scholarly journals Anomalous L-Type Calcium Channels of Rat Spinal Motoneurons

1999 ◽  
Vol 113 (5) ◽  
pp. 679-694 ◽  
Author(s):  
Bruno Hivert ◽  
Siro Luvisetto ◽  
Anacleto Navangione ◽  
Angelita Tottene ◽  
Daniela Pietrobon
Keyword(s):  
Calcium Channels ◽  
Voltage Dependence ◽  
Single Channel ◽  
Spinal Motoneurons ◽  
Relative Probability ◽  
Open Probability ◽  
Voltage Dependent ◽  
The Mean ◽  
Cytosolic Factor ◽  
Rule Out

Single channel patch-clamp recordings show that embryonic rat spinal motoneurons express anomalous L-type calcium channels, which reopen upon repolarization to resting potentials, displaying both short and long reopenings. The probability of reopening increases with increasing voltage of the preceding depolarization without any apparent correlation with inactivation during the depolarization. The probability of long with respect to short reopenings increases with increasing length of the depolarization, with little change in the total number of reopenings and in their delay. With less negative repolarization voltages, the delay increases, while the mean duration of both short and long reopenings decreases, remaining longer than that of the openings during the preceding depolarization. Open times decrease with increasing voltage in the range −60 to +40 mV. Closed times tend to increase at V > 20 mV. The open probability is low at all voltages and has an anomalous bell-shaped voltage dependence. We provide evidence that short and long reopenings of anomalous L-type channels correspond to two gating modes, whose relative probability depends on voltage. Positive voltages favor both the transition from a short-opening to a long-opening mode and the occupancy of a closed state outside the activation pathway within each mode from which the channel reopens upon repolarization. The voltage dependence of the probability of reopenings reflects the voltage dependence of the occupancy of these closed states, while the relative probability of long with respect to short reopenings reflects the voltage dependence of the equilibrium between modes. The anomalous gating persists after patch excision, and therefore our data rule out voltage-dependent block by diffusible ions as the basis for the anomalous gating and imply that a diffusible cytosolic factor is not necessary for voltage-dependent potentiation of anomalous L-type channels.

scholarly journals Voltage and Ca2+ Activation of Single Large-Conductance Ca2+-Activated K+ Channels Described by a Two-Tiered Allosteric Gating Mechanism

2000 ◽  
Vol 116 (1) ◽  
pp. 75-100 ◽  
Author(s):  
Brad S. Rothberg ◽  
Karl L. Magleby
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Open Probability ◽  
Gating Charge ◽  
Gating Mechanism ◽  
Single Channel Analysis ◽  
Voltage Dependent ◽  
The Mean

The voltage- and Ca2+-dependent gating mechanism of large-conductance Ca2+-activated K+ (BK) channels from cultured rat skeletal muscle was studied using single-channel analysis. Channel open probability (Po) increased with depolarization, as determined by limiting slope measurements (11 mV per e-fold change in Po; effective gating charge, qeff, of 2.3 ± 0.6 eo). Estimates of qeff were little changed for intracellular Ca2+ (Ca2+i) ranging from 0.0003 to 1,024 μM. Increasing Ca2+i from 0.03 to 1,024 μM shifted the voltage for half maximal activation (V1/2) 175 mV in the hyperpolarizing direction. V1/2 was independent of Ca2+i for Ca2+i ≤ 0.03 μM, indicating that the channel can be activated in the absence of Ca2+i. Open and closed dwell-time distributions for data obtained at different Ca2+i and voltage, but at the same Po, were different, indicating that the major action of voltage is not through concentrating Ca2+ at the binding sites. The voltage dependence of Po arose from a decrease in the mean closing rate with depolarization (qeff = −0.5 eo) and an increase in the mean opening rate (qeff = 1.8 eo), consistent with voltage-dependent steps in both the activation and deactivation pathways. A 50-state two-tiered model with separate voltage- and Ca2+-dependent steps was consistent with the major features of the voltage and Ca2+ dependence of the single-channel kinetics over wide ranges of Ca2+i (∼0 through 1,024 μM), voltage (+80 to −80 mV), and Po (10−4 to 0.96). In the model, the voltage dependence of the gating arises mainly from voltage-dependent transitions between closed (C-C) and open (O-O) states, with less voltage dependence for transitions between open and closed states (C-O), and with no voltage dependence for Ca2+-binding and unbinding. The two-tiered model can serve as a working hypothesis for the Ca2+- and voltage-dependent gating of the BK channel.

Effect of divalent heavy metals on epithelial Na+ channels in A6 cells

2007 ◽  
Vol 293 (1) ◽  
pp. F236-F244 ◽  
Author(s):  
Ling Yu ◽  
Douglas C. Eaton ◽  
My N. Helms
Keyword(s):  
Heavy Metal ◽  
Voltage Dependence ◽  
Transmembrane Domain ◽  
Single Channel ◽  
Epithelial Cell Line ◽  
Open Probability ◽  
Renal Epithelial Cell ◽  
Histidine Residues ◽  
Voltage Dependent ◽  
Renal Epithelial Cell Line

To better understand how renal Na+ reabsorption is altered by heavy metal poisoning, we examined the effects of several divalent heavy metal ions (Zn2+, Ni2+, Cu2+, Pb2+, Cd2+, and Hg2+) on the activity of single epithelial Na+ channels (ENaC) in a renal epithelial cell line (A6). None of the cations changed the single-channel conductance. However, ENaC activity [measured as the number of channels ( N) × open probability ( Po)] was decreased by Cd2+ and Hg2+ and increased by Cu2+, Zn2+, and Ni2+ but was not changed by Pb2+. Of the cations that induced an increase in Na+ channel function, Zn2+ increased N, Ni2+ increased Po, and Cu2+ increased both. The cysteine modification reagent [2-(trimethylammonium)ethyl]methanethiosulfonate bromide also increased N, whereas diethylpyrocarbonate, which covalently modifies histidine residues, affected neither Po nor N. Cu2+ increased N and stimulated Po by reducing Na+ self-inhibition. Furthermore, we observed that ENaC activity is slightly voltage dependent and that the voltage dependence of ENaC is insensitive to extracellular Na+ concentration; however, apical application of Ni2+ or diethylpyrocarbonate reduced the channel voltage dependence. Thus the voltage sensor of Xenopus ENaC is different from that of typical voltage-gated channels, since voltage appears to be sensed by histidine residues in the extracellular loops of ENaC, rather than by charged amino acids in a transmembrane domain.

scholarly journals A molecular link between activation and inactivation of sodium channels.

1995 ◽  
Vol 106 (4) ◽  
pp. 641-658 ◽  
Author(s):  
M E O'Leary ◽  
L Q Chen ◽  
R G Kallen ◽  
R Horn
Keyword(s):  
Sodium Channels ◽  
Voltage Dependence ◽  
Single Channel ◽  
Critical Role ◽  
Channel Measurements ◽  
Wild Type ◽  
Open Probability ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Normal Coupling

A pair of tyrosine residues, located on the cytoplasmic linker between the third and fourth domains of human heart sodium channels, plays a critical role in the kinetics and voltage dependence of inactivation. Substitution of these residues by glutamine (Y1494Y1495/QQ), but not phenylalanine, nearly eliminates the voltage dependence of the inactivation time constant measured from the decay of macroscopic current after a depolarization. The voltage dependence of steady state inactivation and recovery from inactivation is also decreased in YY/QQ channels. A characteristic feature of the coupling between activation and inactivation in sodium channels is a delay in development of inactivation after a depolarization. Such a delay is seen in wild-type but is abbreviated in YY/QQ channels at -30 mV. The macroscopic kinetics of activation are faster and less voltage dependent in the mutant at voltages more negative than -20 mV. Deactivation kinetics, by contrast, are not significantly different between mutant and wild-type channels at voltages more negative than -70 mV. Single-channel measurements show that the latencies for a channel to open after a depolarization are shorter and less voltage dependent in YY/QQ than in wild-type channels; however the peak open probability is not significantly affected in YY/QQ channels. These data demonstrate that rate constants involved in both activation and inactivation are altered in YY/QQ channels. These tyrosines are required for a normal coupling between activation voltage sensors and the inactivation gate. This coupling insures that the macroscopic inactivation rate is slow at negative voltages and accelerated at more positive voltages. Disruption of the coupling in YY/QQ alters the microscopic rates of both activation and inactivation.

A voltage-dependent and calcium-permeable ion channel in fused presynaptic terminals of Torpedo

1996 ◽  
Vol 75 (5) ◽  
pp. 1858-1870 ◽  
Author(s):  
A. Meir ◽  
R. Rahamimoff
Keyword(s):  
Ion Channel ◽  
Nerve Terminal ◽  
Single Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Voltage Range ◽  
Voltage Dependent ◽  
The Mean ◽  
Presynaptic Nerve Terminals ◽  
Channel Open Time

1. We used a preparation of fused presynaptic nerve terminals of Torpedo electromotor nerve and the patch-clamp technique for characterization of single ion channels. We report here of a large, nonselective ion channel which is highly voltage dependent. 2. The slope conductance of the I-V relation was estimated by either direct measurement of the single-channel current amplitude at different voltages (850 +/- 18 pS (SE); n = 9) or by variance analysis (834 +/- 23 pS; n = 5). 3. The voltage dependence was examined in three ways. At steady-state DC voltage conditions, NPo (the open probability times the number of channels in the patch) was estimated. At potentials < 0 mV, the probability of the channel to open is negligible and increases dramatically, within a very narrow voltage range, to > 50% at +8 mV (n = 8). 4. In pulse experiments, the activation time delay is shorter as the voltage step reaches more positive values. The mean time for half activation (T1/2) decreases from 15 ms at +10 mV to 4 ms at +30 mV (n = 5). 5. Ensemble currents exhibit rectification in response to voltage ramps at negative potentials (n = 10). 6. The channel was found to be nonselective. Its permeability to Na+, K+, Cl-, glutamate, Ba+2, and Ca+2, relative to Na+, was 1.00, 1.00, 1.22, 1.07, 0.85, and 0.62, respectively. 7. Based on the transport number of calcium, the calculated driving force, and the mean channel open time, we estimated the number of calcium ions entering the nerve terminal upon depolarization. This number is not substantially different from the number of ions entering through voltage-dependent, calcium-selective channels in other cells. 8. We speculate that this nonselective ion channel, may serve as a calcium entry route into the nerve terminal and hence be involved in transmitter release.

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.

scholarly journals Modal Gating of Human CaV2.1 (P/Q-type) Calcium Channels

2004 ◽  
Vol 124 (5) ◽  
pp. 463-474 ◽  
Author(s):  
Tommaso Fellin ◽  
Siro Luvisetto ◽  
Michele Spagnolo ◽  
Daniela Pietrobon
Keyword(s):  
Calcium Channels ◽  
Time Scale ◽  
Neuronal Excitability ◽  
Voltage Dependence ◽  
Single Channel ◽  
Preceding Paper ◽  
Hek293 Cells ◽  
Open Probability ◽  
Modal Gating ◽  
Complex Modal

The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing these calcium channels. Human CaV2.1 channels showed a complex modal gating, which is described in this and the preceding paper (Luvisetto, S., T. Fellin, M. Spagnolo, B. Hivert, P.F. Brust, M.M. Harpold, K.A. Stauderman, M.E. Williams, and D. Pietrobon. 2004. J. Gen. Physiol. 124:445–461). Here, we report the characterization of the so-called b gating mode. A CaV2.1 channel in the b gating mode shows a bell-shaped voltage dependence of the open probability, and a characteristic low open probability at high positive voltages, that decreases with increasing voltage, as a consequence of both shorter mean open time and longer mean closed time. Reversible transitions of single human CaV2.1 channels between the b gating mode and the mode of gating in which the channel shows the usual voltage dependence of the open probability (nb gating mode) were much more frequent (time scale of seconds) than those between the slow and fast gating modes (time scale of minutes; Luvisetto et al., 2004), and occurred independently of whether the channel was in the fast or slow mode. We show that the b gating mode produces reversible uncoupling of inactivation in human CaV2.1 channels. In fact, a CaV2.1 channel in the b gating mode does not inactivate during long pulses at high positive voltages, where the same channel in both fast-nb and slow-nb gating modes inactivates relatively rapidly. Moreover, a CaV2.1 channel in the b gating mode shows a larger availability to open than in the nb gating modes. Regulation of the complex modal gating of human CaV2.1 channels could be a potent and versatile mechanism for the modulation of synaptic strength and plasticity as well as of neuronal excitability and other postsynaptic Ca2+-dependent processes.

scholarly journals The weak voltage dependence of pannexin 1 channels can be tuned by N-terminal modifications

2018 ◽  
Vol 150 (12) ◽  
pp. 1758-1768 ◽  
Author(s):  
Kevin Michalski ◽  
Erik Henze ◽  
Phillip Nguyen ◽  
Patrick Lynch ◽  
Toshimitsu Kawate
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Atp Release ◽  
Channel Activity ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
Pannexin 1 ◽  
Voltage Dependent ◽  
Gated Channel

Pannexins are a family of ATP release channels important for physiological and pathological processes like blood pressure regulation, epilepsy, and neuropathic pain. To study these important channels in vitro, voltage stimulation is the most common and convenient tool, particularly for pannexin 1 (Panx1). However, whether Panx1 is a voltage-gated channel remains controversial. Here, we carefully examine the effect of N-terminal modification on voltage-dependent Panx1 channel activity. Using a whole-cell patch-clamp recording technique, we demonstrate that both human and mouse Panx1, with their nativeN termini, give rise to voltage-dependent currents, but only at membrane potentials larger than +100 mV. This weak voltage-dependent channel activity profoundly increases when a glycine–serine (GS) motif is inserted immediately after the first methionine. Single-channel recordings reveal that the addition of GS increases the channel open probability as well as the number of unitary conductance classes. We also find that insertions of other amino acid(s) at the same position mimics the effect of GS. On the other hand, tagging the N terminus with GFP abolishes voltage-dependent channel activity. Our results suggest that Panx1 is a channel with weak voltage dependence whose activity can be tuned by N-terminal modifications.

scholarly journals Interactions of divalent cations with single calcium channels from rat brain synaptosomes.

1986 ◽  
Vol 87 (2) ◽  
pp. 201-222 ◽  
Author(s):  
M T Nelson
Keyword(s):  
Calcium Channels ◽  
Rat Brain ◽  
Divalent Cation ◽  
Single Channel ◽  
Calcium Currents ◽  
Single Channels ◽  
Dependent Manner ◽  
Voltage Dependent ◽  
The Mean ◽  
Single Channel Currents

Voltage-dependent calcium channels from a rat brain membrane preparation ("synaptosomes") were incorporated into planar lipid bilayers. The effects of calcium, barium, strontium, manganese, and cadmium ions on the amplitudes and kinetics of single channel currents were examined. The order of single channel conductances was gBa greater than gSr greater than gMn, which was the inverse of the order of the mean channel open times: TMn greater than TCa = TSr greater than TBa. In contrast, the identity of the charge carrier had little or no effect on the mean closed times of the channel. Manganese, in the absence of other permeant ions, can pass through single channels (gMn = 4 pS). However, when added to a solution that contained another type of permeant divalent cation, manganese reduced the single channel current in a voltage-dependent manner. Cadmium, a potent blocker of macroscopic "ensemble" calcium currents in many preparations, reduced the current through an open channel in a manner consistent with Cd ions both not being measurably permeant and interacting with a single site. The permeant ions competed with cadmium for this site with the following order: Mn greater than Sr = Ca greater than Ba. These results are consistent with the existence of no less than one divalent cation binding site in the channel that regulates ion permeation.

scholarly journals A Dihydropyridine-sensitive Voltage-dependent Calcium Channel in the Sarcolemmal Membrane of Crustacean Muscle

1997 ◽  
Vol 109 (3) ◽  
pp. 313-326 ◽  
Author(s):  
Christian Erxleben ◽  
Werner Rathmayer
Keyword(s):  
Calcium Channels ◽  
Calcium Channel ◽  
Single Channel ◽  
Resting Membrane Potential ◽  
Open Probability ◽  
Cardiac Muscle Cells ◽  
Idotea Baltica ◽  
Voltage Dependent ◽  
Marine Crustacean ◽  
Single Channel Currents

Single-channel currents through calcium channels in muscle of a marine crustacean, the isopod Idotea baltica, were investigated in cell-attached patches. Inward barium currents were strongly voltage-dependent, and the channels were closed at the cell's resting membrane potential. The open probability (Po) increased e-fold for an 8.2 mV (±2.4, n = 13) depolarization. Channel openings were mainly brief (&lt;0.3 ms) and evenly distributed throughout 100-ms pulses. Averaged, quasimacroscopic currents showed fast activation and deactivation and did not inactivate during 100-ms test pulses. Similarly, channel activity persisted at steadily depolarized holding potentials. With 200 mM Ba2+ as charge carrier, the average slope conductance from the unitary currents between +30 and +80 mV, was 20 pS (±2.6, n = 12). The proportion of long openings, which were very infrequent under control conditions, was greatly increased by preincubation of the muscle fibers with the calcium channel agonist, the dihydropyridine Bay K8644 (10–100 μM). Properties of these currents resemble those through the L-type calcium channels of mammalian nerve, smooth muscle, and cardiac muscle cells.

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 &lt; or = 0.25 or &gt; 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 &lt; 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.

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