Biophysical effects of pore mutations of ROMK1

2001 ◽  
Vol 101 (2) ◽  
pp. 121-130 ◽  
Author(s):  
Sunil BHANDARI ◽  
Malcolm HUNTER
Keyword(s):  
Expression System ◽  
Structural Elements ◽  
Loss Of Function ◽  
Wild Type ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Functional Features ◽  
Living Organisms ◽  
Pore Lining ◽  
Channel Properties

Potassium channels are ubiquitous, being present in all living organisms. These proteins share common structural elements, which confer common functional features. In general, all K+ channels have a high selectivity for K+, and are blocked by cations of similar dimensions, such as Cs+ and Ba2+. Mutations in the pore region tend to lead to either the total loss of function or K+ selectivity. We have made mutations to one of the most highly conserved residues of the pore, glycine-143, of the inward rectifier ROMK1 (Kir1.1), and examined the resulting channel properties in the Xenopus oocyte expression system with a two-electrode voltage clamp. Mutations G143A and G143R resulted in failure to express functional channels. Co-injection of wild-type ROMK1 cRNA with these mutants led to rescue of channel function, which was different from wild-type ROMK1. In both mutants, the sensitivity to Ba2+ and Cs+ was increased, the rate of onset of block by Ba2+ was enhanced, and the selectivity to potassium was reduced. Whereas the crystallographic evidence shows that cations bind to the carbonyl backbone of the pore-lining residues, the present results indicate that the side chains of these amino acids, which face away from the pore lining, also affect permeation.

scholarly journals Specific and Nonspecific Effects of Protein Kinase C on the Epithelial Na + Channel

2000 ◽  
Vol 115 (5) ◽  
pp. 559-570 ◽  
Author(s):  
Mouhamed S. Awayda
Keyword(s):  
Membrane Trafficking ◽  
Expression System ◽  
Na Channel ◽  
Specific Effects ◽  
Nonspecific Effects ◽  
Oocyte Expression ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Baseline Values ◽  
Epithelial Na Channel

The Xenopus oocyte expression system was used to explore the mechanisms of inhibition of the cloned rat epithelial Na+ channel (rENaC) by PKC (Awayda, M.S., I.I. Ismailov, B.K. Berdiev, C.M. Fuller, and D.J. Benos. 1996. J. Gen. Physiol. 108:49–65) and to determine whether human ENaC exhibits similar regulation. Effects of PKC activation on membrane and/or channel trafficking were determined using impedance analysis as an indirect measure of membrane area. hENaC-expressing oocytes exhibited an appreciable activation by hyperpolarizing voltages. This activation could be fit with a single exponential, described by a time constant (τ) and a magnitude (ΔI V). A similar but smaller magnitude of activation was also observed in oocytes expressing rENaC. This activation likely corresponds to the previously described effect of hyperpolarizing voltage on gating of the native Na+ channel (Palmer, L.G., and G. Frindt. 1996. J. Gen. Physiol. 107:35–45). Stimulation of PKC with 100 nM PMA decreased ΔIV in hENaC-expressing oocytes to a plateau at 57.1 ± 4.9% (n = 6) of baseline values at 20 min. Similar effects were observed in rENaC-expressing oocytes. PMA decreased the amiloride-sensitive hENaC slope conductance (gNa) to 21.7 ± 7.2% (n = 6) of baseline values at 30 min. This decrease was similar to that previously reported for rENaC. This decrease of g Na was attributed to a decrease of membrane capacitance (C m), as well as the specific conductance (gm/Cm ). The effects on gm/Cm reached a plateau within 15 min, at ∼60% of baseline values. This decrease is likely due to the specific ability of PKC to inhibit ENaC. On the other hand, the decrease of Cm was unrelated to ENaC and is likely an effect of PKC on membrane trafficking, as it was observed in ENaC-expressing as well as control oocytes. At lower PMA concentrations (0.5 nM), smaller changes of Cm were observed in rENaC- and hENaC-expressing oocytes, and were preceded by larger changes of gm and by changes of gm/Cm, indicating specific effects on ENaC. These findings indicate that PKC exhibits multiple and specific effects on ENaC, as well as nonspecific effects on membrane trafficking. Moreover, these findings provide the electrophysiological basis for assessing channel-specific effects of PKC in the Xenopus oocyte expression system.

scholarly journals Investigating the utility of adult zebrafish ex vivo whole hearts to pharmacologically screen hERG channel activator compounds

2019 ◽  
Vol 317 (6) ◽  
pp. R921-R931
Author(s):  
Christina M. Hull ◽  
Christine E. Genge ◽  
Yuki Hobbs ◽  
Kaveh Rayani ◽  
Eric Lin ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Expression System ◽  
Cost Effective ◽  
Channel Blockers ◽  
Loss Of Function ◽  
Adult Zebrafish ◽  
Pharmacological Characterization ◽  
Potential Gain ◽  
Xenopus Oocyte Expression ◽  
Whole Heart

There is significant interest in the potential utility of small-molecule activator compounds to mitigate cardiac arrhythmia caused by loss of function of hERG1a voltage-gated potassium channels. Zebrafish ( Danio rerio) have been proposed as a cost-effective, high-throughput drug-screening model to identify compounds that cause hERG1a dysfunction. However, there are no reports on the effects of hERG1a activator compounds in zebrafish and consequently on the utility of the model to screen for potential gain-of-function therapeutics. Here, we examined the effects of hERG1a blocker and types 1 and 2 activator compounds on isolated zkcnh6a (zERG3) channels in the Xenopus oocyte expression system as well as action potentials recorded from ex vivo adult zebrafish whole hearts using optical mapping. Our functional data from isolated zkcnh6a channels show that under the conditions tested, these channels are blocked by hERG1a channel blockers (dofetilide and terfenadine), and activated by type 1 (RPR260243) and type 2 (NS1643, PD-118057) hERG1a activators with higher affinity than hKCNH2a channels (except NS1643), with differences accounted for by different biophysical properties in the two channels. In ex vivo zebrafish whole hearts, two of the three hERG1a activators examined caused abbreviation of the action potential duration (APD), whereas hERG1a blockers caused APD prolongation. These data represent, to our knowledge, the first pharmacological characterization of isolated zkcnh6a channels and the first assessment of hERG enhancing therapeutics in zebrafish. Our findings lead us to suggest that the zebrafish ex vivo whole heart model serves as a valuable tool in the screening of hKCNH2a blocker and activator compounds.

Bisphosphonates stimulate an endogenous nonselective cation channel in Xenopus oocytes: potential mechanism of action

2005 ◽  
Vol 289 (2) ◽  
pp. C248-C256 ◽  
Author(s):  
Weijian Shao ◽  
Roy C. Orlando ◽  
Mouhamed S. Awayda
Keyword(s):  
Tyrosine Phosphatase ◽  
Expression System ◽  
Mechanisms Of Action ◽  
Gastrointestinal Complications ◽  
Cation Conductance ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Small Inhibition ◽  
Kinase Pathway ◽  
Stimulation Of

The mechanisms of action of bisphosphonates (BPs) have been poorly determined. Besides their actions on osteoclasts, these agents exhibit gastrointestinal complications. They have also recently been described as affecting various preparations that express an epithelial Na+ channel (ENaC). To understand the effects of BP on ion channels and the ENaC in particular, we used the Xenopus oocyte expression system. Alendronate, and similarly risedronate, two aminobisphosphonates, caused a large stimulation of an endogenous nonselective cation conductance (NSCC). This stimulation averaged 63 ± 12 μS ( n = 18) 60 min after the addition of 2 mM alendronate. The effects on the endogenous NSCC were blocked by extracellular acidification to pH 6.4. On the other hand, alendronate caused a small inhibition of ENaC conductance at pH 7.4 and 6.4, but the effects at pH 6.4 were more readily observed in the absence of changes of the endogenous conductance. The effects on membrane capacitance were also markedly different, with a clear decrease at pH 6.4 and no consistent changes at pH 7.4. The effects on the endogenous channel were further augmented by genistein and were inhibited by a tyrosine phosphatase inhibitor, indicating the involvement of the tyrosine kinase pathway. Stimulation of NSCC with BP is expected to cause membrane depolarization and may explain, in part, its mechanisms of action in inhibiting osteoclasts.

A naturally occurring human Nedd4–2 variant displays impaired ENaC regulation in Xenopus laevis oocytes

2004 ◽  
Vol 287 (3) ◽  
pp. F550-F561 ◽  
Author(s):  
Fatemeh Fouladkou ◽  
Rasoul Alikhani-Koopaei ◽  
Bruno Vogt ◽  
Sandra Y. Flores ◽  
Laurence Malbert-Colas ◽  
...  
Keyword(s):  
Expression System ◽  
Donor Site ◽  
Susceptibility Gene ◽  
Xenopus Laevis Oocytes ◽  
Wild Type ◽  
Nonsynonymous Mutation ◽  
Single Strand Conformational Polymorphism ◽  
Phosphorylation Level ◽  
Xenopus Oocyte Expression

The epithelial Na+ channel (ENaC) is regulated by the ubiquitin-protein ligase Nedd4–2 via interaction with ENaC PY-motifs. These PY-motifs are mutated/deleted in Liddle's syndrome, resulting in elevated Na+ reabsorption and hypertension explained partly by impaired ENaC-Nedd4–2 interaction. We hypothesized that Nedd4–2 is a susceptibility gene for hypertension and screened 856 renal patients and healthy controls for mutations in a subset of exons of the human Nedd4–2 gene that are relevant for ENaC regulation by PCR/single-strand conformational polymorphism. Several variants were identified, and one nonsynonymous mutation (Nedd4–2-P355L) was further characterized. This mutation next to the 3′ donor site of exon 15 does not affect in vitro splicing of Nedd4–2 mRNA. However, in the Xenopus oocyte expression system, Nedd4–2-P355L-dependent ENaC inhibition was weaker compared with the wild type (Nedd4–2-WT), and this difference depended on the presence of intact PY-motifs on ENaC. This could not be explained by the amount of wild type or mutant Nedd4–2 coimmunoprecipitating with ENaC. When the phosphorylation level of human Nedd4–2 Ser448 (known to be phosphorylated by the Sgk1 kinase) was determined with a specific anti-pSer448 antibody, we observed stronger basal phosphorylation of Nedd4–2-P355L. Both the phosphorylation level and the accompanying amiloride-sensitive Na+ currents could be further enhanced to approximately the same levels by coexpressing Sgk1. In addition, the role of the two other putative Sgk1 phosphorylation sites (S342 and T367) appears also to be affected by the P355L mutation. The differential phosphorylation status between wild-type and mutant Nedd4–2 provides an explanation for the different potential to inhibit ENaC activity.

scholarly journals Characterization of mouse amino acid transporter B0AT1 (slc6a19)

2005 ◽  
Vol 389 (3) ◽  
pp. 745-751 ◽  
Author(s):  
Christoph Böhmer ◽  
Angelika Bröer ◽  
Michael Munzinger ◽  
Sonja Kowalczuk ◽  
John E. J. Rasko ◽  
...  
Keyword(s):  
Amino Acids ◽  
Membrane Potential ◽  
Amino Acid ◽  
Expression System ◽  
Complex Function ◽  
Aromatic Amino Acids ◽  
Inward Currents ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System

The mechanism of the mouse (m)B0AT1 (slc6a19) transporter was studied in detail using two electrode voltage-clamp techniques and tracer studies in the Xenopus oocyte expression system. All neutral amino acids induced inward currents at physiological potentials, but large neutral non-aromatic amino acids were the preferred substrates of mB0AT1. Substrates were transported with K0.5 values ranging from approx. 1 mM to approx. 10 mM. The transporter mediates Na+–amino acid co-transport with a stoichiometry of 1:1. No other ions were involved in the transport mechanism. An increase in the extracellular Na+ concentration reduced the K0.5 for leucine, and vice versa. Moreover, the K0.5 values and Vmax values of both substrates varied with the membrane potential. As a result, K0.5 and Vmax values are a complex function of the concentration of substrate and co-substrate and the membrane potential. A model is presented assuming random binding order and a positive charge associated with the ternary [Na+–substrate–transporter] complex, which is consistent with the experimental data.

scholarly journals Novel Mutations in the TMPRSS3 Gene May Contribute to Taiwanese Patients with Nonsyndromic Hearing Loss

2020 ◽  
Vol 21 (7) ◽  
pp. 2382
Author(s):  
Swee-Hee Wong ◽  
Yung-Chang Yen ◽  
Shuan-Yow Li ◽  
Jiann-Jou Yang
Keyword(s):  
Hearing Loss ◽  
Serine Protease ◽  
Xenopus Oocyte ◽  
Expression System ◽  
Missense Mutations ◽  
Nonsyndromic Hearing Loss ◽  
Oocyte Expression ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Transmembrane Serine Protease

A previous study indicated that mutations in the transmembrane protease serine 3 (TMPRSS3) gene, which encodes a transmembrane serine protease, cause nonsyndromic hearing loss (NSHL). This was the first description of a serine protease involved in hearing loss (HL). In Taiwan, however, data on the TMPRSS3 gene’s association with NSHL is still insufficient. In this study, we described 10 mutations of TMPRSS3 genes found in 14 patients after screening 230 children with NSHL. The prevalence of the TMPRSS3 mutation appeared to be 6.09% (14/230). Of the 10 mutations, three were missense mutations: c.239G>A (p.R80H), c.551T>C (p.L184S), and 1253C>T (p.A418V); three were silent mutations, and four were mutations in introns. To determine the functional importance of TMPRSS3 mutations, we constructed plasmids carrying TMPRSS3 mutations of p.R80H, p.L184S, and p.A418V. TMPRSS3 function can be examined by secretory genetic assay for site-specific proteolysis (sGASP) and Xenopus oocyte expression system. Our results showed that p.R80H, p.L184S, and p.A418V TMPRSS3 mutations gave ratios of 19.4%, 13.2%, and 27.6%, respectively, via the sGASP system. Moreover, these three TMPRSS3 mutations failed to activate the epithelial sodium channel (ENaC) in the Xenopus oocyte expression system. These results indicate that the p.R80H, p.L184S, and p.A418V missense mutations of TMPRSS3 resulted in greatly diminishing the proteolytic activity of TMPRSS3. Our study provides information for understanding the importance of TMPRSS3 in the NSHL of Taiwanese children and provides a novel molecular explanation for the role of TMPRSS3 in HL.

scholarly journals Signal transduction of rodent opioid μ, δ, κ-receptor in Xenopus oocyte expression system.

1994 ◽  
Vol 64 ◽  
pp. 129
Author(s):  
Takeaki Miyamae ◽  
Nobuyuki Fukushima ◽  
Yoshimi Misu ◽  
Hiroshi Takeshima ◽  
Kazuhiko Fukuda ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Xenopus Oocyte ◽  
Expression System ◽  
Oocyte Expression ◽  
Xenopus Oocyte Expression ◽  
Oocyte Expression System ◽  
Κ Receptor

scholarly journals Gain-of-function variant of the human epithelial sodium channel

2013 ◽  
Vol 304 (2) ◽  
pp. F207-F213 ◽  
Author(s):  
Jingxin Chen ◽  
Thomas R. Kleyman ◽  
Shaohu Sheng
Keyword(s):  
Single Channel ◽  
Expression System ◽  
Surface Expression ◽  
Wild Type ◽  
Open Probability ◽  
Gain Of Function ◽  
Human Disorders ◽  
Human Genome Sequencing ◽  
Xenopus Oocyte Expression ◽  
Salt Sensitive Hypertension

Epithelial Na+ channel (ENaC) mutations are associated with several human disorders, underscoring the importance of these channels in human health. Recent human genome sequencing projects have revealed a large number of ENaC gene variations, several of which have been found in individuals with salt-sensitive hypertension, cystic fibrosis, and other disorders. However, the functional consequences of most variants are unknown. In this study, we used the Xenopus oocyte expression system to examine the functional properties of a human ENaC variant. Oocytes expressing αβγL511Q human ENaCs showed 4.6-fold greater amiloride-sensitive currents than cells expressing wild-type channels. The γL511Q variant did not significantly alter channel surface expression. Single channel recordings revealed that the variant had fourfold higher open probability than wild type. In addition, γL511Q largely eliminated the Na+ self-inhibition response, which reflects a downregulation of ENaC open probability by extracellular Na+. Moreover, γL511Q diminished chymotrypsin-induced activation of the mutant channel. We conclude that γL511Q is a gain-of-function human ENaC variant. Our results suggest that γL511Q enhances ENaC activity by increasing channel open probability and dampens channel regulation by extracellular Na+ and proteases.

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