State‐dependent barium block of wild‐type and inactivation‐deficient HERG channels in
            Xenopus
            oocytes

The antiarrhythmic agent flecainide appears beneficial for painful congenital myotonia and LQT-3/ΔKPQ syndrome. Both diseases manifest small but persistent late Na+ currents in skeletal or cardiac myocytes. Flecainide may therefore block late Na+ currents for its efficacy. To investigate this possibility, we characterized state-dependent block of flecainide in wild-type and inactivation-deficient rNav1.4 muscle Na+ channels (L435W/L437C/A438W) expressed with β1 subunits in Hek293t cells. The flecainide-resting block at −140 mV was weak for wild-type Na+ channels, with an estimated 50% inhibitory concentration (IC50) of 365 μM when the cell was not stimulated for 1,000 s. At 100 μM flecainide, brief monitoring pulses of +30 mV applied at frequencies as low as 1 per 60 s, however, produced an ∼70% use-dependent block of peak Na+ currents. Recovery from this use-dependent block followed an exponential function, with a time constant over 225 s at −140 mV. Inactivated wild-type Na+ channels interacted with flecainide also slowly at −50 mV, with a time constant of 7.9 s. In contrast, flecainide blocked the open state of inactivation-deficient Na+ channels potently as revealed by its rapid time-dependent block of late Na+ currents. The IC50 for flecainide open-channel block at +30 mV was 0.61 μM, right within the therapeutic plasma concentration range; on-rate and off-rate constants were 14.9 μM−1s−1 and 12.2 s−1, respectively. Upon repolarization to −140 mV, flecainide block of inactivation-deficient Na+ channels recovered, with a time constant of 11.2 s, which was ∼20-fold faster than that of wild-type counterparts. We conclude that flecainide directly blocks persistent late Na+ currents with a high affinity. The fast-inactivation gate, probably via its S6 docking site, may further stabilize the flecainide-receptor complex in wild-type Na+ channels.

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Effect of PCMBS on CO2permeability of Xenopus oocytes expressing aquaporin 1 or its C189S mutant

AJP Cell Physiology ◽

10.1152/ajpcell.1998.275.6.c1481 ◽

1998 ◽

Vol 275 (6) ◽

pp. C1481-C1486 ◽

Cited By ~ 135

Author(s):

Gordon J. Cooper ◽

Walter F. Boron

Keyword(s):

Carbonic Anhydrase ◽

Intracellular Ph ◽

Xenopus Oocytes ◽

Membrane Lipid ◽

Wild Type ◽

Aquaporin 1 ◽

Gas Channel ◽

A Cell ◽

Pass Through ◽

Rule Out

A recent study on Xenopus oocytes [N. L. Nakhoul, M. F. Romero, B. A. Davis, and W. F. Boron. Am. J. Physiol. 274 ( Cell Physiol. 43): C543–548, 1998] injected with carbonic anhydrase showed that expressing aquaporin 1 (AQP1) increases by ∼40% the rate at which exposing the cell to CO2 causes intracellular pH to fall. This observation is consistent with several interpretations. Overexpressing AQP1 might increase apparent CO2 permeability by 1) allowing CO2 to pass through AQP1, 2) stimulating injected carbonic anhydrase, 3) enhancing the CO2 solubility of the membrane’s lipid, or 4) increasing the expression of a native “gas channel.” The purpose of the present study was to distinguish among these possibilities. We found that expressing the H2O channel AQP1 in Xenopus oocytes increases the CO2 permeability of oocytes in an expression-dependent fashion, whereas expressing the K+ channel ROMK1 has no effect. The mercury derivative p-chloromercuriphenylsulfonic acid (PCMBS), which inhibits the H2O movement through AQP1, also blocks the AQP1-dependent increase in CO2 permeability. The mercury-insensitive C189S mutant of AQP1 increases the CO2 permeability of the oocyte to the same extent as does the wild-type channel. However, the C189S-dependent increase in CO2permeability is unaffected by treatment with PCMBS. These data rule out options 2–4 listed above. Thus our results suggest that CO2passes through the pore of AQP1 and are the first data to demonstrate that a gas can enter a cell by a means other than diffusing through the membrane lipid.

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Both the wild type and a functional isoform of CFTR are expressed in kidney

AJP Renal Physiology ◽

10.1152/ajprenal.1996.270.6.f1038 ◽

1996 ◽

Vol 270 (6) ◽

pp. F1038-F1048 ◽

Cited By ~ 30

Author(s):

M. M. Morales ◽

T. P. Carroll ◽

T. Morita ◽

E. M. Schwiebert ◽

O. Devuyst ◽

...

Keyword(s):

Cystic Fibrosis ◽

Mammalian Cells ◽

Xenopus Oocytes ◽

Transmembrane Domain ◽

Renal Medulla ◽

Regulatory Domain ◽

Wild Type ◽

Transmembrane Segments ◽

Binding Domains ◽

Cl Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) consists of five domains, two transmembrane-spanning domains, each composed of six transmembrane segments, a regulatory domain, and two nucleotide-binding domains (NBDs). CFTR is expressed in kidney, but its role in overall renal function is not well understood, because mutations in CFTR found in patients with cystic fibrosis are not associated with renal dysfunction. To learn more about the distribution and functional forms of CFTR in kidney, we used a combination of molecular, cell biological, and electrophysiological approaches. These include an evaluation of CFTR mRNA and protein expression, as well as both two-electrode and patch clamping of CFTR expressed either in Xenopus oocytes or mammalian cells. In addition to wild-type CFTR mRNA, an alternate form containing only the first transmembrane domain (TMD), the first NBD, and the regulatory domain (TNR-CFTR) is expressed in kidney. Although missing the second set of TMDs and the second NBD, when expressed in Xenopus oocytes, TNR-CFTR has cAMP-dependent protein kinase A (PKA)-stimulated single Cl- channel characteristics and regulation of PKA activation of outwardly rectifying Cl- channels that are very similar to those of wild-type CFTR. TNR-CFTR mRNA is produced by an unusual mRNA processing mechanism and is expressed in a tissue-specific manner primarily in renal medulla.

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LQT2 nonsense mutations generate trafficking defective NH2-terminally truncated channels by the reinitiation of translation

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00304.2013 ◽

2013 ◽

Vol 305 (9) ◽

pp. H1397-H1404 ◽

Cited By ~ 10

Author(s):

Matthew R. Stump ◽

Qiuming Gong ◽

Zhengfeng Zhou

Keyword(s):

Ventricular Arrhythmias ◽

Premature Termination Codon ◽

Wild Type ◽

Autosomal Dominant Disorder ◽

Nonsense Mutations ◽

Accelerated Degradation ◽

Herg Current ◽

Nonsense Mediated Mrna Decay ◽

Herg Channels

The human ether-a-go-go-related gene ( hERG) encodes a voltage-activated K+ channel that contributes to the repolarization of the cardiac action potential. Long QT syndrome type 2 (LQT2) is an autosomal dominant disorder caused by mutations in hERG, and patients with LQT2 are susceptible to severe ventricular arrhythmias. We have previously shown that nonsense and frameshift LQT2 mutations caused a decrease in mutant mRNA by the nonsense-mediated mRNA decay (NMD) pathway. The Q81X nonsense mutation was recently found to be resistant to NMD. Translation of Q81X is reinitiated at Met124, resulting in the generation of NH2-terminally truncated hERG channels with altered gating properties. In the present study, we identified two additional NMD-resistant LQT2 nonsense mutations, C39X and C44X, in which translation is reinitiated at Met60. Deletion of the first 59 residues of the channel truncated nearly one-third of the highly structured Per-Arnt-Sim domain and resulted in the generation of trafficking-defective proteins and a complete loss of hERG current. Partial deletion of the Per-Arnt-Sim domain also resulted in the accelerated degradation of the mutant channel proteins. The coexpression of mutant and wild-type channels did not significantly disrupt the function and trafficking properties of wild-type hERG. Our present findings indicate that translation reinitiation may generate trafficking-defective as well as dysfunctional channels in patients with LQT2 premature termination codon mutations that occur early in the coding sequence.

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Structural Requirements of Human Ether-a-go-go– related Gene Channels for Block by Bupivacaine

Anesthesiology ◽

10.1097/00000542-200703000-00017 ◽

2007 ◽

Vol 106 (3) ◽

pp. 523-531 ◽

Cited By ~ 14

Author(s):

Cornelia C. Siebrands ◽

Patrick Friederich

Keyword(s):

Aromatic Amino Acids ◽

Selectivity Filter ◽

Site Directed Mutagenesis ◽

Xenopus Laevis Oocytes ◽

Related Gene ◽

Wild Type ◽

Aromatic Residues ◽

Ic50 Value ◽

Herg Channels

Background Local anesthetics interact with human ether-a-go-go-related gene (HERG) channels via the aromatic amino acids Y652 and F656 in the S6 region. This study aimed to establish whether the residues T623, S624, and V625 residing deeper within the pore are also involved in HERG channel block by bupivacaine. In addition, the study aimed to further define the role of the aromatic residues Y652 and F656 in bupivacaine inhibition by mutating these residues to threonine. Methods Alanine and threonine mutants were generated by site-directed mutagenesis. Electrophysiologic and pharmacologic properties of wild-type and mutant HERG channels were established using two-electrode voltage-clamp recordings of Xenopus laevis oocytes expressing HERG channels. Results Tail currents at -120 mV through HERG wild-type channels were inhibited with an IC50 value of 132 +/- 22 microm (n = 33). Bupivacaine (300 microm) inhibited wild-type tail currents by 62 +/- 12% (n = 7). Inhibition of HERG tail currents by bupivacaine (300 microm) was reduced by all mutations (P < 0.001). The effect was largest for F656A (inhibition 5 +/- 2%, n = 6) in the lower S6 region and for T623A (inhibition 13 +/- 4%, n = 9) near the selectivity filter. Introducing threonine at positions 656 and 652 significantly reduced inhibition by bupivacaine compared with HERG wild type (P < 0.001). Conclusions The authors' results indicate that not only the aromatic residues Y652 and F656 but also residues residing deeper within the pore and close to the selectivity filter of HERG channels are involved in inhibition of HERG channels by the low-affinity blocker bupivacaine.

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Different consequences of cataract-associated mutations at adjacent positions in the first extracellular boundary of connexin50

AJP Cell Physiology ◽

10.1152/ajpcell.00384.2010 ◽

2011 ◽

Vol 300 (5) ◽

pp. C1055-C1064 ◽

Cited By ~ 23

Author(s):

Jun-Jie Tong ◽

Peter J. Minogue ◽

Wenji Guo ◽

Tung-Ling Chen ◽

Eric C. Beyer ◽

...

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Hela Cells ◽

Xenopus Oocytes ◽

Dominant Negative ◽

Wild Type ◽

Gap Junction Channels ◽

Dominant Negative Inhibitor ◽

Intercellular Channels ◽

Gap Junctional

Gap junction channels, which are made of connexins, are critical for intercellular communication, a function that may be disrupted in a variety of diseases. We studied the consequences of two cataract-associated mutations at adjacent positions at the first extracellular boundary in human connexin50 (Cx50), W45S and G46V. Both of these mutants formed gap junctional plaques when they were expressed in HeLa cells, suggesting that they trafficked to the plasma membrane properly. However, their functional properties differed. Dual two-microelectrode voltage-clamp studies showed that W45S did not form functional intercellular channels in paired Xenopus oocytes or hemichannel currents in single oocytes. When W45S was coexpressed with wild-type Cx50, the mutant acted as a dominant negative inhibitor of wild-type function. In contrast, G46V formed both functional gap junctional channels and hemichannels. G46V exhibited greatly enhanced currents compared with wild-type Cx50 in the presence of physiological calcium concentrations. This increase in hemichannel activity persisted when G46V was coexpressed with wild-type lens connexins, consistent with a dominant gain of hemichannel function for G46V. These data suggest that although these two mutations are in adjacent amino acids, they have very different effects on connexin function and cause disease by different mechanisms: W45S inhibits gap junctional channel function; G46V reduces cell viability by forming open hemichannels.

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Vigilance state-dependent attenuation of hypercapnic chemoreflex and exaggerated sleep apnea in orexin knockout mice

Journal of Applied Physiology ◽

10.1152/japplphysiol.00679.2006 ◽

2007 ◽

Vol 102 (1) ◽

pp. 241-248 ◽

Cited By ~ 105

Author(s):

Akira Nakamura ◽

Wei Zhang ◽

Masashi Yanagisawa ◽

Yasuichiro Fukuda ◽

Tomoyuki Kuwaki

Keyword(s):

Rem Sleep ◽

Knockout Mice ◽

Slow Wave Sleep ◽

Ventilatory Response ◽

Control Of Breathing ◽

Dependent Manner ◽

Wild Type ◽

Vigilance State ◽

State Dependent ◽

Respiratory Parameters

Exogenous administration of orexin can promote wakefulness and respiration. Here we examined whether intrinsic orexin participates in the control of breathing in a vigilance state-dependent manner. Ventilation was recorded together with electroencephalography and electromyography for 6 h during the daytime in prepro-orexin knockout mice (ORX-KO) and wild-type (WT) littermates. Respiratory parameters were separately determined during quiet wakefulness (QW), slow-wave sleep (SWS), or rapid eye movement (REM) sleep. Basal ventilation was normal in ORX-KO, irrespective of vigilance states. The hypercapnic ventilatory response during QW in ORX-KO (0.19 ± 0.01 ml·min−1·g−1·%CO2−1) was significantly smaller than that in WT mice (0.38 ± 0.04 ml·min−1·g−1·%CO2−1), whereas the responses during SWS and REM in ORX-KO were comparable to those in WT mice. Hypoxic responses during wake and sleep periods were not different between the genotypes. Spontaneous but not postsigh sleep apneas were more frequent in ORX-KO than in WT littermates during both SWS and REM sleep. Our findings suggest that orexin plays a crucial role both in CO2 sensitivity during wakefulness and in preserving ventilation stability during sleep.

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Biochemical and cytological changes associated with expression of deregulated pp60src in Xenopus oocytes.

Molecular and Cellular Biology ◽

10.1128/mcb.12.12.5485 ◽

1992 ◽

Vol 12 (12) ◽

pp. 5485-5498 ◽

Cited By ~ 17

Author(s):

T F Unger ◽

R E Steele

Keyword(s):

Cell Cycle ◽

Xenopus Oocytes ◽

Xenopus Laevis Oocytes ◽

Wild Type ◽

Large Area ◽

Phosphorylated Proteins ◽

Cytoskeletal Structure ◽

Cytological Changes ◽

Pigment Aggregation

We have examined the effects of Xenopus pp60c-src with constitutive kinase activity on the morphology and maturation of Xenopus laevis oocytes. When RNA encoding this deregulated variant was injected into stage VI oocytes, we observed a gross alteration in the cortex of the oocyte. This alteration involved aggregation of pigment and invagination of the cortex in a large area proximal to the site of injection. This phenomenon was not seen in oocytes injected with RNA encoding wild-type pp60c-src. We have correlated this phenomenon with the tyrosine phosphorylation of 84- and 100-kDa proteins. These phosphorylated proteins colocalized with the alteration in the oocyte cortex when assayed by both biochemical and immunocytochemical methods. Neither the pigment aggregation nor phosphorylation of the 84- and 100-kDa proteins was observed in oocytes expressing a nonmyristoylated version of the deregulated pp60c-src. Expression of deregulated Xenopus fyn, a src-family member, resulted in a phenotype similar to that seen with deregulated src. However, in the fyn-injected oocytes, many more proteins were phosphorylated on tyrosine than in the src-injected oocytes. Progesterone stimulation of oocytes expressing deregulated pp60c-src resulted in an increase in the number of tyrosine-phosphorylated proteins. This change may represent the response of pp60src to the resumption of the cell cycle in maturing oocytes. These data suggest that the oocyte may be a particularly useful system for investigating the role of pp60c-src in the regulation of cytoskeletal structure and in the regulation of events associated with the cell cycle.

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Molecular Interaction of Droperidol with Human Ether-a-go-go -related Gene Channels

Anesthesiology ◽

10.1097/01.anes.0000265156.09438.56 ◽

2007 ◽

Vol 106 (5) ◽

pp. 967-976 ◽

Cited By ~ 17

Author(s):

Alexander P. Schwoerer ◽

Carmen Blütner ◽

Sven Brandt ◽

Stephan Binder ◽

Cornelia C. Siebrands ◽

...

Keyword(s):

Amino Acid ◽

Patch Clamp ◽

Cardiac Myocytes ◽

Action Potentials ◽

Amino Acid Residues ◽

Related Gene ◽

Wild Type ◽

Early Afterdepolarizations ◽

High Affinity ◽

Herg Channels

Background The cardiac safety of droperidol given at antiemetic doses is a matter of debate. Although droperidol potently inhibits human ether-a-go-go-related gene (HERG) channels, the molecular mode of this interaction is unknown. The role of amino acid residues typically mediating high-affinity block of HERG channels is unclear. It is furthermore unresolved whether droperidol at antiemetic concentrations induces action potential prolongation and arrhythmogenic early afterdepolarizations in cardiac myocytes. Methods Molecular mechanisms of HERG current inhibition by droperidol were established using two-electrode voltage clamp recordings of Xenopus laevis oocytes expressing wild-type and mutant channels. The mutants T623A, S624A, V625A, Y652A, and F656A were generated by site-directed mutagenesis. The effect of droperidol on action potentials was investigated in cardiac myocytes isolated from guinea pig hearts using the patch clamp technique. Results Droperidol inhibited currents through HERG wild-type channels with a concentration of half-maximal inhibition of 0.6-0.9 microM. Droperidol shifted the channel activation and the steady state inactivation toward negative potentials while channel deactivation was not affected. Current inhibition increased with membrane potential and with increasing duration of current activation. Inhibition of HERG channels was similarly reduced by all mutations. Droperidol at concentrations between 5 and 100 nM prolonged whereas concentrations greater than 300 nm shortened action potentials. Early afterdepolarizations were not observed. Conclusions Droperidol is a high-affinity blocker of HERG channels. Amino acid residues typically involved in high-affinity block mediate droperidol effects. Patch clamp results and computational modeling allow the hypothesis that interaction with calcium currents may explain why droperidol at antiemetic concentrations prolongs the action potential without inducing early afterdepolarizations.

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Clinical and molecular characterisation of the R751L-CFTR mutation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00137.2020 ◽

2020 ◽

Author(s):

Iram J Haq ◽

Mike Althaus ◽

Aaron Ions Gardner ◽

Hui Ying Yeoh ◽

Urjita Joshi ◽

...

Keyword(s):

Xenopus Oocytes ◽

Clinical Phenotype ◽

Chloride Secretion ◽

Biological Properties ◽

Sodium Absorption ◽

Wild Type ◽

Sweat Chloride ◽

Epithelial Cultures ◽

Molecular Phenotypes ◽

Cf Transmembrane Conductance Regulator

Cystic fibrosis (CF) arises from mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in progressive and life-limiting respiratory disease. R751L is a rare CFTR mutation that is poorly characterised. Our aims were to describe the clinical and molecular phenotypes associated with R751L. Relevant clinical data were collected from three heterozygote individuals harbouring R751L (2 patients with G551D/R751L and 1 with F508del/R751L). Assessment of R751L-CFTR function was made in primary human bronchial epithelial cultures (HBEs) and Xenopus oocytes. Molecular properties of R751L-CFTR were investigated in the presence of known CFTR modulators. Although sweat chloride was elevated in all three patients, the clinical phenotype associated with R751L was mild. Chloride secretion in F508del/R751L HBEs was reduced compared to non-CF HBEs and associated with a reduction in sodium absorption by the epithelial sodium channel (ENaC). However, R751L-CFTR function in Xenopus oocytes together with folding and cell surface transport of R751L-CFTR were not different to wild-type CFTR. Overall, R751L-CFTR was associated with reduced sodium chloride absorption but had similar functional properties to wild-type CFTR. This is the first report of R751L-CFTR that combines clinical phenotype with characterisation of functional and biological properties of the mutant channel. Our work will build upon existing knowledge of mutations within this region of CFTR and importantly inform approaches for clinical management. Elevated sweat chloride and reduced chloride secretion in HBEs may be due to alternative non-CFTR factors, which require further investigation.

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