Blockade of lysophosphatidylcholine-modified cardiac Na channels by a lidocaine derivative QX-222

1996 ◽  
Vol 271 (2) ◽  
pp. H790-H797
Author(s):  
A. I. Undrovinas ◽  
J. C. Makielski
Keyword(s):  
Single Channel ◽  
Cardiac Cells ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Ventricular Cells ◽  
Open Time ◽  
Channel Open Time ◽  
Open States ◽  
Inside Out ◽  
Cytoplasmic Side

Single Na channels from rat and rabbit ventricular cells were studied with use of the excised inside-out patch-clamp technique. To investigate local anesthetic interactions with Na channels modified by the ischemic metabolite lysophosphatidylcholine (LPC), the quaternary ammonium lidocaine derivative QX-222 [2-(trimethylamino)-N-(2,6-dimethylphenyl)acetamide] was applied to the cytoplasmic side of patches from untreated cells and from those treated with LPC for approximately 1 h. Single-channel amplitudes and kinetics for unmodified channels were similar to those reported previously for cardiac cells with a single-component, mean-channel open time. LPC-modified channels showed prolonged open channel bursting with a two-component, mean open time, suggesting two open states. Conductance sublevels to the 60-70% level of the main conductance were found in both unmodified and LPC-modified channels and also with and without QX-222 present. QX-222 reversibly shortened the open time of the unmodified channel and for both open times of the LPC-modified channel without decreasing single-channel amplitude. Calculated association rates for QX-222 with the channel were found to be greater for the open states of the modified channel than those for the unmodified channel. Thus the lidocaine analogue QX-222 interacts with and blocks the open state of both unmodified and LPC-modified, cardiac Na channels. The blocking effect on LPC-modified channels would be predicted to be greater both because of the longer dwell time in the high-affinity open states for modified channels and also because of an intrinsically greater association rate in the modified channels.

scholarly journals Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch.

1996 ◽  
Vol 107 (1) ◽  
pp. 35-45 ◽  
Author(s):  
L G Palmer ◽  
G Frindt
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Apical Membrane ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Collecting Tubule ◽  
Excised Patches ◽  
The Mean ◽  
Cortical Collecting Tubule

The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed in cell-attached and inside-out excised patches from split-open tubules using the patch-clamp technique. In patches containing a single channel the open probability (Po) was variable, ranging from 0.05 to 0.9. The average Po was 0.5. However, the individual values were not distributed normally, but were mainly < or = 0.25 or > or = 0.75. Mean open times and mean closed times were correlated directly and inversely, respectively, with Po. In patches where a sufficient number of events could be recorded, two time constants were required to describe the open-time and closed-time distributions. In most patches in which basal Po was < 0.3 the channels could be activated by hyperpolarization of the apical membrane. In five such patches containing a single channel hyperpolarization by 40 mV increased Po by 10-fold, from 0.055 +/- 0.023 to 0.58 +/- 0.07. This change reflected an increase in the mean open time of the channels from 52 +/- 17 to 494 +/- 175 ms and a decrease in the mean closed time from 1,940 +/- 350 to 336 +/- 100 ms. These responses, however, could not be described by a simple voltage dependence of the opening and closing rates. In many cases significant delays in both the activation by hyperpolarization and deactivation by depolarization were observed. These delays ranged from several seconds to several tens of seconds. Similar effects of voltage were seen in cell-attached and excised patches, arguing against a voltage-dependent chemical modification of the channel, such as a phosphorylation. Rather, the channels appeared to switch between gating modes. These switches could be spontaneous but were strongly influenced by changes in membrane voltage. Voltage dependence of channel gating was also observed under whole-cell clamp conditions. To see if mechanical perturbations could also influence channel kinetics or gating mode, negative pressures of 10-60 mm Hg were applied to the patch pipette. In most cases (15 out of 22), this maneuver had no significant effect on channel behavior. In 6 out of 22 patches, however, there was a rapid and reversible increase in Po when the pressure was applied. In one patch, there was a reversible decrease. While no consistent effects of pressure could be documented, membrane deformation could contribute to the variation in Po under some conditions.

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.

scholarly journals Kinetics of 9-aminoacridine block of single Na channels.

1984 ◽  
Vol 84 (3) ◽  
pp. 361-377 ◽  
Author(s):  
D Yamamoto ◽  
J Z Yeh
Keyword(s):  
Rate Constant ◽  
Voltage Dependence ◽  
Single Channel ◽  
Na Channel ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Voltage Dependent ◽  
The Mean ◽  
Kinetics Of

The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA). Following NBA treatment, the current flowing through individual Na channels was manifested by square-wave open events lasting from several to tens of milliseconds. When 9-AA was applied to the cytoplasmic face of Na channels at concentrations ranging from 30 to 100 microM, it caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single channel current. The histograms for the duration of blocked states and the histograms for the duration of open states could be fitted with a single-exponential function. The mean open time (tau o) became shorter as the drug concentration was increased, while the mean blocked time (tau b) was concentration independent. The association (blocking) rate constant, kappa, calculated from the slope of the curve relating the reciprocal mean open time to 9-AA concentration, showed little voltage dependence, the rate constant being on the order of 1 X 10(7) M-1s-1. The dissociation (unblocking) rate constant, l, calculated from the mean blocked time, was strongly voltage dependent, the mean rate constant being 214 s-1 at 0 mV and becoming larger as the membrane being hyperpolarized. The voltage dependence suggests that a first-order blocking site is located at least 63% of the way through the membrane field from the cytoplasmic surface. The equilibrium dissociation constant for 9-AA to block the Na channel, defined by the relation of l/kappa, was calculated to be 21 microM at 0 mV. Both tau -1o and tau -1b had a Q10 of 1.3, which suggests that binding reaction was diffusion controlled. The burst time in the presence of 9-AA, which is the sum of open times and blocked times, was longer than the lifetime of open channels in the absence of drug. All of the features of 9-AA block of single Na channels are compatible with the sequential model in which 9-AA molecules block open Na channels, and the blocked channels could not close until 9-AA molecules had left the blocking site in the channels.

Effects of cell Ca and pH on Na channels from rat cortical collecting tubule

1987 ◽  
Vol 253 (2) ◽  
pp. F333-F339 ◽  
Author(s):  
L. G. Palmer ◽  
G. Frindt
Keyword(s):  
Apical Membrane ◽  
Na Channels ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Collecting Tubule ◽  
Solution Bathing ◽  
The Mean ◽  
Inside Out ◽  
Cytoplasmic Side ◽  
Cortical Collecting Tubule

The patch-clamp technique was used to identify individual Na channels in the apical membrane of the rat cortical collecting tubule and to evaluate the effects of cytoplasmic Ca2+ and pH on channel activity. In excised, inside-out patches, the probability of a channels's being open (P0) increased with alkalinization of the solution bathing the cytoplasmic side of the patch. Estimates of P0 were 0.05 at pH 6.4, 0.19 at pH 6.9, and 0.41 at pH 7.4. Varying the free Ca2+ concentration of the solution bathing the cytoplasmic side of the patch had no measurable effect on P0. In cell-attached patches, addition of the Ca2+ ionophore ionomycin to the solution bathing the tubules to a final concentration of either 1 or 10 microM decreased channel activity measured as the mean number of open channels (no. open) = n X P0 where n is the number of channels in the membrane. (no. open) was significantly decreased at 3 min after addition of ionomycin and fell to less than 10% of control values after 10 min incubation. There was no fall in (no. open) either in time controls or in tubules exposed to ionomycin in the presence of low bath Ca2+ concentrations [no added Ca2+ with 1 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)]. The results suggest that cytoplasmic pH can directly influence channel activity. Cytoplasmic Ca2+ does not interact directly with the channels, but increased cytoplasmic Ca2+ produces a fall in channel activity through an indirect process.

scholarly journals Slow currents through single sodium channels of the adult rat heart.

1985 ◽  
Vol 86 (1) ◽  
pp. 89-104 ◽  
Author(s):  
J B Patlak ◽  
M Ortiz
Keyword(s):  
Time Constant ◽  
Single Channel ◽  
Slow Component ◽  
Reversal Potential ◽  
Careful Examination ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Adult Rat ◽  
Ventricular Cells ◽  
The Mean

The currents through single Na+ channels from the sarcolemma of ventricular cells dissociated from adult rat hearts were studied using the patch-clamp technique. All patches had several Na+ channels; most had 5-10, while some had up to 50 channels. At 10 degrees C, the conductance of the channel was 9.8 pS. The mean current for sets of many identical pulses inactivated exponentially with a time constant of 1.7 +/- 0.6 ms at -40 mV. Careful examination of the mean currents revealed a small, slow component of inactivation at pulse potentials ranging from -60 to -30 mV. The time constant of the slow component was between 8 and 14 ms. The channels that caused the slow component had the same conductance and reversal potential as the fast Na+ currents and were blocked by tetrodotoxin. The slow currents appear to have been caused by repeated openings of one or more channels. The holding potential influenced the frequency with which such channel reopening occurred. The slow component was prominent during pulses from a holding potential of -100 mV, while it was very small during pulses from -140 mV. Ultraslow currents through the Na+ channel were observed occasionally in patches that had large numbers of channels. They consisted of bursts of 10 or more sequential openings of a single channel and lasted for up to 150 ms. We conclude that the single channel data cannot be explained by standard models, even those that have two inactivated states or two open states of the channel. Our results suggest that Na+ channels can function in several different "modes," each with a different inactivation rate.

scholarly journals Stimulation of unitary T-type Ca2+ channel currents by calmodulin-dependent protein kinase II

2000 ◽  
Vol 279 (6) ◽  
pp. C1694-C1703 ◽  
Author(s):  
Paula Q. Barrett ◽  
Hong-Kai Lu ◽  
Roger Colbran ◽  
Andrew Czernik ◽  
Joseph J. Pancrazio
Keyword(s):  
Protein Kinase ◽  
Single Channel ◽  
Low Voltage ◽  
Patch Clamp Technique ◽  
Dependent Protein Kinase ◽  
Protein Kinase Ii ◽  
Dependent Protein ◽  
Channel Open Time ◽  
Channel Currents ◽  
Inside Out

The effect of Ca2+/calmodulin-dependent protein kinase II (CaMKII) stimulation on unitary low voltage-activated (LVA) T-type Ca2+ channel currents in isolated bovine adrenal glomerulosa (AG) cells was measured using the patch-clamp technique. In cell-attached and inside-out patches, LVA channel activity was identified by voltage-dependent inactivation and a single-channel conductance of ∼9 pS in 110 mM BaCl2 or CaCl2. In the cell-attached patch, elevation of bath Ca2+ from 150 nM to 1 μM raised intracellular Ca2+ in K+-depolarized (140 mM) cells and evoked an increase in the LVA Ca2+ channel probability of opening ( NP o) by two- to sixfold. This augmentation was associated with an increase in the number of nonblank sweeps, a rise in the frequency of channel opening in nonblank sweeps, and a 30% reduction in first latency. No apparent changes in the single-channel open-time distribution, burst lengths, or openings/burst were apparent. Preincubation of AG cells with lipophilic or peptide inhibitors of CaMKII in the cell-attached or excised (inside-out) configurations prevented the rise in NP oelicited by elevated Ca2+ concentration. Furthermore, administration of a mutant recombinant CaMKIIα exhibiting cofactor-independent activity in the absence of elevated Ca2+ produced a threefold elevation in LVA channel NP o. These data indicate that CaMKII activity is both necessary and sufficient for LVA channel activation by Ca2+.

Properties of veratridine-modified single Na+ channels in guinea pig ventricular myocytes

1993 ◽  
Vol 264 (2) ◽  
pp. H454-H463 ◽  
Author(s):  
A. Sunami ◽  
T. Sasano ◽  
A. Matsunaga ◽  
Z. Fan ◽  
T. Swanobori ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Voltage Dependence ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Simultaneous Occurrence ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
High Event ◽  
Event Times

Modification of single Na+ channels by the alkaloid neurotoxin veratridine was investigated in guinea pig ventricular myocytes using the cell-attached configuration of the patch-clamp technique. Pipette application of veratridine (50 microM) induced long-lasting openings with two different single-channel conductances of 7.6 and 3.0 pS, in addition to normal type of short openings with a single-channel conductance of 16 pS. The veratridine-modified high- and low-conductance channels appeared commonly, and they could coexist with the normal one in the same patch. The open-time distributions for the high- and low-conductance channels could be fitted by a single exponential. The mean open time for the high- and low-conductance events ranged between 19.1 ms at -120 mV and 86.0 ms at -10 mV and between 4.5 ms at -120 mV and 16.2 ms at -10 mV, respectively. The closed-time distributions for the two conductance channels consisted of at least two components, and their values and voltage dependence were similar. External Ca2+ block resulted in an apparent reduction of unitary current amplitudes with a similar voltage dependence and affinity for Ca2+ in the high- and low-conductance channels. However, the low-conductance channel was more resistant to tetrodotoxin than the high one. The probability of simultaneous occurrence of the high and low events was equal to the product of the probabilities of occurrence of the high event times that of the low event. Furthermore, we observed modified channel openings after a normal opening for the two conductance channels and a modified one turning into a normal one for the high-conductance channel. It is concluded that veratridine induces the two different types of modified Na+ channels in cardiac myocytes and these are correlated with normal openings.

Some properties of acetylcholine receptors in human cultured myotubes

1985 ◽  
Vol 224 (1235) ◽  
pp. 183-196 ◽  
Keyword(s):  
Acetylcholine Receptors ◽  
Single Channel ◽  
Fold Increase ◽  
Resting Potential ◽  
Patch Clamp Technique ◽  
Membrane Hyperpolarization ◽  
Human Myotubes ◽  
Cultured Myotubes ◽  
Channel Open Time ◽  
Ach Receptors

The distribution and single channel properties of acetylcholine (ACh) receptors in human myotubes grown in tissue culture have been examined. Radioautography of myotubes labelled with [ 125 I]α-bungarotoxin showed that ACh receptors are distributed uniformly over the myotube surface at a density of 3.9 ± 0.5 receptors per square micrometre. Ac­cumulations of ACh receptors (hot spots) were found rarely. The conductance and kinetics of ACh-activated channels were investi­gated with the patch-clamp technique. Cell-attached membrane patches were used in all experiments. A single channel conductance in the range 40–45 pS was calculated. No sublevels of conductance (substates) of the activated channel were observed. The distribution of channel open-times varied with ACh concentration. With 100 nM ACh, the distribution was best fitted by the sum of two exponentials, whereas with 1 μM ACh a single exponential could be fitted. The mean channel open-time at the myotube resting potential (ca. — 70 mV, 22°C) was 8.2 ms. The distribution of channel closed-times was complex at all concentrations of ACh studied (100 nM to 10 μm). With desensitizing doses of ACh (10 μM), channel openings occurred in obvious bursts; each burst usually appeared as part of a ‘cluster’ of bursts. Both burst duration and mean interval between bursts increased with membrane hyperpolarization. Individual channel open-times and burst durations showed similar voltage dependence (e-fold increase per 80 mV hyperpolarization), whereas both the channel closed-times within a burst and the number of openings per burst were independent of membrane potential.

Characterization of a newly found stretch-activated KCa,ATP channel in cultured chick ventricular myocytes

1999 ◽  
Vol 276 (6) ◽  
pp. H1827-H1838 ◽  
Author(s):  
Takashi Kawakubo ◽  
Keiji Naruse ◽  
Tatsuaki Matsubara ◽  
Nigishi Hotta ◽  
Masahiro Sokabe
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Heart Cells ◽  
Patch Clamp Technique ◽  
Selective Channel ◽  
New Type ◽  
Inside Out ◽  
Ion Selectivities ◽  
Channel Type

With the use of the patch-clamp technique, five kinds of stretch-activated (SA) ion channels were identified on the basis of their single-channel conductances and ion selectivities in cultured chick ventricular myocytes. Because a high-conductance K+-selective channel predominated among these channels, we concentrated on characterizing its properties mostly using excised inside-out patches. With 145 mM KCl solution in the pipette and the bath, the channel had a conductance of 199.8 ± 8.2 pS ( n = 22). The ion selectivities among K+, Na+, Ca2+, and Cl− as estimated from their permeability ratios were P Na/ P K= 0.03, P Ca/ P K= 0.025, and P Cl/ P K= 0.026. The probability of the channel being open (Po) increased with the Ca2+concentration in the bath ([Ca2+]b; dissociation constant K d = 0.51 μM at +30 mV) and membrane potential (voltage at half-maximal Po= 39.4 mV at 0.35 μM [Ca2+]b). The channel was blocked by gadolinium, tetraethylammonium, and charybdotoxin from the extracellular surface and, consequently, was identified as a Ca2+-activated K+(KCa) channel type. The channel was also reversibly activated by ATP applied to the intracellular surface ( K d = 0.74 mM at 0.10 μM [Ca2+]bat +30 mV). From these data taken together, we concluded that the channel is a new type of KCachannel that could be designated as an “SA KCa,ATP channel.” To our knowledge, this is the first report of KCa channel in heart cells.

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