition). One variant (αA334T) decreased and one (αV481M) increased cell surface expression. Association between these variants and salt sensitivity did not reach statistical significance. This study identifies novel functional human ENaC variants and demonstrates that some variants alter channel cell surface expression and/or Na+ self-inhibition.

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Functional roles of aspartate residues of the proton-coupled folate transporter (PCFT-SLC46A1); a D156Y mutation causing hereditary folate malabsorption

Blood ◽

10.1182/blood-2010-06-291237 ◽

2010 ◽

Vol 116 (24) ◽

pp. 5162-5169 ◽

Cited By ~ 39

Author(s):

Daniel Sanghoon Shin ◽

Sang Hee Min ◽

Laura Russell ◽

Rongbao Zhao ◽

Andras Fiser ◽

...

Keyword(s):

Small Intestine ◽

Critical Role ◽

Surface Expression ◽

Loss Of Function ◽

Wild Type ◽

Intracellular Loop ◽

Folate Transport ◽

Functional Roles ◽

Proximal Small Intestine ◽

Pakistani Origin

Abstract The proton-coupled folate transporter (PCFT; SLC46A1) mediates folate transport into enterocytes in the proximal small intestine; pcft loss-of-function mutations are the basis for hereditary folate malabsorption. The current study explored the roles of Asp residues in PCFT function. A novel, homozygous, loss-of-function mutation, D156Y, was identified in a child of Pakistani origin with hereditary folate malabsorption. Of the 6 other conserved Asp residues, only one, D109, is shown to be required for function. D156Y, along with a variety of other substitutions at this site (Trp, Phe, Val, Asn, or Lys), lacked function due to instability of the PCFT protein. Substantial function was preserved with Glu, Gly, and, to a lesser extent, with Ser, Thr, and Ala substitutions. This correlated with PCFT bio-tinylated at the cell surface. In contrast, all D109 mutants, including D109E, lacked function irrespective of pH (4.5, 5.5, and 7.4) or substrate concentration (0.5-100μM), despite surface expression comparable to wild-type PCFT. Hence, D156 plays a critical role in PCFT protein stability, and D109, located in the first intracellular loop between the second and third transmembrane domains, is absolutely required for PCFT function.

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Enhancement of hERG channel activity by scFv antibody fragments targeted to the PAS domain

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1601116113 ◽

2016 ◽

Vol 113 (35) ◽

pp. 9916-9921 ◽

Cited By ~ 6

Author(s):

Carol A. Harley ◽

Greg Starek ◽

David K. Jones ◽

Andreia S. Fernandes ◽

Gail A. Robertson ◽

...

Keyword(s):

Action Potential ◽

Critical Role ◽

Herg Channel ◽

Pas Domain ◽

Channel Activity ◽

Single Chain ◽

Orphan Receptors ◽

Human Cardiomyocytes ◽

Channel Function ◽

Single Chain Variable Fragments

The human human ether-à-go-go–related gene (hERG) potassium channel plays a critical role in the repolarization of the cardiac action potential. Changes in hERG channel function underlie long QT syndrome (LQTS) and are associated with cardiac arrhythmias and sudden death. A striking feature of this channel and KCNH channels in general is the presence of an N-terminal Per-Arnt-Sim (PAS) domain. In other proteins, PAS domains bind ligands and modulate effector domains. However, the PAS domains of KCNH channels are orphan receptors. We have uncovered a family of positive modulators of hERG that specifically bind to the PAS domain. We generated two single-chain variable fragments (scFvs) that recognize different epitopes on the PAS domain. Both antibodies increase the rate of deactivation but have different effects on channel activation and inactivation. Importantly, we show that both antibodies, on binding to the PAS domain, increase the total amount of current that permeates the channel during a ventricular action potential and significantly reduce the action potential duration recorded in human cardiomyocytes. Overall, these molecules constitute a previously unidentified class of positive modulators and establish that allosteric modulation of hERG channel function through ligand binding to the PAS domain can be attained.

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Loss of secretin results in systemic and pulmonary hypertension with cardiopulmonary pathologies in mice

Scientific Reports ◽

10.1038/s41598-019-50634-x ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Aung Moe Zaw ◽

Revathi Sekar ◽

Sarah O. K. Mak ◽

Helen K. W. Law ◽

Billy K. C. Chow

Keyword(s):

Blood Pressure ◽

Pulmonary Hypertension ◽

Medical Condition ◽

Pulmonary Inflammation ◽

Critical Role ◽

Pressure Control ◽

Pathological Development ◽

Underlying Mechanisms ◽

And Control

Abstract More than 1 billion people globally are suffering from hypertension, which is a long-term incurable medical condition that can further lead to dangerous complications and death if left untreated. In earlier studies, the brain-gut peptide secretin (SCT) was found to be able to control blood pressure by its cardiovascular and pulmonary effects. For example, serum SCT in patients with congestive heart failure was one-third of the normal level. These observations strongly suggest that SCT has a causal role in blood pressure control, and in this report, we used constitutive SCT knockout (SCT−/−) mice and control C57BL/6N mice to investigate differences in the morphology, function, underlying mechanisms and response to SCT treatment. We found that SCT−/− mice suffer from systemic and pulmonary hypertension with increased fibrosis in the lungs and heart. Small airway remodelling and pulmonary inflammation were also found in SCT−/− mice. Serum NO and VEGF levels were reduced and plasma aldosterone levels were increased in SCT−/− mice. Elevated cardiac aldosterone and decreased VEGF in the lungs were observed in the SCT−/− mice. More interestingly, SCT replacement in SCT−/− mice could prevent the development of heart and lung pathologies compared to the untreated group. Taken together, we comprehensively demonstrated the critical role of SCT in the cardiovascular and pulmonary systems and provide new insight into the potential role of SCT in the pathological development of cardiopulmonary and cardiovascular diseases.

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Deletion of α-subunit exon 11 of the epithelial Na+ channel reveals a regulatory module

AJP Renal Physiology ◽

10.1152/ajprenal.00587.2013 ◽

2014 ◽

Vol 306 (5) ◽

pp. F561-F567 ◽

Cited By ~ 7

Author(s):

Jingxin Chen ◽

Thomas R. Kleyman ◽

Shaohu Sheng

Keyword(s):

Surface Expression ◽

Significant Loss ◽

Na Channel ◽

Channel Activity ◽

Wild Type ◽

Open Probability ◽

Α Subunit ◽

Regulatory Module ◽

Channel Expression ◽

Exon 11

Epithelial Na+ channel (ENaC) subunits (α, β, and γ) found in functional complexes are translated from mature mRNAs that are similarly processed by the inclusion of 13 canonical exons. We examined whether individual exons 3–12, encoding the large extracellular domain, are required for functional channel expression. Human ENaCs with an in-frame deletion of a single α-subunit exon were expressed in Xenopus oocytes, and their functional properties were examined by two-electrode voltage clamp. With the exception of exon 11, deletion of an individual exon eliminated channel activity. Channels lacking α-subunit exon 11 were hyperactive. Oocytes expressing this mutant exhibited fourfold greater amiloride-sensitive whole cell currents than cells expressing wild-type channels. A parallel fivefold increase in channel open probability was observed with channels lacking α-subunit exon 11. These mutant channels also exhibited a lost of Na+ self-inhibition, whereas we found similar levels of surface expression of mutant and wild-type channels. In contrast, in-frame deletions of exon 11 from either the β- or γ-subunit led to a significant loss of channel activity, in association with a marked decrease in surface expression. Our results suggest that exon 11 within the three human ENaC genes encodes structurally homologous yet functionally diverse domains and that exon 11 in the α-subunit encodes a module that regulates channel gating.

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