scholarly journals Fast and slow gating are inherent properties of the pore module of the K+ channel Kcv

2009 ◽  
Vol 134 (3) ◽  
pp. 219-229 ◽  
Author(s):  
Alessandra Abenavoli ◽  
Mattia Lorenzo DiFrancesco ◽  
Indra Schroeder ◽  
Svetlana Epimashko ◽  
Sabrina Gazzarrini ◽  
...  
Keyword(s):  
Single Channel ◽  
Negative Slope ◽  
K Channel ◽  
Open Probability ◽  
Channel Current ◽  
Voltage Curve ◽  
Voltage Dependent ◽  
Current Voltage Curve ◽  
Fast Gating ◽  
Basic Features

Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K+ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K+ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K+, single-channel conductance is 114 ± 11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. “Fast” gating, analyzed by β distributions, is responsible for the negative slope conductance in the single-channel current–voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a “slow” gating is revealed by the very low (in the order of 1–4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current.

scholarly journals Calcium currents in the A7r5 smooth muscle-derived cell line. Increase in current and selective removal of voltage-dependent inactivation by intracellular trypsin.

1991 ◽  
Vol 98 (6) ◽  
pp. 1127-1140 ◽  
Author(s):  
C A Obejero-Paz ◽  
S W Jones ◽  
A Scarpa
Keyword(s):  
Smooth Muscle ◽  
Cell Line ◽  
Single Channel ◽  
Calcium Currents ◽  
Open Probability ◽  
Calcium Channel Inactivation ◽  
Voltage Curve ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Current Voltage Curve

We studied the effects of trypsin on L-type calcium current in the A7r5 smooth muscle cell line. Intracellular dialysis with trypsin increased the whole-cell current up to fivefold. The effect was concentration dependent, and was prevented by soybean trypsin inhibitor. Ensemble analysis indicated an increase in the number of functional channels, and possibly a smaller increase in the open probability, with no change in the single channel current. The shape of the current-voltage curve was unaffected. Trypsin also nearly eliminated inactivation of currents carried by Ba2+, but had little or no effect on the rapid inactivation process in Ca2+, This indicates that trypsin removes voltage-dependent but not Ca(2+)-dependent inactivation, suggesting the existence of distinct protein domains for these two mechanisms of calcium channel inactivation.

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.

Two types of K+ channels at the basolateral membrane of proximal tubule: inhibitory effect of taurine

1999 ◽  
Vol 277 (2) ◽  
pp. F290-F297 ◽  
Author(s):  
Jean-François Noulin ◽  
Emmanuelle Brochiero ◽  
Jean-Yves Lapointe ◽  
Raynald Laprade
Keyword(s):  
Potassium Channels ◽  
Time Constant ◽  
Potassium Conductance ◽  
Single Type ◽  
K Channel ◽  
Open Probability ◽  
Open Time ◽  
Voltage Curve ◽  
Current Voltage ◽  
Current Voltage Curve

The cell-attached configuration of the patch-clamp technique was used to investigate the effects of taurine on the basolateral potassium channels of rabbit proximal convoluted tubule. In the absence of taurine, the previously reported ATP-blockable channel, KATP, was observed in 51% of patches. It is characterized by an inwardly rectifying current-voltage curve with an inward slope conductance of 49 ± 5 pS ( n = 15) and an outward slope conductance of 13 ± 6 pS ( n = 15). The KATP channel open probability ( P o) is low, 0.15 ± 0.06 ( n = 15) at a − V p = −100 mV ( V pis the pipette potential), and increases slightly with depolarization. The gating kinetics are characterized by one open time constant (τo = 5.0 ± 1.9 ms, n = 6) and two closed time constants (τC1 = 5.2 ± 1.5 ms, τC2 = 140 ± 40 ms; n = 6). In 34% of patches, a second type of potassium channel, sK, with distinct properties was recorded. Its current-voltage curve is characterized by a sigmoidal shape, with an inward slope conductance of 12 ± 2 pS ( n = 4). Its P o is voltage independent and averages 0.67 ± 0.03 ( n = 4) at − V p = −80 mV. Both its open time and closed time distributions are described by a single time constant (τo = 96 ± 19 ms, τC = 10.5 ± 3.6 ms; n = 4). Extracellular perfusion of 40 mM taurine fails to affect sK channels, whereas KATP channel P o decreases by 75% (from 0.17 ± 0.06 to 0.04 ± 0.02, n = 7, P < 0.05). In conclusion, the absolute basolateral potassium conductance of rabbit proximal tubules is the resulting combination of, at least, two types of potassium channels of roughly equal importance: a high-conductance low-open probability KATP channel and a low-conductance high-open probability sK channel. The previously described decrease in the basolateral absolute potassium conductance by taurine is, however, mediated by a single type of K channel: the ATP-blockable K channel.

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.

Quinidine blocks adenosine 5'-triphosphate-sensitive potassium channels in heart

1990 ◽  
Vol 259 (5) ◽  
pp. H1609-H1612 ◽  
Author(s):  
A. I. Undrovinas ◽  
N. Burnashev ◽  
D. Eroshenko ◽  
I. Fleidervish ◽  
C. F. Starmer ◽  
...  
Keyword(s):  
Single Channel ◽  
Dependent Manner ◽  
Open Probability ◽  
Pipette Solution ◽  
Channel Current ◽  
Closed Intervals ◽  
Ventricular Cells ◽  
Voltage Dependent ◽  
Inside Out ◽  
Ischemic Arrhythmias

The ATP-sensitive potassium channel current (IK-ATP) was studied in excised inside-out patches from rat ventricular cells at 20-23 degrees C. The bath solution contained 140 mM KF, and the pipette solution contained 140 mM KCl and 1.2 mM MgCl2. ATP (0.5 mM) in the bath inhibited IK-ATP. In the absence of ATP, 10 microM quinidine decreased open probability 67 +/- 1% (n = 6) at -50 mV and 28 +/- 12% at -130 mV (n = 5) without affecting single channel conductance (48-52 pS). The block increased with 25 and 50 microM quinidine and could be reversed on washing quinidine for several minutes. Interburst (closed) intervals were increased by quinidine, whereas open and closed time distributions within bursts were not changed. We conclude that quinidine blocks IK-ATP in a "slow" and voltage-dependent manner in clinically relevant concentrations. Because of the postulated role for IK-ATP in cardiac ischemia, quinidine block of this channel may play a role in ischemic arrhythmias.

Permeation and block of dopamine-modulated potassium channels on rat striatal neurons by cesium and barium ions

1996 ◽  
Vol 76 (3) ◽  
pp. 1413-1422 ◽  
Author(s):  
Y. J. Lin ◽  
G. J. Greif ◽  
J. E. Freedman
Keyword(s):  
Dissociation Constant ◽  
Single Channel ◽  
Reversal Potential ◽  
Negative Slope ◽  
K Channel ◽  
Striatal Neurons ◽  
Apparent Dissociation Constant ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Inwardly Rectifying

1. In cell-attached patch-clamp recordings from freshly dissociated rat caudate-putamen neurons, an 85-pS inwardly rectifying K+ channel, which was previously found to be modulated by D2-like dopamine receptors, was blocked by externally applied BaCl2 or CsCl. 2. At concentrations between 100 and 500 microM, Ba2+ blockade was voltage dependent, with a greater block at hyperpolarized voltages, in a manner consistent with blockade of the channel pore. Single-channel currents were flickery, with intervening periods of more complete blockade, and block appeared to be time dependent, with an estimated electrical distance of 0.24 and an apparent dissociation constant of 205 microM at 0 mV. 3. At concentrations between 1 and 3 mM, Cs+ blockade was similarly voltage dependent, but without periods of longer blockade, with an electrical distance of 0.81 and an apparent dissociation constant of 625 microM at 0 mV. Cs+ could also permeate this channel at voltages near the K+ reversal potential. The current-voltage relationship displayed an anomalous negative slope conductance, in a manner inconsistent with a single-ion pore. 4. Smaller-conductance, dopamine-insensitive channels were blocked more potently by both Ba2+ and Cs+ than was the 85-pS channel, reflecting differences between inwardly rectifying K+ channels mediating resting conductance and those mediating dopamine receptor responses in striatal neurons.

scholarly journals AMRP Peptides Modulate a Novel K+ Current in Pleural Sensory Neurons of Aplysia

2002 ◽  
Vol 88 (1) ◽  
pp. 323-332 ◽  
Author(s):  
Jonathan R. McDearmid ◽  
Vladimir Brezina ◽  
Klaudiusz R. Weiss
Keyword(s):  
Sensory Neurons ◽  
Single Channel ◽  
Outward Current ◽  
Negative Slope ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Outward Rectification ◽  
Defensive Behaviors ◽  
Mechanosensory Neurons

Modulation of Aplysia mechanosensory neurons is thought to underlie plasticity of defensive behaviors that are mediated by these neurons. In the past, identification of modulators that act on the sensory neurons and characterization of their actions has been instrumental in providing insight into the functional role of the sensory neurons in the defensive behaviors. Motivated by this precedent and a recent report of the presence of Aplysia Mytilusinhibitory peptide-related (AMRP) neuropeptides in the neuropile and neurons of the pleural ganglia, we sought to determine whether and how pleural sensory neurons respond to the AMRPs. In cultured pleural sensory neurons under voltage clamp, AMRPs elicited a relatively rapidly developing, then partially desensitizing, outward current. The current exhibited outward rectification; in normal 10 mM K+, it was outward at membrane potentials more positive than −80 mV but disappeared without reversing at more negative potentials. When external K+ was elevated to 100 mM, the AMRP-elicited current reversed around −25 mV; the shift in reversal potential was as expected for a current carried primarily by K+. In the high-K+ solution, the reversed current began to decrease at potentials more negative than −60 mV, creating a region of negative slope resistance in the I-V relationship. The AMRP-elicited K+ current was blocked by extremely low concentrations of 4-aminopyridine (4-AP; IC50= 1.7 × 10−7 M) but was not very sensitive to TEA. In cell-attached patches, AMRPs applied outside the patch—thus presumably through a diffusible messenger—increased the activity of a K+ channel that very likely underlies the macroscopic current. The single-channel current exhibited outward rectification, and the open probability of the channel decreased with hyperpolarization; together, these two factors accounted for the outward rectification of the macroscopic current. Submicromolar 4-AP included in the patch pipette blocked the channel by reducing its open probability without altering the single-channel current. Based on the characteristics of the AMRP-modulated K+ current, we conclude that it is a novel current that has not been previously described in Aplysia mechanosensory neurons. In addition to this current, two other AMRP-elicited currents, a slow, 4-AP-resistant outward current and a Na+-dependent inward current, were occasionally observed in the cultured sensory neurons. Responses consistent with all three currents were observed in sensory neurons in situ in intact pleural ganglia.

scholarly journals Voltage-sensitive gating induced by a mutation in the fifth transmembrane domain of CFTR

2002 ◽  
Vol 282 (1) ◽  
pp. L135-L145 ◽  
Author(s):  
Zhi-Ren Zhang ◽  
Shawn Zeltwanger ◽  
Stephen S. Smith ◽  
David C. Dawson ◽  
Nael A. McCarty
Keyword(s):  
Transmembrane Domain ◽  
Single Channel ◽  
Fold Increase ◽  
Open Probability ◽  
Whole Cell ◽  
Channel Current ◽  
Outward Rectification ◽  
Voltage Dependent ◽  
Excised Patches ◽  
Active Channels

A mutation in the fifth transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel (V317E) resulted in whole cell currents that exhibited marked outward rectification on expression in Xenopus oocytes. However, the single-channel unitary current ( i)-voltage ( V) relationship failed to account for the rectification of whole cell currents. In excised patches containing one to a few channels, the time-averaged single-channel current ( I)- V relationship ( I = N × P o × i, where N is the number of active channels and P o is open probability) of V317E CFTR displayed outward rectification, whereas that of wild-type CFTR was linear, indicating that the P o of V317E CFTR is voltage dependent. The decrease in P o at negative potentials was due to both a decreased burst duration and a decreased opening rate that could not be ameliorated by a 10-fold increase in ATP concentration. This behavior appears to reflect a true voltage dependence of the gating process because the P o- V relationship did not depend on the direction of Cl− movement. The results are consistent with the introduction, by a point mutation, of a novel voltage-dependent gating mode that may provide a useful tool for probing the portions of the protein that move in response to ATP-dependent gating.

scholarly journals PIP2 Mediated Inhibition of TREK Potassium Currents by Bradykinin in Mouse Sympathetic Neurons

2020 ◽  
Vol 21 (2) ◽  
pp. 389 ◽  
Author(s):  
Paula Rivas-Ramírez ◽  
Antonio Reboreda ◽  
Lola Rueda-Ruzafa ◽  
Salvador Herrera-Pérez ◽  
J. Antonio Lamas
Keyword(s):  
Single Channel ◽  
Sympathetic Neurons ◽  
K Channel ◽  
Muscarinic Agonists ◽  
Open Probability ◽  
M Current ◽  
Voltage Dependent ◽  
Cervical Ganglion ◽  
Patch Technique ◽  
Single Channel Currents

Bradykinin (BK), a hormone inducing pain and inflammation, is known to inhibit potassium M-currents (IM) and to increase the excitability of the superior cervical ganglion (SCG) neurons by activating the Ca2+-calmodulin pathway. M-current is also reduced by muscarinic agonists through the depletion of membrane phosphatidylinositol 4,5-biphosphate (PIP2). Similarly, the activation of muscarinic receptors inhibits the current through two-pore domain potassium channels (K2P) of the “Tandem of pore-domains in a Weakly Inward rectifying K+ channel (TWIK)-related channels” (TREK) subfamily by reducing PIP2 in mouse SCG neurons (mSCG). The aim of this work was to test and characterize the modulation of TREK channels by bradykinin. We used the perforated-patch technique to investigate riluzole (RIL) activated currents in voltage- and current-clamp experiments. RIL is a drug used in the palliative treatment of amyotrophic lateral sclerosis and, in addition to blocking voltage-dependent sodium channels, it also selectively activates the K2P channels of the TREK subfamily. A cell-attached patch-clamp was also used to investigate TREK-2 single channel currents. We report here that BK reduces spike frequency adaptation (SFA), inhibits the riluzole-activated current (IRIL), which flows mainly through TREK-2 channels, by about 45%, and reduces the open probability of identified single TREK-2 channels in cultured mSCG cells. The effect of BK on IRIL was precluded by the bradykinin receptor (B2R) antagonist HOE-140 (d-Arg-[Hyp3, Thi5, d-Tic7, Oic8]BK) but also by diC8PIP2 which prevents PIP2 depletion when phospholipase C (PLC) is activated. On the contrary, antagonizing inositol triphosphate receptors (IP3R) using 2-aminoethoxydiphenylborane (2-APB) or inhibiting protein kinase C (PKC) with bisindolylmaleimide did not affect the inhibition of IRIL by BK. In conclusion, bradykinin inhibits TREK-2 channels through the activation of B2Rs resulting in PIP2 depletion, much like we have demonstrated for muscarinic agonists. This mechanism implies that TREK channels must be relevant for the capture of information about pain and visceral inflammation.

scholarly journals A Quantitative Description of KcsA Gating I: Macroscopic Currents

2007 ◽  
Vol 130 (5) ◽  
pp. 465-478 ◽  
Author(s):  
Sudha Chakrapani ◽  
Julio F Cordero-Morales ◽  
Eduardo Perozo
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Ph Dependence ◽  
Quantitative Description ◽  
Hill Coefficient ◽  
K Channel ◽  
Open Probability ◽  
Transmembrane Voltage ◽  
Voltage Dependent ◽  
Inactivation Process

The prokaryotic K+ channel KcsA is activated by intracellular protons and its gating is modulated by transmembrane voltage. Typically, KcsA functions have been studied under steady-state conditions, using macroscopic Rb+-flux experiments and single-channel current measurements. These studies have provided limited insights into the gating kinetics of KcsA due to its low open probability, uncertainties in the number of channels in the patch, and a very strong intrinsic kinetic variability. In this work, we have carried out a detailed analysis of KcsA gating under nonstationary conditions by examining the influence of pH and voltage on the activation, deactivation, and slow-inactivation gating events. We find that activation and deactivation gating of KcsA are predominantly modulated by pH without a significant effect of voltage. Activation gating showed sigmoidal pH dependence with a pKa of ∼4.2 and a Hill coefficient of ∼2. In the sustained presence of proton, KcsA undergoes a time-dependent decay of conductance. This inactivation process is pH independent but is modulated by voltage and the nature of permeant ion. Recovery from inactivation occurs via deactivation and also appears to be voltage dependent. We further find that inactivation in KcsA is not entirely a property of the open-conducting channel but can also occur from partially “activated” closed states. The time course of onset and recovery of the inactivation process from these pre-open closed states appears to be different from the open-state inactivation, suggesting the presence of multiple inactivated states with diverse kinetic pathways. This information has been analyzed together with a detailed study of KcsA single-channel behavior (in the accompanying paper) in the framework of a kinetic model. Taken together our data constitutes the first quantitative description of KcsA gating.

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