scholarly journals Human Ether-à-go-go–related Gene K+ Channel Gating Probed with Extracellular Ca2+

1999 ◽  
Vol 113 (4) ◽  
pp. 565-580 ◽  
Author(s):  
J.P. Johnson ◽  
Franklin M. Mullins ◽  
Paul B. Bennett
Keyword(s):  
Voltage Dependence ◽  
Membrane Surface ◽  
Chinese Hamster ◽  
Cell Voltage ◽  
Current Amplitude ◽  
K Channel ◽  
Related Gene ◽  
Surface Charges ◽  
Whole Cell ◽  
Inactivation Gating

Human ether-à-go-go–related gene (HERG) encoded K+ channels were expressed in Chinese hamster ovary (CHO-K1) cells and studied by whole-cell voltage clamp in the presence of varied extracellular Ca2+ concentrations and physiological external K+. Elevation of external Ca2+ from 1.8 to 10 mM resulted in a reduction of whole-cell K+ current amplitude, slowed activation kinetics, and an increased rate of deactivation. The midpoint of the voltage dependence of activation was also shifted +22.3 ± 2.5 mV to more depolarized potentials. In contrast, the kinetics and voltage dependence of channel inactivation were hardly affected by increased extracellular Ca2+. Neither Ca2+ screening of diffuse membrane surface charges nor open channel block could explain these changes. However, selective changes in the voltage-dependent activation, but not inactivation gating, account for the effects of Ca2+ on Human ether-à-go-go–related gene current amplitude and kinetics. The differential effects of extracellular Ca2+ on the activation and inactivation gating indicate that these processes have distinct voltage-sensing mechanisms. Thus, Ca2+ appears to directly interact with externally accessible channel residues to alter the membrane potential detected by the activation voltage sensor, yet Ca2+ binding to this site is ineffective in modifying the inactivation gating machinery.

Developmental change in fast Na channel properties in embryonic chick ventricular heart cells

1995 ◽  
Vol 73 (10) ◽  
pp. 1475-1484 ◽  
Author(s):  
Hideaki Sada ◽  
Takashi Ban ◽  
Takeshi Fujita ◽  
Yoshio Ebina ◽  
Nicholas Sperelakis
Keyword(s):  
Steady State ◽  
Voltage Clamp ◽  
Time Constant ◽  
Voltage Dependence ◽  
Cell Voltage ◽  
Developmental Changes ◽  
Heart Cells ◽  
Embryonic Chick ◽  
Whole Cell ◽  
Whole Cell Voltage Clamp

To assess developmental changes in kinetic properties of the cardiac sodium current, whole-cell voltage-clamp experiments were conducted using 3-, 10-, and 17-day-old embryonic chick ventricular heart cells. Experimental data were quantified according to the Hodgkin–Huxley model. While the Na current density, as examined by the maximal conductance, drastically increased (six- to seven-fold) with development, other current–voltage parameters remained unchanged. Whereas the activation time constant and the steady-state activation characteristics were comparable among the three age groups, the voltage dependence of the inactivation time constant and the steady-state inactivation underwent a shift in the voltage dependence toward negative potentials during embryonic development. Consequently, the steady-state (window current) conductance, which was sufficient to induce automatic activity in the young embryos, was progressively reduced with age.Key words: cardiac electrophysiology, whole-cell voltage-clamp experiments, fast Na currents, heart, development, developmental changes.

GTP modulates run-up of whole-cell Ca2+ channel current in a Ca(2+)-dependent manner

1994 ◽  
Vol 71 (2) ◽  
pp. 814-816 ◽  
Author(s):  
J. J. Wagner ◽  
B. E. Alger
Keyword(s):  
Cell Voltage ◽  
Current Amplitude ◽  
Initial Increase ◽  
Absolute Maximum ◽  
Dependent Manner ◽  
Whole Cell ◽  
Ca1 Neurons ◽  
Channel Current ◽  
Run Up ◽  
Membrane Patch

1. Whole-cell voltage-clamp recordings were obtained from CA1 neurons acutely dissociated from rat hippocampus to study the effects of guanosine 5'-triphosphate (GTP) on the gradual increase in Ca2+ channel current amplitude that takes place over several minutes after breaking in to whole-cell mode ("run-up"). 2. Including GTP (500 microM) in the patch pipette significantly prolonged the duration of run-up of peak Ca2+ channel current to its maximum value compared with controls without GTP when the recording solutions contained Ca2+. On the other hand, GTP significantly enhanced run-up when Mg2+ and Ba2+ were substituted for intracellular and extracellular Ca2+, respectively. 3. The enhancement of run-up of the current in the Mg/Ba condition appeared to be due both to an initial increase in current amplitude that was complete within 30 s after break in and to a more rapid initial rate of run-up when compared with the Ca2+ condition. GTP did not affect the absolute maximum amplitudes of the currents in either Ca2+ or Ba2+ conditions. 4. We conclude that an early GTP-dependent modulation of Ca2+ channel current is qualitatively altered, depending on whether Ca2+ or Ba2+ is used as the charge carrier. Evidence of this modulation is apparent within seconds after rupture of the membrane patch. Conceivably, influences occurring during the period of "equilibration" with electrode contents could alter subsequent regulatory steps.

Na+-induced inward rectification in the two-pore domain K+ channel, TASK-2

2005 ◽  
Vol 288 (1) ◽  
pp. F162-F169 ◽  
Author(s):  
Michael J. Morton ◽  
Sarah Chipperfield ◽  
Abdulrahman Abohamed ◽  
Asipu Sivaprasadarao ◽  
Malcolm Hunter
Keyword(s):  
Single Channel ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Selectivity Filter ◽  
Inward Rectification ◽  
K Channel ◽  
Open Probability ◽  
Whole Cell ◽  
Single Channel Currents ◽  
Pore Domain

TASK-2 is a member of the two-pore domain K+ (K2P) channel family that is expressed at high levels in several epithelia, including the proximal tubule. In common with the other TASK channels, TASK-2 is sensitive to changes in extracellular pH. We have expressed human TASK-2 in Chinese hamster ovary cells and studied whole cell and single-channel activity by patch clamp. The open probability of K2P channels is generally independent of voltage, yielding linear current-voltage ( I- V) curves. Despite these properties, we found that these channels showed distinct inward rectification immediately on the establishment of whole cell clamp, which became progressively less pronounced with time. This rectification was due to intracellular Na+ but was unaffected by polyamines or Mg2+ (agents that cause rectification in Kir channels). Rectification was concentration- and voltage-dependent and could be reversibly induced by switching between Na+-rich and Na+-free bath solutions. In excised inside-out patches, Na+ reduced the amplitude of single-channel currents, indicative of rapid block and unblock of the pore. Mutations in the selectivity filter abolished Na+-induced rectification, suggesting that Na+ binds within the selectivity filter in wild-type channels. This sensitivity to intracellular Na+ may be an additional potential regulatory mechanism of TASK-2 channels.

scholarly journals Interaction of internal Ba2+ with a cloned Ca(2+)-dependent K+ (hslo) channel from smooth muscle.

1996 ◽  
Vol 107 (3) ◽  
pp. 399-407 ◽  
Author(s):  
F Diaz ◽  
M Wallner ◽  
E Stefani ◽  
L Toro ◽  
R Latorre
Keyword(s):  
Time Constant ◽  
Voltage Dependence ◽  
Membrane Surface ◽  
Potential Drop ◽  
Human Myometrium ◽  
K Channel ◽  
Xenopus Laevis Oocytes ◽  
Experimental Conditions ◽  
Internal Solution ◽  
Current Decay

We have studied potassium currents through a cloned Ca(2+)-dependent K+ channel (hslo) from human myometrium. Currents were recorded in inside-out macropatches from membranes of Xenopus laevis oocytes. In particular, the inactivation-like process that these channels show at high positive potentials was assessed in order to explore its molecular nature. This current inhibition conferred a bell shape to the current-voltage curves. The kinetic and voltage dependence of this process suggested the possibility of a Ba2+ block. There were the following similarities between the inactivation process observed at zero-added Ba2+ and the internal Ba2+ block of hslo channels: (a) in the steady state, the voltage dependence of the current inhibition observed at zero-added Ba2+ was the same as the voltage dependence of the Ba2+ block; (b) the time constant for recovery from current decay at zero-added Ba2+ was the same as the time constant for current recovery from Ba2+ blockade; and (c) current decay was largely suppressed in both cases by adding a Ba2+ chelator [(+)-18-crown-6-tetracarboxylic acid] to the internal solution. In our experimental conditions, we determined that the Kd for the complex chelator-Ba2+ is 1.6 x 10(-10) M. We conclude that the current decay observed at zero-added Ba2+ to the internal solution is due to contaminant Ba2+ present in our solutions (approximately 70 nM) and not to an intrinsic gating process. The Ba2+ blocking reaction in hslo channels is bimolecular. Ba2+ binds to a site (Kd = 0.36 +/- 0.05 mM at zero applied voltage) that senses 92 +/- 25% of the potential drop from the internal membrane surface.

Lanthanum blocks a specific component of IK and screens membrane surface change in cardiac cells

1990 ◽  
Vol 259 (6) ◽  
pp. H1881-H1889 ◽  
Author(s):  
M. C. Sanguinetti ◽  
N. K. Jurkiewicz
Keyword(s):  
Ventricular Myocytes ◽  
Membrane Surface ◽  
Surface Membrane ◽  
Cell Voltage ◽  
Cardiac Cells ◽  
Delayed Rectifier ◽  
Class Iii ◽  
Surface Charges ◽  
K Current ◽  
Unidentified Component

Delayed rectifier outward K+ current (IK) in guinea pig ventricular myocytes represents the sum of two currents: a slowly activating delayed rectifier K+ current (IK.s) and a relatively rapidly activating delayed rectifier K+ current (IK.r), which rectifies at positive potentials and is specifically blocked by the class III antiarrhythmic agent, E-4031. La3+ was previously reported to block an unidentified component of IK in these cells. We used the whole cell voltage-clamp technique on isolated myocytes and confirmed these results: we show that the current blocked by La3+ (greater than or equal to 1 microM) is IK.r. This block is not caused by La3+ displacement of surface-bound Ca2+. Thus, in the presence of either E-4031 or La3+, IK represents the activation of a single current, IK.s. La3+ (10 microM-1 mM) also caused a positive shift in the voltage dependence of the activation curve of IK.s. When we assumed that La3+ acts to bind and screen negative surface charges on the outer sarcolemmal membrane, the external surface potential of these cells (in 1.8 mM Ca2+) could be estimated to be -19 mV. A modification of the Gouy-Chapman equation was used to estimate the equilibrium constant for La3+ binding (10.7 mM-1) and the minimum spacing between the negative charges on the surface membrane (22 A).

scholarly journals Extracellular Sodium Interacts with the HERG Channel at an Outer Pore Site

2002 ◽  
Vol 120 (4) ◽  
pp. 517-537 ◽  
Author(s):  
Franklin M. Mullins ◽  
Svetlana Z. Stepanovic ◽  
Reshma R. Desai ◽  
Alfred L. George ◽  
Jeffrey R. Balser
Keyword(s):  
Chinese Hamster ◽  
Cell Voltage ◽  
Channel Structure ◽  
Herg Channel ◽  
K Channel ◽  
Physiological Concentration ◽  
Loop Region ◽  
Herg Current ◽  
Outer Pore ◽  
A Site

Most voltage-gated K+ currents are relatively insensitive to extracellular Na+ (Na+o), but Na+o potently inhibits outward human ether-a-go-go–related gene (HERG)–encoded K+ channel current (Numaguchi, H., J.P. Johnson, Jr., C.I. Petersen, and J.R. Balser. 2000. Nat. Neurosci. 3:429–30). We studied wild-type (WT) and mutant HERG currents and used two strategic probes, intracellular Na+ (Na+i) and extracellular Ba2+ (Ba2+o), to define a site where Na+o interacts with HERG. Currents were recorded from transfected Chinese hamster ovary (CHO-K1) cells using the whole-cell voltage clamp technique. Inhibition of WT HERG by Na+o was not strongly dependent on the voltage during activating pulses. Three point mutants in the P-loop region (S624A, S624T, S631A) with intact K+ selectivity and impaired inactivation each had reduced sensitivity to inhibition by Na+o. Quantitatively similar effects of Na+i to inhibit HERG current were seen in the WT and S624A channels. As S624A has impaired Na+o sensitivity, this result suggested that Na+o and Na+i act at different sites. Extracellular Ba2+ (Ba2+o) blocks K+ channel pores, and thereby serves as a useful probe of K+ channel structure. HERG channel inactivation promotes relief of Ba2+ block (Weerapura, M., S. Nattel, M. Courtemanche, D. Doern, N. Ethier, and T. Hebert. 2000. J. Physiol. 526:265–278). We used this feature of HERG inactivation to distinguish between simple allosteric and pore-occluding models of Na+o action. A remote allosteric model predicts that Na+o will speed relief of Ba2+o block by promoting inactivation. Instead, Na+o slowed Ba2+ egress and Ba2+ relieved Na+o inhibition, consistent with Na+o binding to an outer pore site. The apparent affinities of the outer pore for Na+o and K+o as measured by slowing of Ba2+ egress were compatible with competition between the two ions for the channel pore in their physiological concentration ranges. We also examined the role of the HERG closed state in Na+o inhibition. Na+o inhibition was inversely related to pulsing frequency in the WT channel, but not in the pore mutant S624A.

Voltage dependence of the Ca2+-activated K+channel KCa3.1 in human erythroleukemia cells

2013 ◽  
Vol 304 (9) ◽  
pp. C858-C872 ◽  
Author(s):  
Colin J. Stoneking ◽  
Oshini Shivakumar ◽  
David Nicholson Thomas ◽  
William H. Colledge ◽  
Michael J. Mason
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
K Channel ◽  
Whole Cell ◽  
Whole Cell Patch Clamp ◽  
Erythroleukemia Cells ◽  
Voltage Dependent ◽  
Hel Cells ◽  
Cell Current ◽  
Whole Cell Current

We have isolated a K+-selective, Ca2+-dependent whole cell current and single-channel correlate in the human erythroleukemia (HEL) cell line. The whole cell current was inhibited by the intermediate-conductance KCa3.1 inhibitors clotrimazole, TRAM-34, and charybdotoxin, unaffected by the small-conductance KCa2 family inhibitor apamin and the large-conductance KCa1.1 inhibitors paxilline and iberiotoxin, and augmented by NS309. The single-channel correlate of the whole cell current was blocked by TRAM-34 and clotrimazole, insensitive to paxilline, and augmented by NS309 and had a single-channel conductance in physiological K+gradients of ∼9 pS. RT-PCR revealed that the KCa3.1 gene, but not the KCa1.1 gene, was expressed in HEL cells. The KCa3.1 current, isolated in HEL cells under whole cell patch-clamp conditions, displayed an activated current component during depolarizing voltage steps from hyperpolarized holding potentials and tail currents upon repolarization, consistent with voltage-dependent modulation. This activated current increased with increasing voltage steps above −40 mV and was sensitive to inhibition by clotrimazole, TRAM-34, and charybdotoxin and insensitive to apamin, paxilline, and iberiotoxin. In single-channel experiments, depolarization resulted in an increase in open channel probability ( Po) of KCa3.1, with no increase in channel number. The voltage modulation of Powas an increasing monotonic function of voltage. In the absence of elevated Ca2+, voltage was ineffective at inducing channel activity in whole cell and single-channel experiments. These data indicate that KCa3.1 in HEL cells displays a unique form of voltage dependence modulating Po.

scholarly journals Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp

2020 ◽  
Author(s):  
Jérôme Montnach ◽  
Maxime Lorenzini ◽  
Adrien Lesage ◽  
Isabelle Simon ◽  
Sébastien Nicolas ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Voltage Clamp ◽  
Good Practice ◽  
Cell Voltage ◽  
Current Amplitude ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Clamp Technique ◽  
Whole Cell Voltage Clamp

ABSTRACTThe patch-clamp technique has contributed to major advances in the characterization of ion channels. The recent development of high throughput patch-clamp provides a new momentum to the field. However, whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that current amplitude profoundly impacts the precision of the analyzed characteristics of the ion current under study. For voltagegated channels, the higher the current amplitude is, the less precise the characteristics of voltagedependence are. Similarly, in ion channel pharmacology, the characteristics of dose-response curves are hindered by high current amplitudes. In addition, the recent development of high throughput patch-clamp technique is often associated with the generation of stable cell lines demonstrating high current amplitudes. It is therefore critical to set the limits for current amplitude recordings to avoid inaccuracy in the characterization of channel properties or drug actions, such limits being different from one channel to another. In the present study, we use kinetic models of a voltage-gated sodium channel and a voltage-gated potassium channel to edict simple guidelines for good practice of whole-cell voltage-clamp recordings.

scholarly journals Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jérôme Montnach ◽  
Maxime Lorenzini ◽  
Adrien Lesage ◽  
Isabelle Simon ◽  
Sébastien Nicolas ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Voltage Clamp ◽  
Good Practice ◽  
Cell Voltage ◽  
Current Amplitude ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Clamp Technique ◽  
Whole Cell Voltage Clamp

AbstractThe patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose–response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput patch-clamp technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-clamp recordings.

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