scholarly journals The mammalian Na+/H+ antiporters NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent, but can couple to an H+ conductance.

1995 ◽  
Vol 106 (1) ◽  
pp. 85-111 ◽  
Author(s):  
N Demaurex ◽  
J Orlowski ◽  
G Brisseau ◽  
M Woodside ◽  
S Grinstein
Keyword(s):  
Cho Cells ◽  
Time Course ◽  
Chinese Hamster ◽  
Reversal Potential ◽  
Outward Current ◽  
Rt Pcr ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Transmembrane Currents ◽  
Cytosolic Acidification

Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT-PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time-course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH-sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.

scholarly journals Evidence That the Product of the Human X-Linked Cgd Gene, Gp91-phox, Is a Voltage-Gated H+ Pathway

1999 ◽  
Vol 114 (6) ◽  
pp. 771-786 ◽  
Author(s):  
Lydia M. Henderson ◽  
Robert W. Meech
Keyword(s):  
Cell Membrane ◽  
Time Course ◽  
Lucifer Yellow ◽  
Chinese Hamster ◽  
Reversal Potential ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Histidine Residues ◽  
Voltage Gated

Expression of gp91-phox in Chinese hamster ovary (CHO91) cells is correlated with the presence of a voltage-gated H+ conductance. As one component of NADPH oxidase in neutrophils, gp91-phox is responsible for catalyzing the production of superoxide (O2·2). Suspensions of CHO91 cells exhibit arachidonate-activatable H+ fluxes (Henderson, L.M., G. Banting, and J.B. Chappell. 1995. J. Biol. Chem. 270:5909–5916) and we now characterize the electrical properties of the pathway. Voltage-gated currents were recorded from CHO91 cells using the whole-cell configuration of the patch-clamp technique under conditions designed to exclude a contribution from ions other than H+. As in other voltage-gated proton currents (Byerly, L., R. Meech, and W. Moody. 1984. J. Physiol. 351:199–216; DeCoursey, T.E., and V.V. Cherny. 1993. Biophys. J. 65:1590–1598), a lowered external pH (pHo) shifted activation to more positive voltages and caused the tail current reversal potential to shift in the manner predicted by the Nernst equation. The outward currents were also reversibly inhibited by 200 μM zinc. Voltage-gated currents were not present immediately upon perforating the cell membrane, but showed a progressive increase over the first 10–20 min of the recording period. This time course was consistent with a gradual shift in activation to more negative potentials as the pipette solution, pH 6.5, equilibrated with the cell contents (reported by Lucifer yellow included in the patch pipette). Use of the pH-sensitive dye 2′7′ bis-(2-carboxyethyl)-5(and 6) carboxyfluorescein (BCECF) suggested that the final intracellular pH (pHi) was ∼6.9, as though pHi was largely determined by endogenous cellular regulation. Arachidonate (20 μM) increased the amplitude of the currents by shifting activation to more negative voltages and by increasing the maximally available conductance. Changes in external Cl− concentration had no effect on either the time scale or the appearance of the currents. Examination of whole cell currents from cells expressing mutated versions of gp91-phox suggest that: (a) voltage as well as arachidonate sensitivity was retained by cells with only the NH2-terminal 230 amino acids, (b) histidine residues at positions 111, 115, and 119 on a putative membrane-spanning helical region of the protein contribute to H+ permeation, (c) histidine residues at positions 111 and 119 may contribute to voltage gating, (d) the histidine residue at position 115 is functionally important for H+ selectivity. Mechanisms of H+ permeation through gp91-phox include the possible protonation/deprotonation of His-115 as it is exposed alternatively to the interior and exterior faces of the cell membrane (see Starace, D.M., E. Stefani, and F. Bezanilla. 1997. Neuron. 19:1319–1327) and the transfer of protons across an “H-X-X-X-H-X-X-X-H” motif lining a conducting pore.

Permeability of time-dependent K+ channel in guinea pig ventricular myocytes to Cs+, Na+, NH4+, and Rb+

1990 ◽  
Vol 259 (5) ◽  
pp. H1448-H1454 ◽  
Author(s):  
R. W. Hadley ◽  
J. R. Hume
Keyword(s):  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Reversal Potential ◽  
Outward Current ◽  
Time Dependent ◽  
K Channel ◽  
Delayed Rectifier ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Outward Currents

Currents through time-dependent K+ channels (also referred to as IK or the delayed rectifier) were studied with the whole cell patch-clamp technique in isolated guinea pig ventricular myocytes. IK measurements were restricted to the examination of deactivation tail currents. Substitution of various monovalent cations for external K+ produced shifts of the reversal potential of IK. These shifts were used to calculate permeability ratios relative to K+. The permeability sequence for the IK channels was K+ = Rb+ greater than NH4+ = Cs+ greater than Na+. Time-dependent outward currents were also examined when the myocytes were dialyzed with Cs+ instead of K+. A sizeable time-dependent outward current, quite similar to that seen with K+ dialysis, was demonstrated. This current was primarily carried by intracellular Cs+, as the reversal potential of the current shifted 46 mV per 10-fold change of external Cs+ concentration. The significance of Cs+ permeation through IK channels is discussed with respect to the common use of Cs+ in isolating other currents.

Expression Clone of TMEM16A as a Calcium-Activated Chloride Channels in CHO Cells

2013 ◽  
Vol 709 ◽  
pp. 832-835
Author(s):  
Feng Hao ◽  
Yi Ju Hou ◽  
Chen Zhao ◽  
Li Zhang ◽  
Zhi Qiang Tong ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Cho Cells ◽  
Chloride Channels ◽  
Chinese Hamster ◽  
Green Fluorescence Protein ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Clamp Technique ◽  
Cell Patch Clamp ◽  
Whole Cell Patch Clamp

There is compelling evidence that TMEM16A fuctions as calcium-activated chloride channels (CaCCS), which was discovered by three independent labs in 2008 after Calcium-activated chloride channel current was first recorded in the 1980s. CaCCs are involved in many physiological processes, including transepithelial fluid secretion, smooth muscle contraction , sensory signal transduction and others. CaCCs are considers as potential drug therapy of hypertension, secretoy diarrheas, neuropathic pain, asthma, cystic fibrosis and certain tumors. In our previous study, TMEM16A with green fluorescence protein (GFP) fusion protein were subcloned into pcDNA3.1/Zeo. In this study, TMEM16A transient transfection conditon of Chinese hamster ovary (CHO) cells were optimized through liposome transfection and CHO cells expressing TMEM16A were got by stable transfection in which the classical calcium-activated chloride channels current was recorded by whole cell patch clamp technique. By a comparison between the results in this study and the results in previous study, both CHO and FRT cells are suitable for TMEM16A-pcDNA3.1 expression through liposome transfection and currents were recorded in both FRT and CHO cells by whole cell patch clamp technique, but our results indicated different purposes should require different cell lines and methods. These results were beneficial for the delving into study of TMEM16A-CaCCs by patch clamp technique which is the gold standard for real-time investigation of ion channels and their effectors.

A voltage-gated chloride conductance in rat cultured astrocytes

1986 ◽  
Vol 228 (1252) ◽  
pp. 267-288 ◽  
Keyword(s):  
Time Course ◽  
Reversal Potential ◽  
Outward Current ◽  
Chloride Ions ◽  
Chloride Conductance ◽  
Equilibrium Potential ◽  
Free Calcium ◽  
Patch Clamp Technique ◽  
Bathing Medium ◽  
Cultured Astrocytes

Large voltage-dependent outward currents are recorded with the wholecell patch-clamp technique from rat cultured astrocytes under conditions where an outward movement of potassium ions is excluded (either by blockage of the potassium channels pharmacologically or by replacement of the internal potassium by the impermeant large organic cation N -methyl-( + )-glucamine). The current, which is activated at potentials more positive than —40 to —50 mV, is normally carried by an inward movement of chloride ions. Its reversal potential is the same as the chloride equilibrium potential. With depolarization to +60 mV (for 225 ms) little or no inactivation of the current occurs: with depolarizations to +90 to +110 mV a time-dependent decay is seen. The current, which is often not marked immediately after formation of the whole-cell clamp, generally increases over a period of a few minutes to a maximum (after which it usually declines), as if some as yet unknown intracellular factor keeping the channels closed were being washed away from the membrane. The time course of this phenomenon is not affected by changing of the internal free calcium concentration (from 10 -8 to 10 -6 m) or by an intracellular mixture of cyclic AMP (1 mm), ATP (4 mm) and Mg + (2 mm). The conductance is slightly increased when the chloride of the bathing medium is replaced by bromide; is much reduced on replacement by methylsulphate, sulphate, isethionate, or acetate; and is virtually abolished on replacement by the large anion gluconate. The outward current is inhibited by the disulphonate stilbenes DIDS and SITS; this blocking action was initially partly reversible, although never completely so. It is suggested that the chloride conductance plays a role in the spatial buffering of potassium by astrocytes.

scholarly journals A time-dependent and voltage-sensitive K+ current in single cells from frog atrium.

1986 ◽  
Vol 88 (6) ◽  
pp. 739-755 ◽  
Author(s):  
M A Simmons ◽  
T Creazzo ◽  
H C Hartzell
Keyword(s):  
Single Cells ◽  
Quantitative Description ◽  
Reversal Potential ◽  
Outward Current ◽  
Time Dependent ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
K Current ◽  
And Shifts ◽  
Frog Atrium

A quantitative description of the time-dependent and voltage-sensitive outward currents in heart has been hampered by the complications inherent to the multicellular preparations previously used. We have used the whole-cell patch-clamp technique to record the delayed outward K+ current, IK, in single cells dissociated from frog atrium. Na+ currents were blocked with tetrodotoxin and Ca2+ currents with Mn2+ or Cd2+. After depolarizations from -50 mV to potentials positive to -30 mV, a time-dependent outward current was observed. This current has been characterized according to its steady state activation, kinetics, and ion transfer function. The current is well described as a single Hodgkin-Huxley conductance. The deactivation of the current is a single exponential. Activation of the current is sigmoid and is fitted well by raising the activation variable to the second power. The reversal potential of IK is near EK and shifts by 57 mV/10-fold change in [K+]o. This suggests that the current is carried selectively by K ions. The threshold for activation is near -30 mV. IK is maximally activated positive to +20 mV and shows no inactivation. The fully activated current-voltage relationship is linear between -110 and +50 mV. Neither Ba2+ (250 microM) nor Cd2+ (100 microM) affects IK.

Acute effects of repetitive depolarization on sodium current in chick myocytes

1991 ◽  
Vol 260 (6) ◽  
pp. H1810-H1818
Author(s):  
M. R. Gold ◽  
G. R. Strichartz
Keyword(s):  
Time Course ◽  
Sodium Current ◽  
Complete Recovery ◽  
Acute Effects ◽  
Na Current ◽  
Patch Clamp Technique ◽  
Membrane Hyperpolarization ◽  
Atrial Cells ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation

Acute effects of repetitive depolarization on the inward Na+ current (INa) of cultured embryonic chick atrial cells were studied using the whole cell patch-clamp technique. Stimulation rates of 1 Hz or greater produced a progressive decrement of peak INa. With depolarizations to 0 mV of 150-ms duration, applied at 2 Hz from a holding potential of -100 mV, the steady-state decrement was approximately 20%. The magnitude of this effect increased with stimulation frequency and with test potential depolarization and decreased with membrane hyperpolarization. Analysis of INa kinetics revealed that reactivation was sufficiently slow to preclude complete recovery from inactivation with interpulse intervals less than 1,000 ms. Moreover, reactivation accelerated markedly with membrane hyperpolarization, in parallel with the response to repetitive stimulation. The multiexponential time course of recovery of peak INa from repetitive depolarization was similar to that observed after single stimuli; however, there was a shift toward a greater proportion of current recovering with the slower of two time constants. It is concluded that incomplete recovery from inactivation is responsible for the decrement in INa observed with short interpulse intervals.

BICUCULLINE/BACLOFEN-INSENSITIVE GABA RESPONSE IN CRUSTACEAN NEURONES IN CULTURE

1994 ◽  
Vol 191 (1) ◽  
pp. 167-193
Author(s):  
C Jackel ◽  
W Krenz ◽  
F Nagy
Keyword(s):  
Reversal Potential ◽  
Membrane Conductance ◽  
Gamma Aminobutyric Acid ◽  
Action Potential Firing ◽  
Patch Clamp Technique ◽  
Conformationally Restricted ◽  
Whole Cell Patch Clamp ◽  
Gabac Receptor ◽  
Potential Firing ◽  
Sr 95531

Neurones were dissociated from thoracic ganglia of embryonic and adult lobsters and kept in primary culture. When gamma-aminobutyric acid (GABA) was applied by pressure ejection, depolarizing or hyperpolarizing responses were produced, depending on the membrane potential. They were accompanied by an increase in membrane conductance. When they were present, action potential firing was inhibited. The pharmacological profile and ionic mechanism of GABA-evoked current were investigated under voltage-clamp with the whole-cell patch-clamp technique. The reversal potential of GABA-evoked current depended on the intracellular and extracellular Cl- concentration but not on extracellular Na+ and K+. Blockade of Ca2+ channels by Mn2+ was also without effect. The GABA-evoked current was mimicked by application of the GABAA agonists muscimol and isoguvacine with an order of potency muscimol>GABA>isoguvacine. cis-4-aminocrotonic acid (CACA), a folded and conformationally restricted GABA analogue, supposed to be diagnostic for the vertebrate GABAC receptor, also induced a bicuculline-resistant chloride current, although with a potency about 10 times lower than that of GABA. The GABA-evoked current was largely blocked by picrotoxin, but was insensitive to the GABAA antagonists bicuculline, bicuculline methiodide and SR 95531 at concentrations of up to 100 µmol l-1. Diazepam and phenobarbital did not exert modulatory effects. The GABAB antagonist phaclophen did not affect the GABA-induced current, while the GABAB agonists baclophen and 3-aminopropylphosphonic acid (3-APA) never evoked any response. Our results suggest that lobster thoracic neurones in culture express a chloride-conducting GABA-receptor channel which conforms to neither the GABAA nor the GABAB types of vertebrates but shows a pharmacology close to that of the novel GABAC receptor described in the vertebrate retina.

Regulation of calcium current by voltage and cytoplasmic calcium in canine gastric smooth muscle

1992 ◽  
Vol 262 (3) ◽  
pp. C691-C700 ◽  
Author(s):  
F. Vogalis ◽  
N. G. Publicover ◽  
K. M. Sanders
Keyword(s):  
Time Course ◽  
Fast Component ◽  
Patch Clamp Technique ◽  
Similar Time ◽  
Whole Cell Patch Clamp ◽  
Gastric Smooth Muscle ◽  
Double Exponential ◽  
Circular Layer ◽  
Double Exponential Function ◽  
Ca2 Channels

The regulation of Ca2+ current by intracellular Ca2+ was studied in isolated myocytes from the circular layer of canine gastric antrum. Ca2+ current was measured with the whole cell patch-clamp technique, and changes in cytoplasmic Ca2+ ([Ca2+]i) were simultaneously measured with indo-1 fluorescence. Ca2+ currents were activated by depolarization and inactivated despite maintained depolarization. Ca2+ current inactivation was fit with a double exponential function. Using Ba2+ or Na+ as charge carriers removed the fast component of inactivation, whereas enhanced intracellular buffering of Ca2+ did not remove the fast component. Ca2+ currents were associated with a rise in [Ca2+]i. The decrease in [Ca2+]i following repolarization was exponential, and during the relaxation of [Ca2+]i, Ca2+ current was inactivated. The inward current recovered with a similar time course as the decrease in [Ca2+]i, suggesting that [Ca2+]i regulates the basal availability of Ca2+ channels. These data support the hypothesis that, although [Ca2+]i may influence the resting level of inactivation, it is the "submembrane" compartment of [Ca2+]i that regulates the development of inactivation.

Glycine regulation of synaptic NMDA receptors in hippocampal neurons

1996 ◽  
Vol 76 (5) ◽  
pp. 3415-3424 ◽  
Author(s):  
K. S. Wilcox ◽  
R. M. Fitzsimonds ◽  
B. Johnson ◽  
M. A. Dichter
Keyword(s):  
Nmda Receptor ◽  
Nmda Receptors ◽  
Hippocampal Neurons ◽  
Time Course ◽  
Hippocampal Slices ◽  
Maximal Effect ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Dissociated Cell Culture ◽  
Cultured Hippocampal Neurons

1. Although glycine has been identified as a required coagonist with glutamate at N-methyl-D-aspartate (NMDA) receptors, the understanding of glycine's role in excitatory synaptic neurotransmission is quite limited. In the present study, we used the whole cell patch-clamp technique to examine the ability of glycine to regulate current flow through synaptic NMDA receptors at excitatory synapses between cultured hippocampal neurons and in acutely isolated hippocampal slices. 2. These studies demonstrate that the glycine modulatory site on the synaptic NMDA receptor is not saturated under baseline conditions and that increased glycine concentrations can markedly increased NMDA-receptor-mediated excitatory postsynaptic currents (EPSCs) in hippocampal neurons in both dissociated cell culture and in slice. Saturation of the maximal effect of glycine takes place at different concentrations for different cells in culture, suggesting the presence of heterogenous NMDA receptor subunit compositions. 3. Bath-applied glycine had no effect on the time course of EPSCs in either brain slice or culture, indicating that desensitization of the NMDA receptor is not prevented by glycine over the time course of an EPSC. 4. When extracellular glycine concentration is high, all miniature EPSCs recorded in the cultured hippocampal neurons contained NMDA components, indicating that segregation of non-NMDA receptors at individual synaptic boutons does not occur.

Force measurements from voltage-clamped guinea pig ventricular myocytes

1990 ◽  
Vol 258 (2) ◽  
pp. H452-H459 ◽  
Author(s):  
N. Shepherd ◽  
M. Vornanen ◽  
G. Isenberg
Keyword(s):  
Guinea Pig ◽  
Time Course ◽  
Ventricular Myocytes ◽  
Isometric Force ◽  
Contractile Force ◽  
Patch Clamp Technique ◽  
Thin Glass ◽  
Tonic Component ◽  
Time To Peak ◽  
Whole Cell Patch Clamp

We describe the first observations of isolated mammalian guinea pig ventricular myocytes that combine measurements of contractile force with the voltage-clamp method. The myocytes were attached by poly-L-lysine to the beveled ends of a pair of thin glass rods having a compliance of 0.76 m/N. The contractile force of a cell caused a 1- to 3-microm displacement of the rods; the motion of which was converted to an output voltage by phototransistors. By the use of the whole cell patch-clamp technique, the cells were depolarized at 1 Hz with 200-ms-long clamp pulses from -45 to +5 mV (35 degrees C, 3.6 mM CaCl2). Isometric force began after a latency of 7 +/- 2 ms, peaked at 93 +/- 21 ms, and relaxed (90%) at 235 +/- 63 ms. The time course of force was always faster than that of isotonic shortening (time to peak 154 +/- 18 ms). With 400-ms-long depolarizations, a tonic component was recorded as either sustained force or sustained shortening that decayed on repolarization. Substitution of Ca by Sr in the bath increased the inward current through Ca channels but slowed down the time course of force development. The results are consistent with the hypothesis that activator calcium derives mainly from internal stores and that Ca release needs Ca entry through channels.

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