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 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.

Feedback regulation of Na channels in rat CCT. III. Response to cAMP

1995 ◽  
Vol 268 (3) ◽  
pp. F480-F489 ◽  
Author(s):  
G. Frindt ◽  
R. B. Silver ◽  
E. E. Windhager ◽  
L. G. Palmer
Keyword(s):  
Open Channel ◽  
Single Channel ◽  
Feedback Inhibition ◽  
Feedback Regulation ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Channel Current ◽  
Fluorescence Measurements ◽  
Collecting Tubule ◽  
The Mean

The effects of exogenous adenosine 3',5'-cyclic monophosphate (cAMP) on apical Na channels in the rat cortical collecting tubule were studied using the patch-clamp technique and fura 2 fluorescence measurements of intracellular Ca2+ (Ca2+i). When the permeant analogue, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP, 200 microM), was added to the superfusate during recording from cell-attached patches, both the mean number of open channels (NPo) and the single-channel current (i) decreased within 3 min. When the superfusate also contained amiloride (10 microM), there was no effect of CPT-cAMP on either NPo or i. When CPT-cAMP was added to the bath before formation of the patch, the density of conducting channels was increased from 10 +/- 2 to 37 +/- 6 per patch, as estimated by analysis of channel-induced noise. This suggests that cAMP increases open-channel density in the regions of the apical membrane outside the patch but not within the patch. Channels already active in the patch before stimulation with the nucleotide are subject to feedback inhibition secondary to increased Na entry into the cell. CPT-cAMP increased Ca2+i from 104 to 198 nM. This increase in Ca2+i was abolished by benzamil (0.5 microM) or by low extracellular Ca2+. The cAMP-dependent reduction in NPo was still observed in Ca(2+)-free medium, indicating that a rise in Ca2+i was not essential for the feedback response. The decrease in NPo was attenuated, however, when cAMP was added in the absence of Ca2+ and in the presence of ouabain (1 mM) in the superfusate.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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 Effective Activation of BKCa Channels by QO-40 (5-(Chloromethyl)-3-(Naphthalen-1-yl)-2-(Trifluoromethyl)Pyrazolo [1,5-a]pyrimidin-7(4H)-one), Known to Be an Opener of KCNQ2/Q3 Channels

2021 ◽  
Vol 14 (5) ◽  
pp. 388
Author(s):  
Wei-Ting Chang ◽  
Sheng-Nan Wu
Keyword(s):  
Single Channel ◽  
Slow Component ◽  
Ionic Channel ◽  
Gh3 Cells ◽  
Bkca Channel ◽  
Bkca Channels ◽  
Gating Charge ◽  
Ramp Voltage ◽  
K Current ◽  
The Mean

QO-40 (5-(chloromethyl)-3-(naphthalene-1-yl)-2-(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-7(4H)-one) is a novel and selective activator of KCNQ2/KCNQ3 K+ channels. However, it remains largely unknown whether this compound can modify any other type of plasmalemmal ionic channel. The effects of QO-40 on ion channels in pituitary GH3 lactotrophs were investigated in this study. QO-40 stimulated Ca2+-activated K+ current (IK(Ca)) with an EC50 value of 2.3 μM in these cells. QO-40-stimulated IK(Ca) was attenuated by the further addition of GAL-021 or paxilline but not by linopirdine or TRAM-34. In inside-out mode, this compound added to the intracellular leaflet of the detached patches stimulated large-conductance Ca2+-activated K+ (BKCa) channels with no change in single-channel conductance; however, there was a decrease in the slow component of the mean closed time of BKCa channels. The KD value required for the QO-40-mediated decrease in the slow component at the mean closure time was 1.96 μM. This compound shifted the steady-state activation curve of BKCa channels to a less positive voltage and decreased the gating charge of the channel. The application of QO-40 also increased the hysteretic strength of BKCa channels elicited by a long-lasting isosceles-triangular ramp voltage. In HEK293T cells expressing α-hSlo, QO-40 stimulated BKCa channel activity. Overall, these findings demonstrate that QO-40 can interact directly with the BKCa channel to increase the amplitude of IK(Ca) in GH3 cells.

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.

scholarly journals TOK Homologue in Neurospora crassa: First Cloning and Functional Characterization of an Ion Channel in a Filamentous Fungus

2003 ◽  
Vol 2 (1) ◽  
pp. 181-190 ◽  
Author(s):  
Stephen K. Roberts
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Neurospora Crassa ◽  
Filamentous Fungus ◽  
Single Channel ◽  
Functional Characterization ◽  
Reversal Potential ◽  
Channel Activity ◽  
Biophysical Properties ◽  
Patch Clamp Technique

ABSTRACT In contrast to animal and plant cells, very little is known of ion channel function in fungal physiology. The life cycle of most fungi depends on the “filamentous” polarized growth of hyphal cells; however, no ion channels have been cloned from filamentous fungi and comparatively few preliminary recordings of ion channel activity have been made. In an attempt to gain an insight into the role of ion channels in fungal hyphal physiology, a homolog of the yeast K+ channel (ScTOK1) was cloned from the filamentous fungus, Neurospora crassa. The patch clamp technique was used to investigate the biophysical properties of the N. crassa K+ channel (NcTOKA) after heterologous expression of NcTOKA in yeast. NcTOKA mediated mainly time-dependent outward whole-cell currents, and the reversal potential of these currents indicated that it conducted K+ efflux. NcTOKA channel gating was sensitive to extracellular K+ such that channel activation was dependent on the reversal potential for K+. However, expression of NcTOKA was able to overcome the K+ auxotrophy of a yeast mutant missing the K+ uptake transporters TRK1 and TRK2, suggesting that NcTOKA also mediated K+ influx. Consistent with this, close inspection of NcTOKA-mediated currents revealed small inward K+ currents at potentials negative of EK. NcTOKA single-channel activity was characterized by rapid flickering between the open and closed states with a unitary conductance of 16 pS. NcTOKA was effectively blocked by extracellular Ca2+, verapamil, quinine, and TEA+ but was insensitive to Cs+, 4-aminopyridine, and glibenclamide. The physiological significance of NcTOKA is discussed in the context of its biophysical properties.

Two distinct inactivation processes related to phosphorylation in cardiac L-type Ca2+ channel currents

2000 ◽  
Vol 279 (3) ◽  
pp. C603-C610 ◽  
Author(s):  
Sayaka Mitarai ◽  
Muneshige Kaibara ◽  
Katsusuke Yano ◽  
Kohtaro Taniyama
Keyword(s):  
Membrane Potential ◽  
Protein Kinase ◽  
Time Constant ◽  
Kinase Inhibitor ◽  
Slow Component ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Inward Currents ◽  
Inactivation Process ◽  
Channel Currents

We investigated the inactivation process of macroscopic cardiac L-type Ca2+ channel currents using the whole cell patch-clamp technique with Na+ as the current carrier. The inactivation process of the inward currents carried by Na+ through the channel consisted of two components >0 mV. The time constant of the faster inactivating component (30.6 ± 2.2 ms at 0 mV) decreased with depolarization, but the time constant of the slower inactivating component (489 ± 21 ms at 0 mV) was not significantly influenced by the membrane potential. The inactivation process in the presence of isoproterenol (100 nM) consisted of a single component (538 ± 60 ms at 0 mV). A protein kinase inhibitor, H-89, decreased the currents and attenuated the effects of isoproterenol. In the presence of cAMP (500 μM), the inactivation process consisted of a single slow component. We propose that the faster inactivating component represents a kinetic of the dephosphorylated or partially phosphorylated channel, and phosphorylation converts the kinetics into one with a different voltage dependency.

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.

Feedback regulation of Na channels in rat CCT. I. Effects of inhibition of Na pump

1993 ◽  
Vol 264 (3) ◽  
pp. F557-F564 ◽  
Author(s):  
R. B. Silver ◽  
G. Frindt ◽  
E. E. Windhager ◽  
L. G. Palmer
Keyword(s):  
Feedback Regulation ◽  
Peak Level ◽  
Na Channels ◽  
Channel Activity ◽  
Electrical Measurements ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Collecting Tubule ◽  
Intracellular Ca ◽  
The Mean

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (Po), and currents (i) were measured at 37 degrees C during continuous superfusion of the tubule. Addition of ouabain (1 mM) to the superfusate to increase cell Na resulted in a decrease in the mean number of open channels (NPo) to less than 20% of control values within 2 min. This effect was not reversible within 5 min after removal of ouabain. There was, in addition, a parallel decrease in i. The mechanism of inhibiton appeared to involve increased intracellular Ca (Cai). Cai was measured using the fluorescence of the Ca indicator fura-2 in principal cells of split tubules under conditions identical to those used for electrical measurements. Cai increased from a basal level (153 +/- 36 nM) to a peak level (588 +/- 53 nM) approximately 3 min after the addition of ouabain. When a Ca-free superfusate was used, ouabain did not increase Cai or decrease NPo, although the decrease in i was similar to that observed in Ca-containing solutions. Similar increases in Cai were elicited by the Ca ionophore ionomycin (5 microM) in the presence of 0.1 mM extracellular Ca. This maneuver also resulted in a decrease in NPo which was similar to that observed in the presence of ouabain. Ouabain had no observable effect on cell pH.(ABSTRACT TRUNCATED AT 250 WORDS)

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