scholarly journals Patch recordings from the electrocytes of Electrophorus electricus. Na currents and PNa/PK variability.

1991 ◽  
Vol 97 (5) ◽  
pp. 1013-1041 ◽  
Author(s):  
S Shenkel ◽  
F J Sigworth
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Reversal Potential ◽  
Fluctuation Analysis ◽  
Open Probability ◽  
Na Currents ◽  
Before And After ◽  
Steady State Inactivation ◽  
K Current ◽  
Reversal Potentials

Sodium currents were recorded in cell-attached and inside-out patches from the innervated membrane of Electrophorus electrocytes. Electrocytes from Sachs and main electric organs were prepared as described by Pasquale et al. (1986. J. Membr. Biol. 93:195.). Maximal currents in the Sachs organ, measured with 1-2 microns diameter patch pipettes and at room temperature, were in the range of 20 to 300 pA (27 patches) and were obtained near +10 mV. This range of current corresponds to approximately 70 to 1,300 channels in a patch. Maximal current in main organ cells also occurred near +10 mV and were in the range of 100 to 400 pA. Delayed K current was observed in a few patches. The inactivation phase of the currents during maintained depolarizations appears to be a single-exponential relaxation. The time constant decreases from 1 ms near -55 mV to a minimum of 0.3 ms near 0 mV, and then gradually increases with stronger depolarization. The mean currents are half inactivated near -90 mV with an apparent voltage dependence of e-fold per 6 mV. No apparent differences were observed in the decay time course or steady-state inactivation of the currents in the same patch before and after excision. From ensemble fluctuation analysis the peak open probability was found to be approximately 0.5 at +25 mV and increased only gradually with larger depolarizations. The single channel conductances were approximately 20 pS with 200 mM Na outside and 200 mM K inside, and 40 pS in 400 mM solutions. Reversal potentials in the 200 Na parallel 200 K solutions ranged from +51 to +94 mV in multichannel patches, corresponding to selectivity ratios PNa/PK from 8 to 43. Large differences in reversal potentials were seen even among patches from the same cell. Several controls rule out obvious sources of error in the reversal potential measurements. It is concluded that there is heterogeneity in the selectivity properties of the Na channels.

scholarly journals Potassium channel types in arterial chemoreceptor cells and their selective modulation by oxygen.

1992 ◽  
Vol 100 (3) ◽  
pp. 401-426 ◽  
Author(s):  
M D Ganfornina ◽  
J López-Barneo
Keyword(s):  
Time Course ◽  
Single Channel ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Glomus Cells ◽  
Control Value ◽  
Voltage Dependent ◽  
K Current ◽  
Chemoreceptor Cells

Single K+ channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na+ and Ca2+ channels. We have found three classes of voltage-gated K+ channels that differ in their single-channel conductance (gamma), dependence on internal Ca2+ (Ca2+i), and sensitivity to changes in O2 tension (PO2). Ca(2+)-activated K+ channels (KCa channels) with gamma approximately 210 pS in symmetrical K+ solutions were observed when [Ca2+]i was greater than 0.1 microM. Small conductance channels with gamma = 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (P(open)) was unaffected when exposed to normoxic (PO2 = 140 mmHg) or hypoxic (PO2 approximately 5-10 mmHg) external solutions. A third channel type (referred to as KO2 channel), having an intermediate gamma(approximately 40 pS), was the most frequently recorded. KO2 channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in less than 500 ms, and their P(open) reversibly decreases upon exposure to low PO2. The effect of low PO2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, P(open) diminishes to approximately 40% of the control value. The time course of ensemble current averages of KO2 channels is remarkably similar to that of the O2-sensitive K+ current. In addition, ensemble average and macroscopic K+ currents are affected similarly by low PO2. These observations strongly suggest that KO2 channels are the main contributors to the macroscopic K+ current of glomus cells. The reversible inhibition of KO2 channel activity by low PO2 does not desensitize and is not related to the presence of F-, ATP, and GTP-gamma-S at the internal face of the membrane. These results indicate that KO2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K+ channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule.

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 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.

Single-channel basis for conductance increase induced by isoflurane in Shaker H4 IR K+ channels

2001 ◽  
Vol 280 (5) ◽  
pp. C1130-C1139 ◽  
Author(s):  
Jichang Li ◽  
Ana M. Correa
Keyword(s):  
Time Course ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Noise Analysis ◽  
Single Channels ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Open Probability ◽  
Slowing Down ◽  
Conductance Increase

Volatile anesthetics modulate the function of various K+ channels. We previously reported that isoflurane induces an increase in macroscopic currents and a slowing down of current deactivation of Shaker H4 IR K+ channels. To understand the single-channel basis of these effects, we performed nonstationary noise analysis of macroscopic currents and analysis of single channels in patches from Xenopus oocytes expressing Shaker H4 IR. Isoflurane (1.2% and 2.5%) induced concentration-dependent, partially reversible increases in macroscopic currents and in the time course of tail currents. Noise analysis of currents (70 mV) revealed an increase in unitary current (∼17%) and maximum open probability (∼20%). Single-channel conductance was larger (∼20%), and opening events were more stable, in isoflurane. Tail-current slow time constants increased by 41% and 136% in 1.2% and 2.5% isoflurane, respectively. Our results show that, in a manner consistent with stabilization of the open state, isoflurane increased the macroscopic conductance of Shaker H4 IR K+ channels by increasing the single-channel conductance and the open probability.

Development of muscarinic potassium current in fetal and neonatal rat heart

1997 ◽  
Vol 272 (3) ◽  
pp. H1188-H1195 ◽  
Author(s):  
M. Takano ◽  
A. Noma
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Cell Voltage ◽  
Neonatal Rat ◽  
Receptor Subtypes ◽  
Similar Time ◽  
Adult Rat ◽  
Functional Development ◽  
K Current ◽  
Underlying Mechanisms

Single atrial myocytes were isolated from fetal, neonatal, and adult rat hearts. The muscarinic K+ current activated by rapid application of acetylcholine (ACh) and adenosine (Ado) was recorded under the whole cell voltage clamp. The current density (pA/pF) of ACh-induced K+ current increased from gestation day 12 to the maximum on neonatal day 20 and decreased in the adult myocytes due to greater increase of the membrane capacitance. The development of Ado-induced K+ current followed a similar time course except for a remarkable decrease after neonatal day 10. No significant change was found in single-channel properties during the development. Receptor subtypes were M2 and A1 receptors for ACh and Ado, respectively. In the dose-response relationship, the half-maximal concentration for ACh-induced current markedly decreased with age, from 1.44 (fetus) to 0.17 microM (adult), whereas that for Ado increased from 0.45 (fetus) to 0.99 microM (adult). These changes of the muscarinic K+ current were discussed in relation to the functional development of cardiac myocytes and underlying mechanisms.

scholarly journals Intracellular calcium strongly potentiates agonist-activated TRPC5 channels

2009 ◽  
Vol 133 (5) ◽  
pp. 525-546 ◽  
Author(s):  
Nathaniel T. Blair ◽  
J. Stefan Kaczmarek ◽  
David E. Clapham
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Fold Increase ◽  
Receptor Activation ◽  
Brain Regions ◽  
Repetitive Firing ◽  
Dependent Manner ◽  
Open Probability ◽  
Current Voltage ◽  
Voltage Dependent

TRPC5 is a calcium (Ca2+)-permeable nonselective cation channel expressed in several brain regions, including the hippocampus, cerebellum, and amygdala. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous agonist activation, heterologously expressed TRPC5 currents are potentiated in a voltage-dependent manner (∼5-fold at positive potentials and ∼25-fold at negative potentials). The reversal potential, doubly rectifying current–voltage relation, and permeability to large cations such as N-methyl-d-glucamine remain unchanged during this potentiation. The TRPC5 current potentiation depends on extracellular Ca2+: replacement by Ba2+ or Mg2+ abolishes it, whereas the addition of 10 mM Ca2+ accelerates it. The site of action for Ca2+ is intracellular, as simultaneous fura-2 imaging and patch clamp recordings indicate that potentiation is triggered at ∼1 µM [Ca2+]. This potentiation is prevented when intracellular Ca2+ is tightly buffered, but it is promoted when recording with internal solutions containing elevated [Ca2+]. In cell-attached and excised inside-out single-channel recordings, increases in internal [Ca2+] led to an ∼10–20-fold increase in channel open probability, whereas single-channel conductance was unchanged. Ca2+-dependent potentiation should result in TRPC5 channel activation preferentially during periods of repetitive firing or coincident neurotransmitter receptor activation.

scholarly journals Gating of Recombinant Small-Conductance Ca-activated K+ Channels by Calcium

1998 ◽  
Vol 111 (4) ◽  
pp. 565-581 ◽  
Author(s):  
Birgit Hirschberg ◽  
James Maylie ◽  
John P. Adelman ◽  
Neil V. Marrion
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Cell Types ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Sensitive Clone ◽  
Single Channel Currents ◽  
Open States ◽  
Small Conductance

Small-conductance Ca-activated K+ channels play an important role in modulating excitability in many cell types. These channels are activated by submicromolar concentrations of intracellular Ca2+, but little is known about the gating kinetics upon activation by Ca2+. In this study, single channel currents were recorded from Xenopus oocytes expressing the apamin-sensitive clone rSK2. Channel activity was detectable in 0.2 μM Ca2+ and was maximal above 2 μM Ca2+. Analysis of stationary currents revealed two open times and three closed times, with only the longest closed time being Ca dependent, decreasing with increasing Ca2+ concentrations. In addition, elevated Ca2+ concentrations resulted in a larger percentage of long openings and short closures. Membrane voltage did not have significant effects on either open or closed times. The open probability was ∼0.6 in 1 μM free Ca2+. A lower open probability of ∼0.05 in 1 μM Ca2+ was also observed, and channels switched spontaneously between behaviors. The occurrence of these switches and the amount of time channels spent displaying high open probability behavior was Ca2+ dependent. The two behaviors shared many features including the open times and the short and intermediate closed times, but the low open probability behavior was characterized by a different, long Ca2+-dependent closed time in the range of hundreds of milliseconds to seconds. Small-conductance Ca- activated K+ channel gating was modeled by a gating scheme consisting of four closed and two open states. This model yielded a close representation of the single channel data and predicted a macroscopic activation time course similar to that observed upon fast application of Ca2+ to excised inside-out patches.

scholarly journals Differences in Glycinergic mIPSCs in the Auditory Brain Stem of Normal and Congenitally Deaf Neonatal Mice

2004 ◽  
Vol 91 (2) ◽  
pp. 1006-1012 ◽  
Author(s):  
Richardson N. Leao ◽  
Sharon Oleskevich ◽  
Hong Sun ◽  
Melissa Bautista ◽  
Robert E.W. Fyffe ◽  
...  
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Fluctuation Analysis ◽  
Electron Microscopic ◽  
Early Postnatal Development ◽  
Fundamental Properties ◽  
Medial Nucleus ◽  
Synaptic Structure ◽  
Auditory Brain Stem ◽  
Brain Slice Preparation

We have investigated the fundamental properties of central auditory glycinergic synapses in early postnatal development in normal and congenitally deaf ( dn/dn) mice. Glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded using patch-clamp methods in neurons from a brain slice preparation of the medial nucleus of the trapezoid body (MNTB), at 12-14 days postnatal age. Our results show a number of significant differences between normal and deaf mice. The frequency of mIPSCs is greater (50%) in deaf versus normal mice. Mean mIPSC amplitude is smaller in deaf mice than in normal mice (mean mIPSC amplitude: deaf, 64 pA; normal, 106 pA). Peak-scaled fluctuation analysis of mIPSCs showed that mean single channel conductance is greater in the deaf mice (deaf, 64 pS; normal, 45 pS). The mean decay time course of mIPSCs is slower in MNTB neurons from deaf mice (mean half-width: deaf, 2.9 ms; normal, 2.3 ms). Light- and electron-microscopic immunolabeling results showed that MNTB neurons from deaf mice have more (30%) inhibitory synaptic sites (postsynaptic gephyrin clusters) than MNTB neurons in normal mice. Our results demonstrate substantial differences in glycinergic transmission in normal and congenitally deaf mice, supporting a role for activity during development in regulating both synaptic structure (connectivity) and the fundamental (quantal) properties of mIPSCs at central glycinergic synapses.

scholarly journals Nicotinic acid–adenine dinucleotide phosphate activates the skeletal muscle ryanodine receptor

2002 ◽  
Vol 367 (2) ◽  
pp. 423-431 ◽  
Author(s):  
Martin HOHENEGGER ◽  
Josef SUKO ◽  
Regina GSCHEIDLINGER ◽  
Helmut DROBNY ◽  
Andreas ZIDAR
Keyword(s):  
Skeletal Muscle ◽  
Sarcoplasmic Reticulum ◽  
Nicotinic Acid ◽  
Ryanodine Receptor ◽  
Spatial Information ◽  
Single Channel ◽  
Reversal Potential ◽  
Open Probability ◽  
Release Channel

Calcium is a universal second messenger. The temporal and spatial information that is encoded in Ca2+-transients drives processes as diverse as neurotransmitter secretion, axonal outgrowth, immune responses and muscle contraction. Ca2+-release from intracellular Ca2+ stores can be triggered by diffusible second messengers like InsP3, cyclic ADP-ribose or nicotinic acid—adenine dinucleotide phosphate (NAADP). A target has not yet been identified for the latter messenger. In the present study we show that nanomolar concentrations of NAADP trigger Ca2+-release from skeletal muscle sarcoplasmic reticulum. This was due to a direct action on the Ca2+-release channel/ryanodine receptor type-1, since in single channel recordings, NAADP increased the open probability of the purified channel protein. The effects of NAADP on Ca2+-release and open probability of the ryanodine receptor occurred over a similar concentration range (EC5030nM) and were specific because (i) they were blocked by Ruthenium Red and ryanodine, (ii) the precursor of NAADP, NADP, was ineffective at equimolar concentrations, (iii) NAADP did not affect the conductance and reversal potential of the ryanodine receptor. Finally, we also detected an ADP-ribosyl cyclase activity in the sarcoplasmic reticulum fraction of skeletal muscle. This enzyme was not only capable of synthesizing cyclic GDP-ribose but also NAADP, with an activity of 0.25nmol/mg/min. Thus, we conclude that NAADP is generated in the vicinity of type 1 ryanodine receptor and leads to activation of this ion channel.

scholarly journals Charybdotoxin selectively blocks small Ca-activated K channels in Aplysia neurons.

1987 ◽  
Vol 90 (1) ◽  
pp. 27-47 ◽  
Author(s):  
A Hermann ◽  
C Erxleben
Keyword(s):  
Single Channel ◽  
Delayed Rectifier ◽  
Pacemaker Activity ◽  
Dependent Manner ◽  
Open Probability ◽  
Apparent Dissociation Constant ◽  
External Application ◽  
K Channels ◽  
Voltage Dependent ◽  
K Current

The action of charybdotoxin (ChTX), a peptide component isolated from the venom of the scorpion Leiurus quinquestriatus, was investigated on membrane currents of identified neurons from the marine mollusk, Aplysia californica. Macroscopic current recordings showed that the external application of ChTX blocks the Ca-activated K current in a dose- and voltage-dependent manner. The apparent dissociation constant is 30 nM at V = -30 mV and increases e-fold for a +50- to +70-mV change in membrane potential, which indicates that the toxin molecule is sensitive to approximately 35% of the transmembrane electric field. The toxin is bound to the receptor with a 1:1 stoichiometry and its effect is reversible after washout. The toxin also suppresses the membrane leakage conductance and a resting K conductance activated by internal Ca ions. The toxin has no significant effect on the inward Na or Ca currents, the transient K current, or the delayed rectifier K current. Records from Ca-activated K channels revealed a single channel conductance of 35 +/- 5 pS at V = 0 mV in asymmetrical K solution. The channel open probability increased with the internal Ca concentration and with membrane voltage. The K channels were blocked by submillimolar concentrations of tetraethylammonium ions and by nanomolar concentrations of ChTX, but were not blocked by 4-aminopyridine if applied externally on outside-out patches. From the effects of ChTX on K current and on bursting pacemaker activity, it is concluded that the termination of bursts is in part controlled by a Ca-activated K conductance.

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