Ammonium interaction with the epithelial sodium channel

2001 ◽  
Vol 281 (3) ◽  
pp. F493-F502 ◽  
Author(s):  
Nazih L. Nakhoul ◽  
Kathleen S. Hering-Smith ◽  
Solange M. Abdulnour-Nakhoul ◽  
L. Lee Hamm
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Sodium Channel ◽  
Outward Current ◽  
Intracellular Acidification ◽  
Positive Potential ◽  
Steady State Current ◽  
Electrode Voltage ◽  
Ion Activities ◽  
Cl Permeability

The purpose of this study was to investigate the direct effect of NH3/NH[Formula: see text] on mouse epithelial Na+ channels (mENaC) expressed in Xenopusoocytes. Two-electrode voltage-clamp and ion-selective microelectrodes were used to measure the Na+ current, intracellular pH (pHi), and ion activities in oocytes expressing mENaC. In oocytes expressing mENaC, removal of external Na+reversibly hyperpolarized membrane potential by 129 ± 5.3 mV in the absence of 20 mM NH4Cl but only by 100 ± 7.8 mV in its presence. Amiloride completely inhibited the changes in membrane potential. In oocytes expressing mENaC, butyrate (20 mM) caused a decrease in pHi (0.43 ± 0.07) similar to the NH4Cl-induced pHi decrease (0.47 ± 0.12). Removal of Na+ in the presence of butyrate caused hyperpolarization that was not significantly different from that in the absence of butyrate at high pHi (in the absence of NH4Cl). Removal of external Na+ resulted in an outward current of 3.7 ± 0.8 μA (at −60 mV). The magnitude of this change in current was only 2.7 ± 0.7 μA when Na+ was removed in the presence of NH4Cl. In oocytes expressing mENaC, NH4Cl also caused a decrease in whole cell conductance at negative potential and an outward current at positive potential. In the presence of amiloride, steady-state current and the change in current caused by removal of Na+ were not different from zero. These results indicate that NH4Cl inhibits Na+ transport when mENaC is expressed in oocytes. The inhibition of voltage changes is not due to intracellular acidification caused by NH4Cl. Permeability and selectivity of ENaC to NH[Formula: see text] may play a role.

scholarly journals Extracellular Hco3− Dependence of Electrogenic Na/Hco3 Cotransporters Cloned from Salamander and Rat Kidney

2000 ◽  
Vol 115 (5) ◽  
pp. 533-546 ◽  
Author(s):  
Irina I. Grichtchenko ◽  
Michael F. Romero ◽  
Walter F. Boron
Keyword(s):  
Membrane Potential ◽  
Voltage Clamp ◽  
Xenopus Oocytes ◽  
Rat Kidney ◽  
Outward Current ◽  
Membrane Vesicles ◽  
Disulfonic Acid ◽  
Ambystoma Tigrinum ◽  
Ph Titration ◽  
Electrode Voltage

We studied the extracellular [HCOabstract 3 −] dependence of two renal clones of the electrogenic Na/HCO3 cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (ΔVm) elicited by the NBC cloned from the kidney of the salamander Ambystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (ΔI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract 3 −/CO2 (0.33–99 mM HCOabstract 3−, pHo 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na+-dependent hyperpolarization (or outward current). In ΔVm experiments, the apparent Km for HCOabstract 3− of akNBC (10.6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent Km for HCOabstract 3− of rkNBC was less (6.5 mM). Because it has been reported that SOabstract 3=/HSO abstract 3− stimulates Na/HCO3 cotransport in renal membrane vesicles (a result that supports the existence of a COabstract 3= binding site with which SOabstract 3= interacts), we examined the effect of SOabstract 3=/HSO abstract 3− on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract 4= nor 33 mM SOabstract 3 =/HSOabstract 3− substantially affects the apparent Km for HCO abstract 3−. We also used microelectrodes to monitor intracellular pH (pHi) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract 3 −/0.5% CO2. We found that SO abstract 3=/HSOabstract 3 − did not significantly affect the DIDS-sensitive component of the pHi recovery from the initial CO2 -induced acidification. We also monitored the rkNBC current while simultaneously varying [CO2]o, pHo, and [COabstract 3=]o at a fixed [HCOabstract 3−]o of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract 3=]o . However, a pH titration curve nicely fitted the data expressed as current versus pHo. Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SOabstract 3 =, HSOabstract 3−, or COabstract 3=.

Effects of sevoflurane on inward rectifier K+ current in guinea pig ventricular cardiomyocytes

1997 ◽  
Vol 273 (1) ◽  
pp. H324-H332 ◽  
Author(s):  
A. Stadnicka ◽  
Z. J. Bosnjak ◽  
J. P. Kampine ◽  
W. M. Kwok
Keyword(s):  
Steady State ◽  
Guinea Pig ◽  
Outward Current ◽  
Inward Rectifier ◽  
Equilibrium Potential ◽  
Ventricular Cardiomyocytes ◽  
Steady State Current ◽  
Voltage Dependent ◽  
Current Activation ◽  
Extracellular Side

The effects of sevoflurane on the inward rectifier potassium current (IKIR) were examined in guinea pig ventricular cardiomyocytes using the whole cell patch-clamp methodology. Sevoflurane had a unique dual effect on the steady-state current amplitude, producing a reversible, concentration- and voltage-dependent block of the inward current at potentials negative to the potassium equilibrium potential (EK) but enhancing the outward current positive to EK. Accordingly, the steady-state conductance negative to EK was reduced by sevoflurane, but conductance positive to EK was increased. The chord conductance-voltage relationship showed depolarizing shifts at 0.7, 1.3, and 1.6 mM sevoflurane. When the myocytes were dialyzed with 10 mM Mg2+, but not with 1.0 mM Mg2+, sevoflurane further slowed current activation kinetics. With 10 mM intracellular Mg2+, the outward current enhancement by sevoflurane and the associated shifts in half-activation potential were abolished. Polyamines abolished all effects of sevoflurane on IKIR. With the use of the Woodhull model for voltage-dependent block, we determined the sevoflurane interaction site with the inward rectifier potassium channel to be at an electrical distance of 0.2 from the extracellular side.

scholarly journals The mechanism of inward rectification of potassium channels: "long-pore plugging" by cytoplasmic polyamines.

1995 ◽  
Vol 106 (5) ◽  
pp. 923-955 ◽  
Author(s):  
A N Lopatin ◽  
E N Makhina ◽  
C G Nichols
Keyword(s):  
Steady State ◽  
Outward Current ◽  
Inward Rectifier ◽  
Time Dependent ◽  
Inward Rectification ◽  
Voltage Sensitivity ◽  
Intact Cells ◽  
Time Constants ◽  
Steady State Current ◽  
Activation Phase

The mechanism of inward rectification was examined in cell-attached and inside-out membrane patches from Xenopus oocytes expressing the cloned strong inward rectifier HRK1. Little or no outward current was measured in cell-attached patches. Inward currents reach their maximal value in two steps: an instantaneous phase followed by a time-dependent "activation" phase, requiring at least two exponentials to fit the time-dependent phase. After an activating pulse, the quasi-steady state current-voltage (I-V) relationship could be fit with a single Boltzmann equation (apparent gating charge, Z = 2.0 +/- 0.1, n = 3). Strong rectification and time-dependent activation were initially maintained after patch excision into high [K+] (K-INT) solution containing 1 mM EDTA, but disappeared gradually, until only a partial, slow inactivation of outward current remained. Biochemical characterization (Lopatin, A. N., E. N. Makhina, and C. G. Nichols, 1994. Nature. 372:366-396.) suggests that the active factors are naturally occurring polyamines (putrescine, spermidine, and spermine). Each polyamine causes reversible, steeply voltage-dependent rectification of HRK1 channels. Both the blocking affinity and the voltage sensitivity increased as the charge on the polyamine increased. The sum two Boltzmann functions is required to fit the spermine and spermidine steady state block. Putrescine unblock, like Mg2+ unblock, is almost instantaneous, whereas the spermine and spermidine unblocks are time dependent. Spermine and spermidine unblocks (current activation) can each be fit with single exponential functions. Time constants of unblock change e-fold every 15.0 +/- 0.7 mV (n = 3) and 33.3 +/- 6.4 mV (n = 5) for spermine and spermidine, respectively, matching the voltage sensitivity of the two time constants required to fit the activation phase in cell-attached patches. It is concluded that inward rectification in intact cells can be entirely accounted for by channel block. Putrescine and Mg2+ ions can account for instantaneous rectification; spermine and spermidine provide a slower rectification corresponding to so-called intrinsic gating of inward rectifier K channels. The structure of spermine and spermidine leads us to suggest a specific model in which the pore of the inward rectifier channel is plugged by polyamines that enter deeply into the pore and bind at sites within the membrane field. We propose a model that takes into account the linear structure of the natural polyamines and electrostatic repulsion between two molecules inside the pore. Experimentally observed instantaneous and steady state rectification of HRK1 channels as well as the time-dependent behavior of HRK1 currents are then well fit with the same set of parameters for all tested voltages and concentrations of spermine and spermidine.

Physiological role of Ca(2+)-activated and voltage-dependent K+ currents in rabbit coronary myocytes

1994 ◽  
Vol 266 (6) ◽  
pp. C1523-C1537 ◽  
Author(s):  
N. Leblanc ◽  
X. Wan ◽  
P. M. Leung
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Physiological Role ◽  
Cell Voltage ◽  
Resting Membrane Potential ◽  
Physiological Level ◽  
Steady State Current ◽  
Voltage Dependent ◽  
A Minor ◽  
And Function

The properties and function of Ca(2+)-activated K+ (KCa) and voltage-dependent K+ (IK) currents of rabbit coronary myocytes were studied under whole cell voltage-clamp conditions (22 degrees C). Inhibition of KCa by tetraethylammonium chloride (1-10 mM) or charybdotoxin (50-100 nM) suppressed noisy outward rectifying current elicited by 5-s voltage steps or ramp at potentials > 0 mV, reduced the hump of the biphasic ramp current-voltage relation, and shifted by less than +5 mV the potential at which no net steady-state current is recorded (Enet; index of resting membrane potential). Inhibition of steady-state inward Ca2+ currents [ICa(L)] by nifedipine (1 microM) displaced Enet by -11 mV. Analysis of steady-state voltage dependence of IK supported the existence of a "window" current between -50 and 0 mV. 4-Aminopyridine (2 mM) blocked a noninactivating component of IK evoked between -30 and -40 mV, abolished the hump current during ramps, and shifted Enet by more than +15 mV; hump current persisted during 2-min ramp depolarizations and peaked near the maximum overlap of the steady-state activation and inactivation curves of IK (about -22 mV). A threefold rise in extracellular Ca2+ concentration (1.8-5.4 mM) enhanced time-dependent outward K+ current (6.7-fold at +40 mV) and shifted Enet by -30 mV. It is concluded that, under steady-state conditions, IK and ICa(L) play a major role in regulating resting membrane potential at a physiological level of intracellular Ca2+ concentration, with a minor contribution from KCa. However, elevation of intracellular Ca2+ concentration enhances KCa and hyperpolarizes the myocyte to limit Ca2+ entry through ICa(L).

scholarly journals Current- and Voltage-Clamped Studies on Myxicola Giant Axons

1969 ◽  
Vol 54 (6) ◽  
pp. 730-740 ◽  
Author(s):  
L. Binstock ◽  
L. Goldman
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Action Potential ◽  
Voltage Clamp ◽  
Transient Current ◽  
Resting Membrane Potential ◽  
Action Potential Amplitude ◽  
Giant Axons ◽  
Potential Amplitude ◽  
Steady State Current

A new dissection procedure for preparing Myxicola giant axons for observation under voltage clamp is described. Preparation time is generally 40–45 min. 65–70% of the preparations attempted may be brought through the entire procedure, including insertion of the long internal electrode, and support an initial action potential amplitude of 100 mv or greater. Mean values for axon diameter, resting membrane potential, action potential amplitude, maximum peak inward transient current, and resting membrane resistance are 560 µ, —66.5 mv, 112 mv, 0.87 ma/cm2 and 1.22 KΩ cm 2 respectively. Cut branches do not seem to be a problem in this preparation. Behavior under voltage clamp is reasonably stable over several hours. Reductions in maximum inward transient current of 10% and in steady-state current of 5–10% are expected in the absence of any particular treatment. Tetrodotoxin blocks the action potential and both the inward and outward transient current, but has no effect on either the resting membrane potential or the steady-state current. This selective action of tetrodotoxin on the transient current is taken as an indication that this current component is probably carried by Na.

scholarly journals Na Self Inhibition of Human Epithelial Na Channel

2002 ◽  
Vol 120 (2) ◽  
pp. 133-145 ◽  
Author(s):  
Ahmed Chraïbi ◽  
Jean-Daniel Horisberger
Keyword(s):  
Steady State ◽  
Reversal Potential ◽  
Outward Current ◽  
Inactivation Rate ◽  
Na Channel ◽  
Temperature Dependent ◽  
Open Probability ◽  
Inward Current ◽  
Steady State Current ◽  
Epithelial Na Channel

The regulation of the open probability of the epithelial Na+ channel (ENaC) by the extracellular concentration of Na+, a phenomenon called “Na+ self inhibition,” has been well described in several natural tight epithelia, but its molecular mechanism is not known. We have studied the kinetics of Na+ self inhibition on human ENaC expressed in Xenopus oocytes. Rapid removal of amiloride or rapid increase in the extracellular Na+ concentration from 1 to 100 mM resulted in a peak inward current followed by a decline to a lower quasi-steady-state current. The rate of current decline and the steady-state level were temperature dependent and the current transient could be well explained by a two-state (active-inactive) model with a weakly temperature-dependent (Q10act = 1.5) activation rate and a strongly temperature-dependant (Q10inact = 8.0) inactivation rate. The steep temperature dependence of the inactivation rate resulted in the paradoxical decrease in the steady-state amiloride-sensitive current at high temperature. Na+ self inhibition depended only on the extracellular Na+ concentration but not on the amplitude of the inward current, and it was observed as a decrease of the conductance at the reversal potential for Na+ as well as a reduction of Na+ outward current. Self inhibition could be prevented by exposure to extracellular protease, a treatment known to activate ENaC or by treatment with p-CMB. After protease treatment, the amiloride-sensitive current displayed the expected increase with rising temperature. These results indicate that Na+ self inhibition is an intrinsic property of sodium channels resulting from the expression of the α, β, and γ subunits of human ENaC in Xenopus oocyte. The extracellular Na+-dependent inactivation has a large energy of activation and can be abolished by treatment with extracellular proteases.

scholarly journals Native and recombinant Slc26a3 (downregulated in adenoma, Dra) do not exhibit properties of 2Cl−/1HCO3− exchange

2011 ◽  
Vol 300 (2) ◽  
pp. C276-C286 ◽  
Author(s):  
Seth L. Alper ◽  
Andrew K. Stewart ◽  
David H. Vandorpe ◽  
Jeffrey S. Clark ◽  
R. Zachary Horack ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Ex Vivo ◽  
Short Circuit ◽  
Outward Current ◽  
Anion Channel ◽  
Short Circuit Current ◽  
Surface Epithelium ◽  
Steady State Current ◽  
Health And Disease ◽  
Secondary Membrane

The recent proposal that Dra/Slc26a3 mediates electrogenic 2Cl−/1HCO3− exchange suggests a required revision of classical concepts of electroneutral Cl− transport across epithelia such as the intestine. We investigated 1) the effect of endogenous Dra Cl−/HCO3− activity on apical membrane potential ( Va) of the cecal surface epithelium using wild-type (WT) and knockout (KO) mice; and 2) the electrical properties of Cl−/(OH−)HCO3− exchange by mouse and human orthologs of Dra expressed in Xenopus oocytes. Ex vivo 36Cl− fluxes and microfluorometry revealed that cecal Cl−/HCO3− exchange was abolished in the Dra KO without concordant changes in short-circuit current. In microelectrode studies, baseline Va of Dra KO surface epithelium was slightly hyperpolarized relative to WT but depolarized to the same extent as WT during luminal Cl− substitution. Subsequent studies indicated that Cl−-dependent Va depolarization requires the anion channel Cftr. Oocyte studies demonstrated that Dra-mediated exchange of intracellular Cl− for extracellular HCO3− is accompanied by slow hyperpolarization and a modest outward current, but that the steady-state current-voltage relationship is unaffected by Cl− removal or pharmacological blockade. Further, Dra-dependent 36Cl− efflux was voltage-insensitive in oocytes coexpressing the cation channels ENaC or ROMK. We conclude that 1) endogenous Dra and recombinant human/mouse Dra orthologs do not exhibit electrogenic 2Cl−/1HCO3− exchange; and 2) acute induction of Dra Cl−/HCO3− exchange is associated with secondary membrane potential changes representing homeostatic responses. Thus, participation of Dra in coupled NaCl absorption and in uncoupled HCO3− secretion remains compatible with electroneutrality of these processes, and with the utility of electroneutral transport models for predicting epithelial responses in health and disease.

scholarly journals Chloride channels in mast cells: block by DIDS and role in exocytosis.

1994 ◽  
Vol 104 (6) ◽  
pp. 1099-1111 ◽  
Author(s):  
J Dietrich ◽  
M Lindau
Keyword(s):  
Steady State ◽  
Mast Cells ◽  
Chloride Transport ◽  
Chloride Concentration ◽  
Outward Current ◽  
Chloride Current ◽  
Hill Coefficient ◽  
Disulfonic Acid ◽  
Intact Cells ◽  
Steady State Current

In rat peritoneal mast cells, we have investigated the influence of the chloride transport blocker 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) and the extracellular chloride concentration on the chloride current induced by intracellular application of cyclic AMP (cAMP) and on hexosaminidase secretion from intact cells stimulated with compound 48/80. The inhibition of the Cl-current by extracellular DIDS is voltage and time dependent. Upon depolarization from -10 to +70 mV, the outward current diminishes with millisecond kinetics. The size of the steady state current and the time constant of the decrease both decrease with increasing DIDS concentrations. The steady state current at +70 mV is blocked by DIDS with an IC50 of 2.3 microM. The number of open channels at -10 mV is reduced with an IC50 of 22 microM. The electrophysiological and pharmacological properties of this current are most similar to those of the Cl- current in T lymphocytes activated by osmotic stress (Lewis, R. S., P. E. Ross, and M. D. Cahalan. 1993. Journal of General Physiology. 101:801-826). Extracellular DIDS also inhibits exocytosis. At optimal stimulation with 10 micrograms/ml compound 48/80 secretion is inhibited with an IC50 = 50 microM and a Hill coefficient n = 10. At half optimal stimulation with 1 microgram/ml inhibition occurs with an IC50 = 10 microM and n = 1. Substitution of extracellular chloride by glutamate has only very small effects on secretion stimulated with 10 micrograms/ml compound 48/80. We conclude that activation of the chloride current in mast cells is not essential for stimulation of exocytosis but may enhance secretion at suboptimal stimulation. Alternatively, the channel may play a role in volume regulation following degranulation.

Four different components contribute to outward current in rat ventricular myocytes

1999 ◽  
Vol 277 (1) ◽  
pp. H107-H118 ◽  
Author(s):  
Herbert M. Himmel ◽  
Erich Wettwer ◽  
Qi Li ◽  
Ursula Ravens
Keyword(s):  
Steady State ◽  
Ventricular Myocytes ◽  
Outward Current ◽  
Cell Voltage ◽  
Differential Sensitivity ◽  
Resting Potential ◽  
Residual Fraction ◽  
Voltage Range ◽  
Rat Ventricular Myocytes ◽  
Steady State Current

In rat ventricle, two Ca2+-insensitive components of K+ current have been distinguished kinetically and pharmacologically, the transient, 4-aminopyridine (4-AP)-sensitive I to and the sustained, tetraethylammonium (TEA)-sensitive I K. However, a much greater diversity of depolarization-activated K+ channels has been reported on the level of mRNA and protein. In the search for electrophysiological evidence of further current components, the whole cell voltage-clamp technique was used to analyze steady-state inactivation of outward currents by conditioning potentials in a wide voltage range. Peak ( I peak) and late ( I late) currents during the test pulse were analyzed by Boltzmann curve fitting, producing three fractions each. Fractions a and b had different potentials of half-maximum inactivation ( V 0.5); the third residual fraction, r, did not inactivate. Fractions a for I peak and I late had similar relative amplitudes and V 0.5 values, whereas size and V 0.5 of fractions b differed significantly between I peak and I late. Only b of I peak was transient, suggesting a relation with I to, whereas a, b, and r of I late appeared to be three different sustained currents. Therefore, four individual outward current components were distinguished: I to( b of I peak), I K( a), the steady-state current I ss( r), and the novel current I Kx( b of I late). This was further supported by differential sensitivity to TEA, 4-AP, clofilium, quinidine, dendrotoxin, heteropodatoxin, and hanatoxin. With the exception of I to, none of the currents exhibited a marked transmural gradient. Availability of I K was low at resting potential; nevertheless, I K contributed to action potential shortening in hyperpolarized subendocardial myocytes. In conclusion, on the basis of electrophysiological and pharmacological evidence, at least four components contribute to outward current in rat ventricular myocytes.

Long-Term Effects of Prior Heat Shock on Neuronal Potassium Currents Recorded in a Novel Insect Ganglion Slice Preparation

1999 ◽  
Vol 81 (2) ◽  
pp. 795-802 ◽  
Author(s):  
J. M. Ramirez ◽  
F. P. Elsen ◽  
R. M. Robertson
Keyword(s):  
Steady State ◽  
Heat Shock ◽  
Outward Current ◽  
Potassium Currents ◽  
Respiratory Neurons ◽  
Long Term Effects ◽  
Slice Preparation ◽  
Population Activity ◽  
Steady State Current

Long-term effects of prior heat shock on neuronal potassium currents recorded in a novel insect ganglion slice preparation. Brief exposure to high temperatures (heat shock) induces long-lasting adaptive changes in the molecular biology of protein interactions and behavior of poikilotherms. However, little is known about heat shock effects on neuronal properties. To investigate how heat shock affects neuronal properties we developed an insect ganglion slice from locusts. The functional integrity of neuronal circuits in slices was demonstrated by recordings from rhythmically active respiratory neurons and by the ability to induce rhythmic population activity with octopamine. Under these “functional” in vitro conditions we recorded outward potassium currents from neurons of the ventral midline of the A1 metathoracic neuromere. In control neurons, voltage steps to 40 mV from a holding potential of −60 mV evoked in control neurons potassium currents with a peak current of 10.0 ± 2.5 nA and a large steady state current of 8.5 ± 2.6 nA, which was still activated from a holding potential of −40 mV. After heat shock most of the outward current inactivated rapidly (peak amplitude: 8.4 ± 2.4 nA; steady state: 3.6 ± 2.0 nA). This current was inactivated at a holding potential of −40 mV. The response to temperature changes was also significantly different. After changing the temperature from 38 to 42°C the amplitude of the peak and steady-state current was significantly lower in neurons obtained from heat-shocked animals than those obtained from controls. Our study indicates that not only heat shock can alter neuronal properties, but also that it is possible to investigate ion currents in insect ganglion slices.

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