scholarly journals On the effect of hyperaldosteronism-inducing mutations in Na/K pumps

2017 ◽  
Vol 149 (11) ◽  
pp. 1009-1028 ◽  
Author(s):  
Dylan J. Meyer ◽  
Craig Gatto ◽  
Pablo Artigas
Keyword(s):  
Xenopus Oocytes ◽  
Turnover Rate ◽  
Outward Current ◽  
Zona Glomerulosa ◽  
Common Mechanism ◽  
Functional Evaluation ◽  
Loss Of Function ◽  
Wild Type ◽  
Inward Current ◽  
Inward Currents

Primary aldosteronism, a condition in which too much aldosterone is produced and that leads to hypertension, is often initiated by an aldosterone-producing adenoma within the zona glomerulosa of the adrenal cortex. Somatic mutations of ATP1A1, encoding the Na/K pump α1 subunit, have been found in these adenomas. It has been proposed that a passive inward current transported by several of these mutant pumps is a "gain-of-function" activity that produces membrane depolarization and concomitant increases in aldosterone production. Here, we investigate whether the inward current through mutant Na/K pumps is large enough to induce depolarization of the cells that harbor them. We first investigate inward currents induced by these mutations in Xenopus Na/K pumps expressed in Xenopus oocytes and find that these inward currents are similar in amplitude to wild-type outward Na/K pump currents. Subsequently, we perform a detailed functional evaluation of the human Na/K pump mutants L104R, delF100-L104, V332G, and EETA963S expressed in Xenopus oocytes. By combining two-electrode voltage clamp with [3H]ouabain binding, we measure the turnover rate of these inward currents and compare it to the turnover rate for outward current through wild-type pumps. We find that the turnover rate of the inward current through two of these mutants (EETA963S and L104R) is too small to induce significant cell depolarization. Electrophysiological characterization of another hyperaldosteronism-inducing mutation, G99R, reveals the absence of inward currents under many different conditions, including in the presence of the regulator FXYD1 as well as with mammalian ionic concentrations and body temperatures. Instead, we observe robust outward currents, but with significantly reduced affinities for intracellular Na+ and extracellular K+. Collectively, our results point to loss-of-function as the common mechanism for the hyperaldosteronism induced by these Na/K pump mutants.

scholarly journals Potentiated L-type Ca2+ Channels Rectify

2003 ◽  
Vol 121 (6) ◽  
pp. 541-550 ◽  
Author(s):  
Valérie Leuranguer ◽  
Robert T. Dirksen ◽  
Kurt G. Beam
Keyword(s):  
Outward Current ◽  
Complete Removal ◽  
Inward Rectification ◽  
Wild Type ◽  
Strong Suppression ◽  
Inward Current ◽  
Outward Currents ◽  
Inward Currents ◽  
Bayk 8644 ◽  
Ca2 Channels

Strong depolarization and dihydropyridine agonists potentiate inward currents through native L-type Ca2+ channels, but the effect on outward currents is less clear due to the small size of these currents. Here, we examined potentiation of wild-type α1C and two constructs bearing mutations in conserved glutamates in the pore regions of repeats II and IV (E2A/E4A-α1C) or repeat III (E3K-α1C). With 10 mM Ca2+ in the bath and 110 mM Cs+ in the pipette, these mutated channels, expressed in dysgenic myotubes, produced both inward and outward currents of substantial amplitude. For both the wild-type and mutated channels, we observed strong inward rectification of potentiation: strong depolarization had little effect on outward tail currents but caused the inward tail currents to be larger and to decay more slowly. Similarly, exposure to DHP agonist increased the amplitude of inward currents and decreased the amplitude of outward currents through both E2A/E4A-α1C and E3K-α1C. As in the absence of drug, strong depolarization in the presence of dihydropyridine agonist had little effect on outward tail currents but increased the amplitude and slowed the decay of inward tail currents. We tested whether cytoplasmic Mg2+ functions as the blocking particle responsible for the rectification of potentiated L-type Ca2+ channels. However, even after complete removal of cytoplasmic Mg2+, (−)BayK 8644 still potentiated inward current and partially blocked outward current via E2A/E4A-α1C. Although zero Mg2+ did not reveal potentiation of outward current by DHP agonist, it did have two striking effects, (a) a strong suppression of decay of both inward and outward currents via E2A/E4A-α1C and (b) a nearly complete elimination of depolarization-induced potentiation of inward tail currents. These results can be explained by postulating that potentiation exposes a binding site in the pore to which an intracellular blocking particle can bind and produce inward rectification of the potentiated channels.

scholarly journals Effect of Auxin (IAA) on the Fast Vacuolar (FV) Channels in Red Beet (Beta vulgaris L.) Taproot Vacuoles

2020 ◽  
Vol 21 (14) ◽  
pp. 4876
Author(s):  
Zbigniew Burdach ◽  
Agnieszka Siemieniuk ◽  
Waldemar Karcz
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
K Channel ◽  
Single Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Inward Current ◽  
Bath Solution ◽  
Outward Currents ◽  
Inward Currents

In contrast to the well-studied effect of auxin on the plasma membrane K+ channel activity, little is known about the role of this hormone in regulating the vacuolar K+ channels. Here, the patch-clamp technique was used to investigate the effect of auxin (IAA) on the fast-activating vacuolar (FV) channels. It was found that the macroscopic currents displayed instantaneous currents, which at the positive potentials were about three-fold greater compared to the one at the negative potentials. When auxin was added to the bath solution at a final concentration of 1 µM, it increased the outward currents by about 60%, but did not change the inward currents. The imposition of a ten-fold vacuole-to-cytosol KCl gradient stimulated the efflux of K+ from the vacuole into the cytosol and reduced the K+ current in the opposite direction. The addition of IAA to the bath solution with the 10/100 KCl gradient decreased the outward current and increased the inward current. Luminal auxin reduced both the outward and inward current by approximately 25% compared to the control. The single channel recordings demonstrated that cytosolic auxin changed the open probability of the FV channels at the positive voltages to a moderate extent, while it significantly increased the amplitudes of the single channel outward currents and the number of open channels. At the positive voltages, auxin did not change the unitary conductance of the single channels. We suggest that auxin regulates the activity of the fast-activating vacuolar (FV) channels, thereby causing changes of the K+ fluxes across the vacuolar membrane. This mechanism might serve to tightly adjust the volume of the vacuole during plant cell expansion.

Ca2+-activated Cl− channels in corpus cavernosum smooth muscle: a novel mechanism for control of penile erection

2003 ◽  
Vol 94 (1) ◽  
pp. 301-313 ◽  
Author(s):  
Tom Karkanis ◽  
Ling DeYoung ◽  
Gerald B. Brock ◽  
Stephen M. Sims
Keyword(s):  
Smooth Muscle ◽  
Penile Erection ◽  
Corpus Cavernosum ◽  
Outward Current ◽  
Channel Blockers ◽  
Inward Current ◽  
Inward Currents ◽  
Cl Channels ◽  
Patch Clamp Electrophysiology

Little is known of the excitatory mechanisms that contribute to the tonic contraction of the corpus cavernosum smooth muscle in the flaccid state. We used patch-clamp electrophysiology to investigate a previously unidentified inward current in freshly isolated rat and human corporal myocytes. Phenylephrine (PE) contracted cells and activated whole cell currents. Outward current was identified as large-conductance Ca2+-activated K+ current. The inward current elicited by PE was dependent on the Cl− gradient and was inhibited by niflumic acid, indicative of a Ca2+-activated Cl− (ClCa) current. Furthermore, spontaneous transient outward and inward currents (STOCs and STICs, respectively) were identified in both rat and human corporal myocytes and derived from large-conductance Ca2+-activated K+ and ClCa channel activity. STICs and STOCs were inhibited by PE and A-23187, and combined 8-bromoadenosine cAMP and 8-bromoadenosine cGMP decreased their frequency. When studied in vivo, chloride channel blockers transiently increased intracavernosal pressure and prolonged nerve-evoked erections. This report reveals for the first time ClCa current in rat and human corpus cavernosum smooth muscle cells and demonstrates its key functional role in the regulation of penile erection.

Potassium channels regulate tone in rat pulmonary veins

2001 ◽  
Vol 280 (6) ◽  
pp. L1138-L1147 ◽  
Author(s):  
Evangelos D. Michelakis ◽  
E. Kenneth Weir ◽  
Xichen Wu ◽  
Ali Nsair ◽  
Ross Waite ◽  
...  
Keyword(s):  
Low Dose ◽  
Venous Return ◽  
Pulmonary Veins ◽  
Outward Current ◽  
Inward Rectifier ◽  
Inward Current ◽  
Voltage Gated ◽  
Disease States ◽  
Inward Currents ◽  
Vascular Beds

Intrapulmonary veins (PVs) contribute to pulmonary vascular resistance, but the mechanisms controlling PV tone are poorly understood. Although smooth muscle cell (SMC) K+ channels regulate tone in most vascular beds, their role in PV tone is unknown. We show that voltage-gated (KV) and inward rectifier (Kir) K+ channels control resting PV tone in the rat. PVs have a coaxial structure, with layers of cardiomyocytes (CMs) arrayed externally around a subendothelial layer of typical SMCs, thus forming spinchterlike structures. PVCMs have both an inward current, inhibited by low-dose Ba2+, and an outward current, inhibited by 4-aminopyridine. In contrast, PVSMCs lack inward currents, and their outward current is inhibited by tetraethylammonium (5 mM) and 4-aminopyridine. Several KV, Kir, and large-conductance Ca2+-sensitive K+channels are present in PVs. Immunohistochemistry showed that Kir channels are present in PVCMs and PV endothelial cells but not in PVSMCs. We conclude that K+ channels are present and functionally important in rat PVs. PVCMs form sphincters rich in Kir channels, which may modulate venous return both physiologically and in disease states including pulmonary edema.

Segment-specific effects of FMRFamide on membrane properties of heart interneurons in the leech

1995 ◽  
Vol 74 (4) ◽  
pp. 1485-1497 ◽  
Author(s):  
J. Schmidt ◽  
S. Gramoll ◽  
R. L. Calabrese
Keyword(s):  
Outward Current ◽  
Membrane Conductance ◽  
Membrane Properties ◽  
Inward Current ◽  
Specific Effects ◽  
Outward Currents ◽  
Voltage Dependent ◽  
Inward Currents ◽  
K Current ◽  
Conductance Increase

1. The effects of Phe-Met-Arg-Phe (FMRF)amide (10(-6) M) on membrane properties of heart interneurons in the third, fourth, and fifth segmental ganglia [HN(3), HN(4), and HN(5) cells, respectively] of the leech were studied using discontinuous current-clamp and single-electrode voltage-clamp techniques. FMRFamide was focally applied onto the soma of the cell under investigation. 2. Application of FMRFamide depolarized HN(3) and HN(4) cells by evoking an inward current. These responses were subject to pronounced desensitization. The inward currents evoked by application of FMRFamide were associated with an increase in membrane conductance and appeared to be voltage dependent. Currents were enhanced at more depolarized potentials. 3. The responsiveness of the HN(3) and HN(4) cells was not affected when the Ca2+ concentration in the bath saline was reduced from normal (1.8 mM) to 0.1 mM. The depolarizing response on application of FMRFamide was blocked when Co2+ was substituted for Ca2+. 4. HN(3) and HN(4) cells did not respond to FMRFamide application in Na(+)-free solution. Inward currents were largely reduced when bath saline with 30% of the normal Na+ concentration was used. When Li+ was substituted for Na+ in the saline, application of FMRFamide still evoked depolarizing responses in HN(3) and HN(4) cells. 5. We conclude that focal application of FMRFamide onto the somata of HN(3) and HN(4) cells evokes a voltage-dependent inward current, carried largely by Na+. 6. Focal application of FMRFamide onto somata of HN(5) cells hyperpolarized these cells by activating a voltage-dependent outward current. 7. HN(5) cells were loaded with Cl- until inhibitory postsynaptic potentials carried by Cl- reversed. Cl(-)-loaded cells still responded with a hyperpolarization when FMRFamide was applied onto their somata. Therefore the outward current evoked by FMRFamide appears to be mediated by a K+ conductance increase. 8. Application of FMRFamide onto the somata of HN(5) cells enhanced outward currents that were evoked by depolarizing voltage steps from a holding potential of -45 mV. 9. We conclude that the hyperpolarizing response of HN(5) cells to focal application of FMRFamide onto their somata is the result of an up-regulation of a voltage-dependent K+ current.

scholarly journals Voltage-dependent conductances in Limulus ventral photoreceptors.

1982 ◽  
Vol 79 (2) ◽  
pp. 187-209 ◽  
Author(s):  
J E Lisman ◽  
G L Fain ◽  
P M O'Day
Keyword(s):  
Outward Current ◽  
Delayed Rectifier ◽  
Molluscan Neurons ◽  
Inward Current ◽  
Transient Component ◽  
Outward Currents ◽  
Voltage Dependent ◽  
Inward Currents ◽  
K Current ◽  
A Current

The voltage-dependent conductances of Limulus ventral photoreceptors have been investigated using a voltage-clamp technique. Depolarization in the dark induces inward and outward currents. The inward current is reduced by removing Na+ or Ca2+ and is abolished by removing both ions. These results suggest that both Na+ and Ca2+ carry voltage-dependent inward current. Inward current is insensitive to tetrodotoxin but is blocked by external Ni2+. The outward current has a large transient component that is followed by a smaller maintained component. Intracellular tetraethylammonium preferentially reduces the maintained component, and extracellular 4-amino pyridine preferentially reduces the transient component. Neither component is strongly affected by removal of extracellular Ca2+ or by intracellular injection of EGTA. It is concluded that the photoreceptors contain at least three separate voltage-dependent conductances: 1) a conductance giving rise to inward currents; 2) a delayed rectifier giving rise to maintained outward K+ current; and 3) a rapidly inactivating K+ conductance similar to the A current of molluscan neurons.

Hereditary Stomatocytosis Associated with a Loss of Function Mutation In Rh-Associated Glycoprotein (RhAG)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2040-2040
Author(s):  
Connie M Westhoff ◽  
Seth Alper
Keyword(s):  
Xenopus Oocytes ◽  
Collecting Duct ◽  
Monovalent Cation ◽  
Band 3 ◽  
Cell Permeability ◽  
Red Cell ◽  
Loss Of Function ◽  
Wild Type ◽  
Water Control ◽  
Glycophorin B

Abstract Abstract 2040 The erythroid Rh family of proteins includes RhCE and RhD which carry the common Rh antigens, and the related Rh-associated glycoprotein, RhAG. RhAG is required for trafficking of the blood group proteins to the membrane and forms the core of a macro-complex in the membrane which includes glycophorin B, Band 3, CD47, and LW. The Rh proteins are structurally and functionally related to the Amt superfamily of NH3/NH4+ transport proteins, and RhAG and its nonerythroid paralogs, RhCG and RhBG, have been shown to mediate NH3/NH4+ transport. RhCG is responsible for part of renal collecting duct epithelial cell NH3/NH4+ secretion, and Rhcg-/- mice exhibit incomplete distal renal tubular acidosis due to impaired urinary NH4+ excretion. The Rhag-/- mouse is grossly normal, and the significance of RhAG-mediated NH3/NH4+ transport in human erythrocytes remains unclear. Over-hydrated hereditary stomatocytosis (OHSt) is a rare dominant disorder characterized by moderate hemolytic anemia, increased mean red cell volumes, stomatocytes and echinocytes, and increased red cell permeability to the monovalent cations, Na+ and K+. Six of the seven OHSt kindred studied by Bruce et al. (Blood. 2009;113:1350) displayed a heterozygous Phe65Ser mutation in RhAG. Expression studies of the mutant 65Ser-RhAG in Xenopus oocytes induced a monovalent cation flux compatible with the cation leak seen in RBCs. The increased Na+ and decreased K+ contents of mutant RhAG-expressing oocytes suggested that F65S is a gain-of-function mutation that opens a cation leak, likely within the RhAG polypeptide. In this study the ammonia transport properties of the OHSt mutant 65Ser-RhAG were investigated. Xenopus oocytes were injected with cRNA encoding wild-type RhAG, the OHSt mutant 65Ser-RhAG, and 65Val-RhAG, an engineered mutation with a smaller hydrophobic side chain at position 65. Wild-type and mutant RhAG polypeptides were well-expressed in the oocyte membrane as measured by quantitative immunoblotting. Uptake of the NH3/NH4+ substrate analog 14C-methylammonium (MA), was assayed in oocytes previously injected with water (control) or with cRNA. Expression of wild-type RhAG mediated MA uptake at rates 6-fold greater than that of water-injected controls. Uptake of MA by oocytes expressing 65Val-RhAG was equivalent to that of wild type RhAG. However, MA uptake by oocytes expressing OHSt mutant 65Ser-RhAG was greatly reduced to less than 20% that of oocytes expressing wild-type RHAG or 65Val-RhAG, and was only 1.5-fold greater than that of water-injected control oocytes. Co-expression with other, individual Rh complex members glycophorin B, RhD, RhCE, or Band 3 did not alter MA-mediated uptake by RhAG-expressing oocytes. Importantly, this study reveals that the RhAG mutation Phe65Ser found in patients with type 1 over-hydrated stomatocytosis is a loss of function mutation. Further study is required to define the relationship between loss of NH3/NH4+ transport and erythrocyte Na+ and K+ cation content. Disclosures: Westhoff: Immucor: Scientific Advisor.

Connexin46 mutations linked to congenital cataract show loss of gap junction channel function

2000 ◽  
Vol 279 (3) ◽  
pp. C596-C602 ◽  
Author(s):  
Jay D. Pal ◽  
Xiaoqin Liu ◽  
Donna Mackay ◽  
Alan Shiels ◽  
Viviana M. Berthoud ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
Dominant Negative ◽  
Metabolic Labeling ◽  
Loss Of Function ◽  
Wild Type ◽  
Reading Frame ◽  
Gap Junction Channel ◽  
Intercellular Channels ◽  
Normal Lens ◽  
Gap Junctional

Human connexin46 (hCx46) forms gap junctional channels interconnecting lens fiber cells and appears to be critical for normal lens function, because hCx46 mutations have been linked to congenital cataracts. We studied two hCx46 mutants, N63S, a missense mutation in the first extracellular domain, and fs380, a frame-shift mutation that shifts the translational reading frame at amino acid residue 380. We expressed wild-type Cx46 and the two mutants in Xenopus oocytes. Production of the expressed proteins was verified by SDS-PAGE after metabolic labeling with [35S]methionine or by immunoblotting. Dual two-microelectrode voltage-clamp studies showed that hCx46 formed both gap junctional channels in paired Xenopus oocytes and hemi-gap junctional channels in single oocytes. In contrast, neither of the two cataract-associated hCx46 mutants could form intercellular channels in paired Xenopus oocytes. The hCx46 mutants were also impaired in their ability to form hemi-gap-junctional channels. When N63S or fs380 was coexpressed with wild-type connexins, both mutations acted like “loss of function” rather than “dominant negative” mutations, because they did not affect the gap junctional conductance induced by either wild-type hCx46 or wild-type hCx50.

Role of Na-Ca exchange in the action potential changes caused by drive in cardiac myocytes exposed to different Ca2+ loads

1999 ◽  
Vol 77 (6) ◽  
pp. 383-397
Author(s):  
Qi-Ying Liu ◽  
Mario Vassalle
Keyword(s):  
Action Potentials ◽  
Ventricular Myocytes ◽  
Outward Current ◽  
Time Dependent ◽  
Inward Current ◽  
Tail Current ◽  
Outward Currents ◽  
Inward Currents ◽  
Single Ventricular Myocytes

The role of Na-Ca exchange in the membrane potential changes caused by repetitive activity ("drive") was studied in guinea pig single ventricular myocytes exposed to different [Ca2+]o. The following results were obtained. (i) In 5.4 mM [Ca2+]o, the action potentials (APs) gradually shortened during drive, and the outward current during a train of depolarizing voltage clamp steps gradually increased. (ii) The APs shortened more and were followed by a decaying voltage tail during drive in the presence of 5 mM caffeine; the outward current became larger and there was an inward tail current on repolarization during a train of depolarizing steps. (iii) These effects outlasted drive so that immediately after a train of APs, currents were already bigger and, after a train of steps, APs were already shorter. (iv) In 0.54 mM [Ca2+]o, the above effects were much smaller. (v) In high [Ca2+]o APs were shorter and outward currents larger than in low [Ca2+]o. (vi) In 10.8 mM [Ca2+]o, both outward and inward currents during long steps were exaggerated by prior drive, even with steps (+80 and +120 mV) at which there was no apparent inward current identifiable as ICa. (vii) In 0.54 mM [Ca2+]o, the time-dependent outward current was small and prior drive slightly increased it. (viii) During long steps, caffeine markedly increased outward and inward tail currents, and these effects were greatly decreased by low [Ca2+]o. (ix) After drive in the presence of caffeine, Ni2+ decreased the outward and inward tail currents. It is concluded that in the presence of high [Ca2+]o drive activates outward and inward Na-Ca exchange currents. During drive, the outward current participates in the plateau shortening and the inward tail current in the voltage tail after the action potential.Key words: ventricular myocytes, repetitive activity, outward and inward Na-Ca exchange currents, caffeine, nickel.

scholarly journals Divalent Cation Block of Inward Currents and Low-Affinity K+ Uptake in Saccharomyces cerevisiae

1999 ◽  
Vol 181 (1) ◽  
pp. 291-297 ◽  
Author(s):  
Stephen K. Roberts ◽  
Marc Fischer ◽  
Graham K. Dixon ◽  
Dale Sanders
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Patch Clamp ◽  
Divalent Cation ◽  
Divalent Cations ◽  
Outward Current ◽  
Alkali Cations ◽  
Patch Clamp Technique ◽  
Inward Current ◽  
Negative Membrane Potential ◽  
Inward Currents

ABSTRACT We have used the patch clamp technique to characterize whole-cell currents in spheroplasts isolated from a trk1Δ trk2Δstrain of Saccharomyces cerevisiae which lacks high- and moderate-affinity K+ uptake capacity. In solutions in which extracellular divalent cation concentrations were 0.1 mM, cells exhibited a large inward current. This current was not the result of increasing leak between the glass pipette and membrane, as there was no effect on the outward current. The inward current comprised both instantaneous and time-dependent components. The magnitude of the inward current increased with increasing extracellular K+and negative membrane potential but was insensitive to extracellular anions. Replacing extracellular K+ with Rb+, Cs+, or Na+ only slightly modulated the magnitude of the inward current, whereas replacement with Li+ reduced the inward current by approximately 50%, and tetraethylammonium (TEA+) and choline were relatively impermeant. The inward current was blocked by extracellular Ca2+ and Mg2+ with apparentKi s (at −140 mV) of 363 ± 78 and 96 ± 14 μM, respectively. Furthermore, decreasing cytosolic K+ increased the magnitude of the inward current independently of the electrochemical driving force for K+influx, consistent with regulation of the inward current by cytosolic K+. Uptake of 86Rb+ by intacttrk1Δ trk2Δ cells was inhibited by extracellular Ca2+ with a Ki within the range observed for the inward current. Furthermore, increasing extracellular Ca2+ from 0.1 to 20 mM significantly inhibited the growth of these cells. These results are consistent with those of the patch clamp experiments in suggesting that low-affinity uptake of alkali cations in yeast is mediated by a transport system sensitive to divalent cations.

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