Single potassium channels blocked by lidocaine and quinidine in isolated turtle colon epithelial cells

1986 ◽  
Vol 251 (1) ◽  
pp. C85-C89 ◽  
Author(s):  
N. W. Richards ◽  
D. C. Dawson
Keyword(s):  
Epithelial Cells ◽  
Single Channel ◽  
Reversal Potential ◽  
Cell Swelling ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Colon Cells ◽  
High K ◽  
A Cell ◽  
Single Channel Currents

The patch-clamp technique for recording single-channel currents across cell membranes was applied to single turtle colon epithelial cells isolated with hyaluronidase. With electrodes fabricated from Corning #7052 glass, high-resistance seals were consistently formed to these cells. In on-cell patches with low K (2.5 mM) in the pipette and high K (114.5 mM) in the bath, outward K currents were recorded that had a slope conductance of 17 pS and a reversal potential greater than -70 mV. Currents through this K channel were blocked by lidocaine, quinidine, and barium. These agents also block a cell swelling-induced K conductance identified by macroscopic current measurements in the basolateral membranes of the intact colonic epithelium, suggesting that the 17 pS K channel identified by single-channel recording in isolated turtle colon cells may be responsible for this macroscopically defined K conductance.

scholarly journals A whole-cell and single-channel study of the voltage-dependent outward potassium current in avian hepatocytes.

1988 ◽  
Vol 91 (2) ◽  
pp. 255-274 ◽  
Author(s):  
C Marchetti ◽  
R T Premont ◽  
A M Brown
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Outward Current ◽  
Single Type ◽  
K Channel ◽  
Delayed Rectifier ◽  
Patch Clamp Technique ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
K Currents

Voltage-dependent membrane currents were studied in dissociated hepatocytes from chick, using the patch-clamp technique. All cells had voltage-dependent outward K+ currents; in 10% of the cells, a fast, transient, tetrodotoxin-sensitive Na+ current was identified. None of the cells had voltage-dependent inward Ca2+ currents. The K+ current activated at a membrane potential of about -10 mV, had a sigmoidal time course, and did not inactivate in 500 ms. The maximum outward conductance was 6.6 +/- 2.4 nS in 18 cells. The reversal potential, estimated from tail current measurements, shifted by 50 mV per 10-fold increase in the external K+ concentration. The current traces were fitted by n2 kinetics with voltage-dependent time constants. Omitting Ca2+ from the external bath or buffering the internal Ca2+ with EGTA did not alter the outward current, which shows that Ca2+-activated K+ currents were not present. 1-5 mM 4-aminopyridine, 0.5-2 mM BaCl2, and 0.1-1 mM CdCl2 reversibly inhibited the current. The block caused by Ba was voltage dependent. Single-channel currents were recorded in cell-attached and outside-out patches. The mean unitary conductance was 7 pS, and the channels displayed bursting kinetics. Thus, avian hepatocytes have a single type of K+ channel belonging to the delayed rectifier class of K+ channels.

Permeation and gating properties of a cloned renal K+ channel

1995 ◽  
Vol 268 (2) ◽  
pp. C389-C401 ◽  
Author(s):  
S. Chepilko ◽  
H. Zhou ◽  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Cation Binding ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Channel Current ◽  
Inward Currents ◽  
Kinetics Of

The renal K+ channel (ROMK2) was expressed in Xenopus oocytes, and the patch-clamp technique was used to assess its conducting and gating properties. In cell-attached patches with 110 mM K+ in the bath and pipette, the reversal potential was near zero and the inward conductance (36 pS) was larger than the outward conductance (17 pS). In excised inside-out patches the channels showed rectification in the presence of 5 mM Mg2+ on the cytoplasmic side but not in Mg(2+)-free solution. Inward currents were also observed when K+ was replaced in the pipette by Rb+, NH4+, or thallium (Tl+). The reversal potentials under these conditions yielded a selectivity sequence of Tl+ > K+ > Rb+ > NH4+. On the other hand, the slope conductances for inward current gave a selectivity sequence of K+ = NH4+ > Tl+ > Rb+. The differences in the two sequences can be explained by the presence of cation binding sites within the channel, which interact with Rb+ and Tl+ more strongly and with NH4+ less strongly than with K+. Two other ions, Ba2+ and Cs+, blocked the channel from the outside. The effect of Ba2+ (1 mM) was to reduce the open probability of the channels, whereas Cs+ (10 mM) reduced the apparent single-channel current. The effects of both blockers are enhanced by membrane hyperpolarization. The kinetics of the channel were also studied in cell-attached patches. With K+ in the pipette the distribution of open times could be described by a single exponential (tau 0 = 25 ms), whereas two exponentials (tau 1 = 1 ms, tau 2 = 30 ms) were required to describe the closed-time distribution. Hyperpolarization of the oocyte membrane decreased the open probability and tau 0, and increased tau 1, tau 2, and the number of long closures. The presence of Tl+ in the pipette significantly altered the kinetics, reducing tau 0 and eliminating the long-lived closures. These results suggest that the gating of the channel may depend on the nature of the ion in the pore.

Swelling-induced and depolarization-induced C1-channels in normal and cystic fibrosis epithelial cells

1991 ◽  
Vol 261 (4) ◽  
pp. C658-C674 ◽  
Author(s):  
C. K. Solc ◽  
J. J. Wine
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Current Density ◽  
Single Channel ◽  
Cell Swelling ◽  
Disulfonic Acid ◽  
Channel Activity ◽  
Whole Cell ◽  
Cl Channels ◽  
Single Channel Currents

Cl- currents induced by cell swelling were characterized at the whole cell and single-channel levels in primary cultures of normal and cystic fibrosis (CF) epithelial cells and in the T84 cell line. Currents recorded in normal and CF cells were indistinguishable. At 22-24 degrees C with isotonic CsCl in the pipette, initial whole cell outward current density at 100 mV in unswollen cells was 2-4 pA/pF. The current density increased with time during whole cell recording up to 100 pA/pF in isotonic solutions and up to 200 pA/pF in a hypotonic bath, though values typically ranged between 10 and 70 pA/pF. Currents were outwardly rectifying, active at negative voltages, started to inactivate above approximately 40 mV, and were blocked by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Single Cl- channels (approximately 50 pS near 0 mV) with an outwardly rectifying current-voltage relation were recorded in cell-attached and outside-out patches from swollen cells. The channels were mostly open at negative voltages and inactivated at positive voltages with a voltage dependence similar to the whole cell currents. Channel activity decreased rapidly (channel rundown) after seal formation. After swelling-induced channel activity had ceased, outwardly rectifying, depolarization-induced Cl- channels (ORDIC channels) were activated in some patches. The swelling-induced and ORDIC single-channel currents were similar, but some consistent differences were observed. ORDIC channels were often closed at resting voltages (-70 to -50 mV), while swelling-induced channels were always open in this voltage range. In addition, ORDIC channels started to inactivate at more positive voltages (approximately 90 vs. approximately 50 mV), rectified more, and had smaller conductances (approximately 25 pS near 0 mV), shorter mean open durations (approximately 70 vs. approximately 350 ms), and more open-channel noise than swelling-induced channels. The two types of currents might arise from separate channel proteins or from a single channel molecule in different states.

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.

Permeation and block of dopamine-modulated potassium channels on rat striatal neurons by cesium and barium ions

1996 ◽  
Vol 76 (3) ◽  
pp. 1413-1422 ◽  
Author(s):  
Y. J. Lin ◽  
G. J. Greif ◽  
J. E. Freedman
Keyword(s):  
Dissociation Constant ◽  
Single Channel ◽  
Reversal Potential ◽  
Negative Slope ◽  
K Channel ◽  
Striatal Neurons ◽  
Apparent Dissociation Constant ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Inwardly Rectifying

1. In cell-attached patch-clamp recordings from freshly dissociated rat caudate-putamen neurons, an 85-pS inwardly rectifying K+ channel, which was previously found to be modulated by D2-like dopamine receptors, was blocked by externally applied BaCl2 or CsCl. 2. At concentrations between 100 and 500 microM, Ba2+ blockade was voltage dependent, with a greater block at hyperpolarized voltages, in a manner consistent with blockade of the channel pore. Single-channel currents were flickery, with intervening periods of more complete blockade, and block appeared to be time dependent, with an estimated electrical distance of 0.24 and an apparent dissociation constant of 205 microM at 0 mV. 3. At concentrations between 1 and 3 mM, Cs+ blockade was similarly voltage dependent, but without periods of longer blockade, with an electrical distance of 0.81 and an apparent dissociation constant of 625 microM at 0 mV. Cs+ could also permeate this channel at voltages near the K+ reversal potential. The current-voltage relationship displayed an anomalous negative slope conductance, in a manner inconsistent with a single-ion pore. 4. Smaller-conductance, dopamine-insensitive channels were blocked more potently by both Ba2+ and Cs+ than was the 85-pS channel, reflecting differences between inwardly rectifying K+ channels mediating resting conductance and those mediating dopamine receptor responses in striatal neurons.

scholarly journals Hypotonicity-induced cell swelling activates TRPA1

2017 ◽  
Vol 68 (4) ◽  
pp. 431-440 ◽  
Author(s):  
Fumitaka Fujita ◽  
Kunitoshi Uchida ◽  
Yasunori Takayama ◽  
Yoshiro Suzuki ◽  
Masayuki Takaishi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Single Channel ◽  
Cell Swelling ◽  
Imaging Method ◽  
Channel Activation ◽  
Patch Pipette ◽  
Physical Stimuli ◽  
A Cell ◽  
Single Channel Currents ◽  
Channel Currents

Abstract Hypotonic solutions can cause painful sensations in nasal and ocular mucosa through molecular mechanisms that are not entirely understood. We clarified the ability of human TRPA1 (hTRPA1) to respond to physical stimulus, and evaluated the response of hTRPA1 to cell swelling under hypotonic conditions. Using a Ca2+-imaging method, we found that modulation of AITC-induced hTRPA1 activity occurred under hypotonic conditions. Moreover, cell swelling in hypotonic conditions evoked single-channel activation of hTRPA1 in a cell-attached mode when the patch pipette was attached after cell swelling under hypotonic conditions, but not before swelling. Single-channel currents activated by cell swelling were also inhibited by a known hTRPA1 blocker. Since pre-application of thapsigargin or pretreatment with the calcium chelator BAPTA did not affect the single-channel activation induced by cell swelling, changes in intracellular calcium concentrations are likely not related to hTRPA1 activation induced by physical stimuli.

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.

scholarly journals Competitive Mg2+ block of a large-conductance, Ca(2+)-activated K+ channel in rat skeletal muscle. Ca2+, Sr2+, and Ni2+ also block.

1991 ◽  
Vol 98 (1) ◽  
pp. 163-181 ◽  
Author(s):  
W B Ferguson
Keyword(s):  
Skeletal Muscle ◽  
Single Channel ◽  
Competitive Inhibition ◽  
Reversal Potential ◽  
K Channel ◽  
Dependent Manner ◽  
Rat Skeletal Muscle ◽  
Patch Clamp Technique ◽  
Channel Current ◽  
Single Channel Current

The patch-clamp technique was used to investigate the effect of intracellular Mg2+ (Mgi2+) on the conductance of the large-conductance, Ca(2+)-activated K+ channel in cultured rat skeletal muscle. Measurements of single-channel current amplitudes indicated that Mgi2+ decreased the K+ currents in a concentration-dependent manner. Increasing Mgi2+ from 0 to 5, 10, 20, and 50 mM decreased channel currents by 34%, 44%, 56%, and 73%, respectively, at +50 mV. The magnitude of the Mgi2+ block increased with depolarization. For membrane potentials of -50, +50, and +90 mV, 20 mM Mgi2+ reduced the currents 22%, 56%, and 70%, respectively. Mgi2+ did not change the reversal potential, indicating that Mg2+ does not permeate the channel. The magnitude of the Mgi2+ block decreased as the concentration of K+ was increased. At a membrane potential of +50 mv, 20 mM Mgi2+ reduced the currents 71%, 56%, and 25% for Ki+ of 75, 150, and 500 mM. These effects of Mgi2+, voltage, and K+ were totally reversible. Although the Woodhull blocking model could approximate the voltage and concentration effects of the Mgi2+ block (Kd approximately 30 mM with 150 mM symmetrical K+; electrical distance approximately 0.22 from the inner surface), the Woodhull model could not account for the effects of K+. Double reciprocal plots of 1/single channel current vs. 1/[K+] in the presence and absence of Mgi2+, indicated that the Mgi2+ block is consistent with apparent competitive inhibition between Mgi2+ and Ki+. Cai2+, Nii2+, and Sri2+ were found to have concentration- and voltage-dependent blocking effects similar, but not identical, to those of Mgi2+. These observations suggest the blocking by Mgi2+ of the large-conductance, Ca(2+)-activated K+ channel is mainly nonspecific, competitive with K+, and at least partially electrostatic in nature.

Ca2+ inhibits outward current through single K+ channels in canine atrial myocyte sarcolemma

1988 ◽  
Vol 254 (3) ◽  
pp. C397-C403 ◽  
Author(s):  
J. K. Bubien ◽  
H. Van Der Heyde ◽  
W. T. Woods
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
K Channel ◽  
Transient Outward Current ◽  
Single Channels ◽  
Bathing Solution ◽  
Voltage Sensitivity ◽  
Patch Clamp Technique ◽  
K Channels ◽  
Single Channel Currents

Single-channel currents in canine atrial cells were recorded by the patch-clamp technique in a bathing solution containing 150 mM [K+] and pipette solutions containing 5 mM [K+]. One kind of current was observed in 56% of 178 cell-free patches and in 3% of 60 patches in the cell-attached configuration. Single-channel amplitude varied in direct proportion to the bath [K+]. Openings of these single channels were prevented when bath [Ca2+] exceeded 1 microM. Below this concentration single-channel percent open time was inversely proportional to log [Ca2+]. Inward current was observed at hyperpolarized membrane potentials in some patches. There was no apparent steady-state voltage sensitivity. These properties suggest that the K+ channel described in this study (gK+LF), a low transition frequency K+ conductor, may be distinct from single K+ channels previously studied in cardiac myocyte sarcolemmae. The single-channel response to "intracellular" free [Ca2+] and the single-channel kinetic characteristics described in this study are similar to the macroscopic "long-lasting transient outward current" (IIO) described by Escande et al. [Am. J. Physiol. 252 (Heart Circ. Physiol. 21): H142-H148, 1987] in human atrial myocytes (tau open = 29.6 ms, tau inactivation = 35.7 ms, respectively). This suggests that gK+LF channels may carry IIO.

KCNA10: a novel ion channel functionally related to both voltage-gated potassium and CNG cation channels

2000 ◽  
Vol 278 (6) ◽  
pp. F1013-F1021 ◽  
Author(s):  
Rainer Lang ◽  
George Lee ◽  
Weimin Liu ◽  
Shulan Tian ◽  
Hamid Rafi ◽  
...  
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Amino Acid Identity ◽  
Cation Channels ◽  
K Channel ◽  
Xenopus Laevis Oocytes ◽  
Selectivity Ratio ◽  
Channel Blockers ◽  
Voltage Gated ◽  
Single Channel Currents

Our laboratory previously cloned a novel rabbit gene ( Kcn1), expressed in kidney, heart, and aorta, and predicted to encode a protein with 58% amino acid identity with the K channel Shaker Kv1.3 (Yao X et al. Proc Natl Acad Sci USA 92: 11711–11715, 1995). Because Kcn1 did not express well (peak current in Xenopus laevis oocytes of 0.3 μA at +60 mV), the human homolog (KCNA10) was isolated, and its expression was optimized in oocytes. KCNA10 mediates voltage-gated K+currents that exhibit minimal steady-state inactivation. Ensemble currents of 5–10 μA at +40 mV were consistently recorded from injected oocytes. Channels are closed at the holding potential of −80 mV but are progressively activated by depolarizations more positive than −30 mV, with half-activation at +3.5 ± 2.5 mV. The channel displays an unusual inhibitor profile because, in addition to being blocked by classical K channel blockers (barium tetraethylammonium and 4-aminopyridine), it is also sensitive to inhibitors of cyclic nucleotide-gated (CNG) cation channels (verapamil and pimozide). Tail-current analysis shows a reversal potential shift of 47 mV/decade change in K concentration, indicating a K-to-Na selectivity ratio of at least 15:1. The phorbol ester phorbol 12-myristate 13-acetate, an activator of protein kinase C, inhibited whole cell current by 42%. Analysis of single-channel currents reveals a conductance of ∼11 pS. We conclude KCNA10 is a novel human voltage-gated K channel with features common to both K-selective and CNG cation channels. Given its distribution in renal blood vessels and heart, we speculate that KCNA10 may be involved in regulating the tone of renal vascular smooth muscle and may also participate in the cardiac action potential.

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