scholarly journals Gating characteristics of a steeply voltage-dependent gap junction channel in rat Schwann cells.

1993 ◽  
Vol 102 (5) ◽  
pp. 925-946 ◽  
Author(s):  
M Chanson ◽  
K J Chandross ◽  
M B Rook ◽  
J A Kessler ◽  
D C Spray
Keyword(s):  
Steady State ◽  
Gap Junction ◽  
Schwann Cells ◽  
Voltage Dependence ◽  
Single Channel ◽  
Neonatal Rat ◽  
Order Kinetic ◽  
Patch Clamp Technique ◽  
Gap Junctional ◽  
Closing Rate

The gating properties of macroscopic and microscopic gap junctional currents were compared by applying the dual whole cell patch clamp technique to pairs of neonatal rat Schwann cells. In response to transjunctional voltage pulses (Vj), macroscopic gap junctional currents decayed exponentially with time constants ranging from < 1 to < 10 s before reaching steady-state levels. The relationship between normalized steady-state junctional conductance (Gss) and (Vj) was well described by a Boltzmann relationship with e-fold decay per 10.4 mV, representing an equivalent gating charge of 2.4. At Vj > 60 mV, Gss was virtually zero, a property that is unique among the gap junctions characterized to date. Determination of opening and closing rate constants for this process indicated that the voltage dependence of macroscopic conductance was governed predominantly by the closing rate constant. In 78% of the experiments, a single population of unitary junctional currents was detected corresponding to an unitary channel conductance of approximately 40 pS. The presence of only a limited number of junctional channels with identical unitary conductances made it possible to analyze their kinetics at the single channel level. Gating at the single channel level was further studied using a stochastic model to determine the open probability (Po) of individual channels in a multiple channel preparation. Po decreased with increasing Vj following a Boltzmann relationship similar to that describing the macroscopic Gss voltage dependence. These results indicate that, for Vj of a single polarity, the gating of the 40 pS gap junction channels expressed by Schwann cells can be described by a first order kinetic model of channel transitions between open and closed states.

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.

Opioid Receptor-Mediated Inhibition of ω-Conotoxin GVIA-Sensitive Calcium Channel Currents in Rat Intracardiac Neurons

1998 ◽  
Vol 79 (2) ◽  
pp. 753-762 ◽  
Author(s):  
David J. Adams ◽  
Carlo Trequattrini
Keyword(s):  
Calcium Channel ◽  
Opioid Receptor ◽  
Voltage Dependence ◽  
Neonatal Rat ◽  
Patch Clamp Technique ◽  
Receptor Agonists ◽  
Channel Current ◽  
Opioid Receptor Agonists ◽  
Channel Currents ◽  
Intracardiac Neurons

Adams, David J. and Carlo Trequattrini. Opioid receptor-mediated inhibition of ω-conotoxin GVIA-sensitive calcium channel currents in rat intracardiac neurons. J. Neurophysiol. 79: 753–762, 1998. Modulation of depolarization-activated ionic conductances by opioid receptor agonists was investigated in isolated parasympathetic neurons from neonatal rat intracardiac ganglia by using the whole cell perforated patch clamp technique. Met-enkephalin (10 μM) altered the action potential waveform, reducing the maximum amplitude and slowing the rate of rise and repolarization but the afterhyperpolarization was not appreciably altered. Under voltage clamp, 10 μM Met-enkephalin selectively and reversibly inhibited the peak amplitude of high-voltage–activated Ca2+ channel currents elicited at 0 mV by ∼52% and increased three- to fourfold the time to peak. Met-enkephalin had no effect on the voltage dependence of steady-state inactivation but shifted the voltage dependence of activation to more positive membrane potentials whereby stronger depolarization was required to open Ca2+ channels. Half-maximal inhibition of Ba2+ current ( I Ba) amplitude was obtained with 270 nM Met-enkephalin or Leu-enkephalin. The opioid receptor subtype selective agonists, DAMGO and DADLE, but not DPDPE, inhibited I Ba and were antagonized by the opioid receptor antagonists, naloxone and naltrindole with IC50s of 84 nM and 1 μM, respectively. The κ-opioid receptor agonists, bremazocine and dynorphin A, did not affect Ca2+ channel current amplitude or kinetics. Taken together, these data suggest that enkephalin-induced inhibition of Ca2+ channels in rat intracardiac neurons is mediated primarily by the μ-opioid receptor type. Addition of Met-enkephalin after exposure to 300 nM ω-conotoxin GVIA, which blocked ∼75% of the total Ca2+ channel current, failed to cause a further decrease of the residual current. Met-enkephalin inhibited the ω-conotoxin GVIA-sensitive but not the ω-conotoxin-insensitive I Ba in rat intracardiac neurons. Dialysis of the cell with a GTP-free intracellular solution or preincubation of the neurons in Pertussis toxin (PTX) abolished the attenuation of I Ba by Met-enkephalin, suggesting the involvement of a PTX-sensitive Gprotein in the signal transduction pathway. The activation of μ-opioid receptors and subsequent inhibition of N-type Ca2+ channels in the soma and terminals of postganglionic intracardiac neurons is likely to inhibit the release of ACh and thereby regulate vagal transmission to the mammalian heart.

Changes in lens connexin expression lead to increased gap junctional voltage dependence and conductance

1995 ◽  
Vol 269 (3) ◽  
pp. C590-C600 ◽  
Author(s):  
P. J. Donaldson ◽  
Y. Dong ◽  
M. Roos ◽  
C. Green ◽  
D. A. Goodenough ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Gap Junction ◽  
Voltage Dependence ◽  
Expression Patterns ◽  
Fiber Cell ◽  
Lens Epithelial Cells ◽  
Connexin Expression ◽  
Fiber Cells ◽  
Gap Junctional ◽  
Mouse Lens

The differentiation of mouse lens epithelial cells into fiber cells is a useful model for studying the changes of the electrical properties of gap junction (cell-to-cell) channels that are induced by an alteration in connexin expression patterns. In this model, cuboidal lens epithelial cells differentiate into elongated fiber cells, and the expression of connexin43 (Cx43) in the epithelial cells is replaced with the production of high levels of Cx50 and Cx46 in the fiber cells. We now report a new procedure to isolate mouse lens fiber cell pairs suitable for double whole cell patch-clamp analysis. Analysis was also performed for fiberlike cell pairs differentiated from epithelial cells in culture. Voltage dependence and unitary conductance of fiber cell gap junction channels were determined and compared with the corresponding values previously measured for the channels joining lens epithelial cells and for lens connexin channels formed in Xenopus oocyte pairs. Our results support a differentiation-induced shift toward stronger gap junctional voltage dependence and larger unitary conductances in the fiber cells. Our data further reflect a balanced functional contribution of Cx50 and Cx46 in the fiber cell-to-cell channels rather than a predominance of a single connexin.

A-Type K+ Current in Neurons Cultured From Neonatal Rat Hypothalamus and Brain Stem: Modulation by Angiotensin II

1997 ◽  
Vol 78 (2) ◽  
pp. 1021-1029 ◽  
Author(s):  
Desuo Wang ◽  
Colin Sumners ◽  
Philip Posner ◽  
Craig H. Gelband
Keyword(s):  
Angiotensin Ii ◽  
Brain Stem ◽  
Single Channel ◽  
Neonatal Rat ◽  
Ang Ii ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Voltage Step ◽  
Rat Hypothalamus ◽  
Type K

Wang, Desuo, Colin Sumners, Philip Posner, and Craig H. Gelband. A-type K+ current in neurons cultured from neonatal rat hypothalamus and brain stem: modulation by angiotensin II. J. Neurophysiol. 78: 1021–1029, 1997. The regulation of A-type K+ current ( I A) and the single channel underlying I A in neonatal rat hypothalamus/brain stem cultured neurons were studied with the use of the patch-clamp technique. I A had a threshold of activation between −30 and −25 mV ( n = 14). Steady-state inactivation of I A occurred between −80 and −70 mV and had a membrane voltage at which I A was half-maximum of −52.2 mV ( n = 14). The mean values for the activation and inactivation (decay) time constants during a voltage step to +20 mV were 2.1 ± 0.3 (SE) ms ( n = 8) and 13.6 ± 1.9 ms ( n = 8), respectively. Single-channel recordings from outside-out patches revealed A-type K+ channels with voltage-dependent activation, 4-aminopyridine (4-AP) sensitivity, and inactivation kinetics similar to those of I A. The single-channel conductance obtained from cell-attached patches was15.8 ± 1.3 pS ( n = 4) in a physiological K+ gradient and 41.2 ± 3.7 pS ( n = 5) in symmetrical 140 mM K+. Angiotensin II (Ang II, 100 nM) reduced peak I A by ∼20% during a voltage step to +20 mV ( n = 8). Similarly, Ang II (100 nM) markedly reduced single A-type K+ channel activity by decreasing open probability ( n = 4). The actions of Ang II on I A and single A-type K+ channels were reversible either by addition of the selective angiotensin type 1 (AT1) receptor antagonist losartan (1 μM) or on washout of the peptide. Thus the activation of AT1 receptors inhibits a tetraethylammonium-chloride-resistant, 4-AP-sensitive I A and single A-type K+ channels, and this may underlie some of the actions of Ang II on electrical activity of the brain.

Properties of gap junction channels formed of connexin 45 endogenously expressed in human hepatoma (SKHep1) cells

1995 ◽  
Vol 268 (2) ◽  
pp. C356-C365 ◽  
Author(s):  
A. P. Moreno ◽  
J. G. Laing ◽  
E. C. Beyer ◽  
D. C. Spray
Keyword(s):  
Gap Junction ◽  
Voltage Dependence ◽  
Hepatoma Cell ◽  
Hepatoma Cell Line ◽  
Voltage Sensitivity ◽  
Gap Junction Channels ◽  
Unitary Conductance ◽  
Junction Protein ◽  
Voltage Dependent ◽  
Gap Junctional

We have evaluated the voltage dependence and unitary conductance of gap junctional channels that were recorded in a clone isolated from the hepatoma cell line SKHep1. In this clonal population (designated SKHep1A), Northern blots, immunoprecipitation, and immunohistochemical staining demonstrated the expression of connexin (Cx) 45; no other gap junction protein was identified by these techniques, although weak hybridization with Cx40 was detected. Macroscopic junctional conductance (gj) in these cells was low, averaging 1.3 nS, and was steeply voltage dependent. Parameters of voltage sensitivity were as follows: voltage at which voltage-sensitive conductance is reduced by 50%, 13.4 mV; steepness of relation, 0.115 (corresponding to 2.7 gating charges), and voltage-insensitive fraction of residual to total conductance approximately 0.06. Unitary conductance (gamma j) of these junctional channels averaged 32 +/- 8 pS; although gamma j was independent of transjunctional voltage (Vj), at high Vj values (> 50 mV), smaller conductance values were also detected. Open probabilities of the 30-pS channels at various Vj values closely matched the predicted voltage-dependent component of macroscopic gj, the residual conductance at high Vj might be attributable to the smaller conductance events. The voltage dependence of human Cx45 gap junction channels is as steep as that seen for channels formed by Xenopus Cx38 and is much steeper than that previously reported for channels formed of the highly homologous chick Cx45 and for other mammalian connexins expressed either endogenously or exogenously.

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.

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.

Voltage-dependent gating of gap junction channels in embryonic chick ventricular cell pairs

1990 ◽  
Vol 258 (4) ◽  
pp. C662-C672 ◽  
Author(s):  
R. D. Veenstra
Keyword(s):  
Steady State ◽  
Gap Junction ◽  
Time Constant ◽  
Single Channel ◽  
Boltzmann Distribution ◽  
Embryonic Chick ◽  
Residual Voltage ◽  
Gap Junction Channels ◽  
Enzymatic Dissociation ◽  
Time Courses

The dependence of macroscopic gap junctional conductance (Gj) on transjunctional voltage (Vj) was studied in paired myocytes after enzymatic dissociation of 7-day-old embryonic chick ventricles. The membrane voltage of both cells was independently controlled by separate patch-clamp circuits in the whole cell configuration. Two distinctive unitary junctional conductances were identified in recordings from seven different cell pairs. The larger channel had a mean conductance of 166 +/- 37 pS (n = 6 pairs), whereas a second channel averaged 58 +/- 10 pS (n = 3). Instantaneous Gj remained linear over a Vj range of -100 to +100 mV, whereas the steady-state Gj declined when voltages exceeded +/- 30 mV. Both decay and recovery phases of Gj follow exponential time courses, with the recovery time constant being four times slower than inactivation, requiring 1.1 s at 80 mV. The normalized steady-state Gj-Vj curve could be defined by a two-state Boltzmann distribution, assuming an effective gating charge of 1.72, a half-inactivation voltage of 45 mV, and a residual voltage-insensitive Gj of 27% of maximum. Single-channel recordings revealed closure of 160-pS channels on a Vj step to 80 mV, and the ensemble average of five such records produced an exponentially decaying junctional current with a time constant of 184 ms. The single-channel current-voltage relationship remains linear with a slope of 145 pS over the entire Vj range. The results support the hypothesis that a population of 160-pS gap junction channels is gated by transjunctional potentials.

Cardiac gap junction channel activity in embryonic chick ventricle cells

1988 ◽  
Vol 254 (1) ◽  
pp. H170-H180 ◽  
Author(s):  
R. D. Veenstra ◽  
R. L. DeHaan
Keyword(s):  
Gap Junction ◽  
Single Channel ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Gap Junction Channel ◽  
Whole Cell Patch Clamp ◽  
Junctional Conductance ◽  
Low Levels ◽  
Active Channels ◽  
Channel Currents

We have recorded single-gap junction-channel currents from pairs of 7-day chick embryo ventricle cells, using the double whole cell patch-clamp technique. Junctional conductance (Gj) was variable from one preparation to the next, ranging from 0.15 to 35.0 nS. Single-channel conductance (gamma j) of the main junctional channel was 166 +/- 51 pS and was independent of Gj; a second conductance level of 60–80 pS was also seen in favorable records. The transition time from the closed to the open state was 285 +/- 153 microseconds, with some slow transitions lasting 1–5 ms. Channels opened and closed stochastically; Gj could be defined by the product of the number of active channels in the junction (N), the mean open-state probability (Po) of the channels, and gamma j. Channel activity was unaffected by cell membrane potential or by transjunctional potential. Po and Gj were reversibly reduced to low levels by 1-octanol or by elevated [Cai], whereas gamma j was unchanged by these agents. The 60–80 pS conductance mechanism was octanol- and Ca-resistant, but it is not clear whether this represents a subconductance level of the main channel or a separate class of smaller channels.

Asymmetric voltage dependence of embryonic cardiac gap junction channels

1996 ◽  
Vol 270 (1) ◽  
pp. C276-C285 ◽  
Author(s):  
Y. H. Chen ◽  
R. L. DeHaan
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Cardiac Development ◽  
Heart Cells ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Voltage Dependent ◽  
Chick Heart ◽  
Embryonic Chick Heart ◽  
Conductance States

The voltage dependence of junctional conductance (Gj) and the unitary channel behavior of junctions in most pairs of 3-day, 7-day, and 18-day embryonic chick heart cells are symmetrical, i.e., they are independent of the direction of polarization of junctional potential (Vj). With either cell depolarized relative to its neighbor, unitary channel events have a maximal unit conductance (yj) near 240 pS and five substates at nearly equal 40-pS increments down to near 40 pS (6, 9). Using the dual patch-clamp technique, we demonstrate here that, in a fraction of such cell pairs, Vj-dependent channel kinetics are asymmetric. Depolarization of one cell causes a larger and faster voltage-dependent decline in Gj than the same depolarization of the other cell. In a typical asymmetric preparation, depolarization of the strongly Vj-dependent side caused an immediate series of 47 +/- 16 pS closing steps in single-channel current (ij), followed by virtual cessation of channel activity. After depolarization of the less Vj-sensitive side, channel activity (56 +/- 13 pS) continues for many seconds. The large-conductance states (160-240 pS) observed in the electrically symmetric junctions were absent from the asymmetric preparations. In these cell pairs, connexin (Cx) 42, Cx43, and Cx45 could be immunolocalized at the junctional surfaces. We postulate that the asymmetry of voltage dependence in some cell pairs results from a preponderance of heterochannels formed from these different connexins. The frequency of asymmetric pairs obtained from 3-day, 7-day, and 18-day embryonic hearts was 50% (4/8), 24% (6/25), and 12.5% (1/8), suggesting that the fraction of heterochannels in the junctions decreases with cardiac development.

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