Nonselective cation and Cl channels in luminal membrane of the marginal cell

1993 ◽  
Vol 265 (1) ◽  
pp. C72-C78 ◽  
Author(s):  
H. Sunose ◽  
K. Ikeda ◽  
Y. Saito ◽  
A. Nishiyama ◽  
T. Takasaka
Keyword(s):  
Single Channel ◽  
Patch Clamp Technique ◽  
Cl Channel ◽  
Luminal Membrane ◽  
Cl Channels ◽  
Cytoplasmic Acidification ◽  
Marginal Cells ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Linear Conductance

Single-channel currents of the luminal membrane of marginal cells dissected from the guinea pig cochlea were investigated using the patch-clamp technique. Nonselective cation channels having a linear conductance of 27 pS were activated by depolarization, cytoplasmic Ca2+, and cytoplasmic acidification. Cytoplasmic ATP inactivated the channel. A mixture of 3-isobutyl-1-methylxanthine and forskolin activated a small-conductance Cl channel in the cell-attached mode. On excision in the inside-out mode, the Cl channel was inactivated, but it was reactivated by a cytoplasmic catalytic subunit of protein kinase A with ATP. This Cl channel had a linear conductance of 12 pS, and its activity was little affected by voltage. The sequence of permeation by anions was Br- > Cl > I-. The Cl channel blocker diphenylamine-2-carboxylic acid (3 mM) completely blocked the channel, but 5-nitro-2-(3-phenylpropylamino)-benzoic acid (50 microM) blocked it only partially. The above-mentioned characteristics are similar to those of the well-demonstrated Cl- channel, cystic fibrosis transmembrane regulator.

Anion channels for amino acids in MDCK cells

1992 ◽  
Vol 263 (6) ◽  
pp. C1200-C1207 ◽  
Author(s):  
U. Banderali ◽  
G. Roy
Keyword(s):  
Amino Acids ◽  
Single Channel ◽  
Mdck Cells ◽  
Reversal Potential ◽  
Patch Clamp Technique ◽  
Excised Patches ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Inside Out ◽  
Cytoplasmic Side

Large losses of amino acids by diffusion were previously observed in Madin-Darby canine kidney (MDCK) cells during volume regulation. Also, an outward rectifying anion channel was activated. Because this channel was not selective among anions, it was suggested that it could be permeable to amino acids. Its permeability to aspartate, glutamate, and taurine was studied using the patch-clamp technique in the inside-out configuration. Solutions containing 500 mM aspartate or glutamate were used on the cytoplasmic side of excised patches to detect single-channel currents carried by these anions. Permeability ratios were estimated in two different ways: 1) from the shift in reversal potential of current-voltage curves after anion replacement in the bath solution and 2) from comparisons of amplitudes of single-channel currents carried by tested anions and chloride, respectively. The values of aspartate-to-chloride and glutamate-to-chloride permeability ratios obtained with both methods were quite consistent and were of the order of 0.2 for both amino acids. Taurine in solutions at physiological pH 7.3 is a zwitterionic molecule and bears no net charge. To detect single-channel currents carried by taurine, solutions containing 500 mM taurine at pH 8.2 were used in inside-out experiments. Under these conditions 120 mM of negatively charged taurine was present in the solutions bathing the cytoplasmic side of excised patches. The permeability ratio estimated from the shift in reversal potential was 0.75. These results showed that some of the organic compounds released by cells during regulatory volume decrease could diffuse through this outwardly rectifying anionic channel.

Ca(2+)-dependent Cl- channels in undifferentiated human colonic cells (HT-29). I. Single-channel properties

1993 ◽  
Vol 264 (4) ◽  
pp. C968-C976 ◽  
Author(s):  
A. P. Morris ◽  
R. A. Frizzell
Keyword(s):  
Single Channel ◽  
Voltage Sensitivity ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Channel Activation ◽  
Whole Cell ◽  
Multiple Channel ◽  
Cl Channel ◽  
Cl Channels ◽  
Ht 29

The patch-clamp technique was combined with camera-based intracellular Ca2+ concentration ([Ca2+]i) imaging to identify the single-channel basis of the Ca(2+)-dependent Cl- conductance in human colonic adenocarcinoma cells (HT-29). Cl- channels were activated when membrane patches were excised into solutions containing high (1 microM) Ca2+ concentrations. Their single-channel conductance, measured by amplitude histogram analysis, averaged 13 pS at -90 mV and 16 pS at +90 mV membrane potential (MP). In multiple channel patches, Cl- currents showed properties similar to Ca(2+)-activated whole cell currents: outward rectification and time-dependent activation at depolarizing MP. Channel activity disappeared shortly after patch excision from the cell. In cell-attached patches, Cl- channel opening was infrequent at resting [Ca2+]i values (96 +/- 18 nM), but when [Ca2+]i was increased by the Ca2+ ionophore ionomycin (1 microM), Cl- channels were activated with a time course that paralleled the [Ca2+]i rise. Repetitive ionophore exposure produced equivalent rises in [Ca2+]i, but the corresponding Cl- channel activity became progressively reduced. The Ca(2+)-mediated agonist neurotensin (50 nM) elicited a transient Cl- channel activation that preceded the generalized cellular [Ca2+]i rise. Channel activation with neurotensin occurred in the absence of pipette Ca2+ but was abolished by preloading cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Thus, in response to the Ca(2+)-mediated agonist neurotensin, Cl- channel activation results from Ca2+ mobilization from intracellular pools localized within the vicinity of the plasma membrane. The Ca2+ dependency, voltage sensitivity, and kinetics of this 15-pS Cl- channel indicate that it is the basis of the whole cell Ca(2+)-activated Cl- current.

scholarly journals Ion channels activated by light in Limulus ventral photoreceptors.

1986 ◽  
Vol 87 (1) ◽  
pp. 73-89 ◽  
Author(s):  
J Bacigalupo ◽  
K Chinn ◽  
J E Lisman
Keyword(s):  
Light Adaptation ◽  
Voltage Dependence ◽  
Single Channel ◽  
Patch Clamp Technique ◽  
Channel Activation ◽  
Voltage Range ◽  
Secondary Result ◽  
Channel Open Time ◽  
Single Channel Currents ◽  
Channel Currents

The light-activated conductance of Limulus ventral photoreceptors was studied using the patch-clamp technique. Channels (40 pS) were observed whose probability of opening was greatly increased by light. In some cells the latency of channel activation was nearly the same as that of the macroscopic response, while in other cells the channel latency was much greater. Like the macroscopic conductance, channel activity was reduced by light adaptation but enhanced by the intracellular injection of the calcium chelator EGTA. The latter observation indicates that channel activation was not a secondary result of the light-induced rise in intracellular calcium. A two-microelectrode voltage-clamp method was used to measure the voltage dependence of the light-activated macroscopic conductance. It was found that this conductance is constant over a wide voltage range more negative than zero, but it increases markedly at positive voltages. The single channel currents measured over this same voltage range show that the single channel conductance is independent of voltage, but that channel gating properties are dependent on voltage. Both the mean channel open time and the opening rate increase at positive voltages. These properties change in a manner consistent with the voltage dependence of the macroscopic conductance. The broad range of similarities between the macroscopic and single channel currents supports the conclusion that the 40-pS channel that we have observed is the principal channel underlying the response to light in these photoreceptors.

Characterization of single non-inactivating potassium channels in primary neuronal cultures of Drosophila

1989 ◽  
Vol 145 (1) ◽  
pp. 173-184
Author(s):  
D. Yamamoto ◽  
N. Suzuki
Keyword(s):  
Time Distribution ◽  
Single Channel ◽  
K Channel ◽  
Delayed Rectifier ◽  
Neuronal Cultures ◽  
Patch Clamp Technique ◽  
Primary Neuronal Cultures ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Cytoplasmic Side

Permeability and gating properties of single, non-inactivating, K+ channel currents in cultured Drosophila neurons were studied using the gigaohm-seal patch-clamp technique. The non-inactivating K+ currents were activated by depolarizing the membrane to −30 mV or to more positive potentials. The slope conductance of the channel was estimated to be 17.6 +/− 3.70 pS when the cytoplasmic side of the inside-out membrane patch was perfused with solutions containing 145 mmoll-1 K+. The single-channel conductance was temperature-sensitive, with a Q10 of 1.44 between 10 and 20 degrees C. Single-channel currents could be recorded when the cytoplasmic K+ was replaced with NH4+, Rb+ or Na+, but not with Cs+. The conductance ratio of the channel for these cations was: K+ (1) greater than NH4+(0.53) greater than Rb+ (0.47) greater than Na+ (0.44). Tetraethylammonium (TEA+) ions applied at a concentration of 10 mmoll-1 to the cytoplasmic side of the membrane increased the frequency of ‘blank’ traces which contained no channel openings during repetitive depolarization. In addition, single-channel amplitude was reduced by about 20%. The open-time distribution was fitted by a single exponential function, whereas the closed-time distribution required a three-exponential fit. Permeability and gating properties of single, non-inactivating K+ channel currents in neurons of eag, a mutant which has defects in the delayed rectifier K+ channel, were indistinguishable from those recorded from wild-type neurons.

PKC activity modulates availability and long openings of L-type Ca2+ channels in A7r5 cells

1998 ◽  
Vol 275 (2) ◽  
pp. C535-C543 ◽  
Author(s):  
C. A. Obejero-Paz ◽  
M. Auslender ◽  
A. Scarpa
Keyword(s):  
Okadaic Acid ◽  
Single Channel ◽  
Inhibitory Effect ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Whole Cell ◽  
A7r5 Cells ◽  
High Level ◽  
Single Channel Currents ◽  
Channel Currents

The possibility that protein kinase C (PKC) could control the activity of L-type Ca2+ channels in A7r5 vascular smooth muscle-derived cells in the absence of agonist stimulation was investigated using the patch-clamp technique. Consistent with the possibility that L-type Ca2+ channels are maximally phosphorylated by PKC under these conditions, we show that 1) activation of PKC with the phorbol ester phorbol 12,13-dibutyrate was ineffective in modulating whole cell and single-channel currents, 2) inhibition of PKC activity with staurosporine or chelerythrine inhibited channel activity, 3) inhibition of protein phosphatases by intracellular dialysis of okadaic acid did not affect whole cell currents, and 4) the inhibitory effect of staurosporine was absent in the presence of okadaic acid. The inhibition of Ca2+ currents by PKC inhibitors was due to a decrease in channel availability and long open events, whereas the voltage dependence of the open probability and the single-channel conductance were not affected. The evidence suggests that in resting, nonstimulated A7r5 cells there is a high level of PKC activity that modulates the gating of L-type Ca2+ channels.

Calcium-activated chloride channels in cultured embryonic Xenopus spinal neurons

1992 ◽  
Vol 68 (6) ◽  
pp. 2042-2050 ◽  
Author(s):  
N. Hussy
Keyword(s):  
Single Channel ◽  
Developmental Change ◽  
Reversal Potential ◽  
Equilibrium Potential ◽  
Ionophore A23187 ◽  
Spinal Neurons ◽  
Patch Clamp Technique ◽  
Cl Channels ◽  
Single Channel Currents

1. Single-channel currents were recorded from Xenopus spinal neurons developing in vitro using the patch-clamp technique, to identify the channels underlying the large and small macroscopic Ca(2+)-activated Cl- currents (ICl(Ca)) present in these cells. 2. Channels of large (maxi-channels; 310 pS) and smaller conductance (mini-channels; 50-60 pS) are activated by elevation of cytoplasmic Ca2+ concentration. Channel activity is not altered by subsequent removal of Ca2+ from the bath, arguing against a direct ligand-type Ca2+ dependence. The much higher incidence of channel activation in cell-attached patches from cells permeabilized with the Ca2+ ionophore A23187 than in excised patches also suggests the involvement of some unidentified intracellular factor. 3. The reversal potential of maxi-Cl- channels is not altered by changes in Na+ concentration, but is shifted in the negative direction by the substitution of Cl- by methanesulfonate on the intracellular side of the patch, indicating their anionic selectivity. 4. Maxi-Cl- channels exhibited the presence of multiple probable subconductance states and showed marked voltage-dependent inactivation above and below +/- 20 mV. 5. Examination of maxi-Cl- channels at early times in culture (6-9 h) and 24 h later did not reveal any developmental change in the characteristics described above. However, the mean open duration of the channel was found to increase twofold during this period of time. 6. The simultaneous presence of maxi- and mini-Cl- channels prevented detailed characterization of the latter. The anionic selectivity of mini-Cl- channels is suggested by their reversal potential that lies close to the Cl- equilibrium potential.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Phagocytosis by human macrophages is accompanied by changes in ionic channel currents.

1988 ◽  
Vol 106 (6) ◽  
pp. 1873-1878 ◽  
Author(s):  
C Ince ◽  
J M Coremans ◽  
D L Ypey ◽  
P C Leijh ◽  
A A Verveen ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Single Channel ◽  
Ionic Channel ◽  
Mononuclear Phagocytes ◽  
Patch Clamp Technique ◽  
Intact Cells ◽  
Latex Particles ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Particle Attachment

The present study has shown that changes in ionic channel currents accompany the phagocytosis of particles by mononuclear phagocytes. The patch-clamp technique in the cell-attached configuration was applied to human monocyte-derived macrophages to measure the activity of single transmembrane ionic channels in intact cells. During such measurements, IgG-opsonized and non-opsonized latex particles were offered for phagocytosis under continuous video-microscopical observation. Single particles were presented to the phagocytes at a membrane location some distance from that of the patch electrode. After a lag period following particle attachment, enhanced inward and outward time-variant single channel currents coinciding with particle engulfment were observed. On the basis of current-voltage characteristics and membrane potential measurements, the outward-directed channels were identified as K+ channels. Phagocytosis was also accompanied by slow transient changes in background membrane currents, probably due to changes in the membrane potential of the phagocytosing cell. Phagocytosis of IgG-coated latex particles differed from phagocytosis of uncoated or albumin-coated particles by a shorter lag time between particle attachment and the onset of enhanced ionic channel activity.

Oxantel-activated single channel currents in the muscle membrane of Ascaris suum

Parasitology ◽  
1995 ◽  
Vol 110 (4) ◽  
pp. 437-448 ◽  
Author(s):  
V. M. E. Dale ◽  
R. J. Martin
Keyword(s):  
Single Channel ◽  
Ascaris Suum ◽  
Muscle Membrane ◽  
Kinetic Properties ◽  
Voltage Sensitivity ◽  
Channel Block ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Single Channel Currents ◽  
Channel Currents

SUMMARYThe patch clamp technique was used to investigate the action of the anthelmintic drug, oxantel, on nicotinic acetylcholine receptor (nAChR) currents recorded from vesicles of the somatic muscle cells of the nematode parasite Ascaris suum. The amplitudes of the currents were analysed at different membrane potentials to determine the single channel conductance. Also the open and closed durations were measured to determine the kinetic properties of the activated channel. Oxantel activated single nAChR currents throughout a concentration range 10–100 μM, these currents were not observed with oxantel-free pipette solutions. The mean open time of the activated channels at a membrane potential of –75 mV and a concentration of 10 μM was 1·34 ms. At higher concentrations the open times were shorter and voltage sensitive, decreasing in duration on hyperpolarization, thus suggesting open channel block. The kinetics were analysed using a simple channel block model. The forward block rate, K + B, increased with increasing oxantel concentration but showed little increase as the membrane was hyperpolarized. K + B was 2·41×107 M−1s−1 – 50 mV and 2·64 × 107 M−1s−1 at – 100mV. The unblocking rate constant, K – B, did exhibit voltage sensitivity being 443·6 s−1 at – 50 mV and 86·8 s−1 at –100 mV. Thus the blocking dissociation constant KB (= K – B/K + B) was 18·5 μM at –50 mV and 3·3 μM at –100 mV. The simple channel block scheme was found to be insufficient to explain fully the observations made; reasons for this are discussed.

Negative shift of cardiac Na+ channel kinetics in cell-attached patch recordings

1990 ◽  
Vol 258 (1) ◽  
pp. H247-H254 ◽  
Author(s):  
T. Kimitsuki ◽  
T. Mitsuiye ◽  
A. Noma
Keyword(s):  
Patch Clamp ◽  
Single Channel ◽  
Resting Potential ◽  
Na Channel ◽  
Channel Kinetics ◽  
Patch Clamp Technique ◽  
Inactivation Curve ◽  
Pipette Solution ◽  
Single Channel Currents ◽  
Channel Currents

Na+ channel kinetics were studied by recording single-channel currents in the cell-attached patch configuration of the patch-clamp technique in single ventricular cells isolated from guinea pig hearts. The inactivation time course of ensemble currents was accelerated, and the peak amplitude increased temporarily and then decreased within a few minutes after the gigaohm seal formation. After reaching a new steady state, the inactivation-voltage relation was found to have shifted to more negative potentials. The potential of half-maximal inactivation was more negative by 20–31 mV from the resting potential or between -96 and -112 mV. The voltage dependency of the channel activation also shifted. Although the cell membrane was depolarized using the whole cell patch-clamp electrode and single-channel currents were recorded with an independent cell-attached electrode, the shift of the inactivation curve was also evident. Complete removal of Ca2+ using 5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the pipette solution failed to prevent the shift. Increasing Ca2+ to 10 mM, however, reduced magnitude of the shift significantly. Involvement of an increased membrane fluidity and surface potential of the glass pipette to the shift is discussed.

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