Voltage clamp analysis of the kinetics of piperidine-induced chloride current in isolated Aplysia neurons

1990 ◽  
Vol 342 (5) ◽  
Author(s):  
K. Takahama ◽  
M.R. Klee
Keyword(s):  
Voltage Clamp ◽  
Chloride Current ◽  
Aplysia Neurons ◽  
Voltage Clamp Analysis ◽  
Kinetics Of

Voltage-clamp analysis of nodes of Ranvier in regenerated rat sciatic nerve

1987 ◽  
Vol 409 (2) ◽  
pp. 227-235 ◽  
Author(s):  
T. Brismar ◽  
C. Hildebrand ◽  
S. Berglund
Keyword(s):  
Sciatic Nerve ◽  
Voltage Clamp ◽  
Nodes Of Ranvier ◽  
Rat Sciatic Nerve ◽  
Voltage Clamp Analysis

Voltage-clamp analysis of a Ca2+- and voltage-dependent chloride conductance in cultured mouse spinal neurons

1986 ◽  
Vol 55 (6) ◽  
pp. 1115-1135 ◽  
Author(s):  
D. G. Owen ◽  
M. Segal ◽  
J. L. Barker
Keyword(s):  
Voltage Clamp ◽  
Reversal Potential ◽  
Gamma Aminobutyric Acid ◽  
Spinal Neurons ◽  
Exponential Process ◽  
Voltage Clamp Analysis ◽  
Voltage Dependent ◽  
Inward Currents ◽  
Ca 50 ◽  
O 10

Current and voltage-clamp recordings were made at room temperature from cultured mouse spinal neurons using conventional two-electrode voltage-clamp techniques and electrodes filled with either 3 M KCl, 3 M CsCl, or 3 M Cs2SO4. In the presence of tetraethylammonium and tetrodotoxin, “fast” (rapidly rising and falling) action potentials (FAP) of variable duration were recorded in most neurons. “Slow” (slowly rising and falling) depolarizing potentials (SDP) occurred in 23% of the cells, when using KCl-filled electrodes, and in 82% of the cells with CsCl-filled electrodes. The SDP was frequently preceded by an FAP, although in some cells activation of the SDP occurred before the FAP threshold was reached and in a graded fashion. Both the FAP and SDP were abolished by Cd2+ and other Ca2+ antagonists. In cells exhibiting SDPs, voltage-clamp analysis revealed a sustained (noninactivating) inward current (Isin) during depolarizing steps to potentials more positive than -45 mV. Repolarizing steps resulted in slowly decaying inward tail currents (Itail). Both Isin and Itail were abolished in solutions nominally free of Cao2+, or containing Ca2+-channel antagonists. Bao2+ did not support Isin. The data indicated a U-shaped activation curve for Isin, peaking at about -10 mV. Activation of Isin occurred exponentially with a time constant of approximately 140 ms at -23 mV, becoming faster at more depolarized potentials (ca. 50 ms at -2 mV). Deactivation was slow, giving rise to tail currents lasting seconds. In some cases deactivation could be described by a single exponential process, although frequently the kinetics were more complex. Deactivation was faster at hyperpolarized potentials and sensitive to extracellular ([Ca2+]o), duration of activating voltage steps, and the degree of activation of Isin. Using CsCl-filled electrodes, the reversal potential (Erev) for Isin was -1.7 mV (SEM 3.5 mV, n = 20). Erev always corresponded to the reversal potential for gamma-aminobutyric acid-evoked currents in the same cell. In experiments in which Cs2SO4-filled electrodes were used, Erev was estimated to be -44 mV (SEM 2.3 mV, n = 9). Neither complete substitution of Nao+ with choline ions nor elevation of [K+]o 10-fold significantly affected the estimated Erev. However, substitution of Cl0- with isethionate or methanesulphonate increased the amplitude of inward currents (recorded with CsCl-filled electrodes) and shifted Erev to more depolarized potentials. The results indicate that Cl- are the primary charge carriers for this current and that Cai2+ is required for its activation, leading us to identify it as ICl(Ca).(ABSTRACT TRUNCATED AT 400 WORDS)

Serotonin and forskolin increase an inwardly rectifying potassium conductance in cultured identified Aplysia neurons

1987 ◽  
Vol 58 (5) ◽  
pp. 909-921 ◽  
Author(s):  
D. P. Lotshaw ◽  
I. B. Levitan
Keyword(s):  
Voltage Clamp ◽  
Potassium Conductance ◽  
Phosphodiesterase Inhibitor ◽  
Identified Neurons ◽  
Inward Rectification ◽  
Current Response ◽  
Aplysia Neurons ◽  
Voltage Dependent ◽  
K Concentration ◽  
Inwardly Rectifying

1. The effect of serotonin (5-HT) and forskolin on an inwardly rectifying K+ conductance (IKR) was studied using voltage-clamp techniques in several identified Aplysia neurons isolated and maintained in primary cell culture. 2. Inward rectification was observed in the current-voltage relationship of the identified neurons R15, R2, B1, and B2 and was predominately due to IKR, as demonstrated by the dependence of inward rectification on the extracellular K+ concentration, instantaneous kinetics of the membrane current response to hyperpolarizing voltage clamp pulses, and voltage-dependent Ba2+ block of the inwardly rectifying current. 3. 5-HT increased IKR conductance between 100 and 400% in the identified neuron R15 in culture and increased IKR conductance approximately 50% in the identified neurons B1, B2, and R2 in culture. The adenylate cyclase activator, forskolin, plus a phosphodiesterase inhibitor, Ro 20-1724, also increased IKR conductance in these neurons. 4. 5-HT and forskolin modulated other ion conductances as well in all of these cultured neurons.

Opposite effects of Ni2+ on Xenopus and rat ENaCs expressed in Xenopus oocytes

2005 ◽  
Vol 289 (4) ◽  
pp. C946-C958 ◽  
Author(s):  
Dana Cucu ◽  
Jeannine Simaels ◽  
Jan Eggermont ◽  
Willy Van Driessche ◽  
Wolfgang Zeiske
Keyword(s):  
Voltage Clamp ◽  
Serine Proteases ◽  
Metal Ion ◽  
Xenopus Oocytes ◽  
Noise Analysis ◽  
Inhibitory Effect ◽  
Rat Colon ◽  
Current Response ◽  
Α Subunit ◽  
Kinetics Of

Opposite effects of Ni2+ on Xenopus and rat ENaCs expressed in Xenopus oocytes. Am J Physiol Cell Physiol 289: C946–C958, 2005. First published June 8, 2005; .—The epithelial Na+ channel (ENaC) is modulated by various extracellular factors, including Na+, organic or inorganic cations, and serine proteases. To identify the effect of the divalent Ni2+ cation on ENaCs, we compared the Na+ permeability and amiloride kinetics of Xenopus ENaCs (xENaCs) and rat ENaCs (rENaCs) heterologously expressed in Xenopus oocytes. We found that the channel cloned from the kidney of the clawed toad Xenopus laevis [wild-type (WT) xENaC] was stimulated by external Ni2+, whereas the divalent cation inhibited the channel cloned from the rat colon (WT rENaC). The kinetics of amiloride binding were determined using noise analysis of blocker-induced fluctuation in current adapted for the transoocyte voltage-clamp method, and Na+ conductance was assessed using the dual electrode voltage-clamp (TEVC) technique. The inhibitory effect of Ni2+ on amiloride binding is not species dependent, because Ni2+ decreased the affinity (mainly reducing the association rate constant) of the blocker in both species in competition with Na+. Importantly, using the TEVC method, we found a prominent difference in channel conductance at hyperpolarizing voltage pulses. In WT xENaCs, the initial ohmic current response was stimulated by Ni2+, whereas the secondary voltage-activated current component remained unaffected. In WT rENaCs, only a voltage-dependent block by Ni2+ was obtained. To further study the origin of the xENaC stimulation by Ni2+, and based on the rationale of the well-known high affinity of Ni2+ for histidine residues, we designed α-subunit mutants of xENaCs by substituting histidines that were expressed in oocytes, together with WT β- and γ-subunits. Changing His215 to Asp in one putative amiloride-binding domain (WYRFHY) in the extracellular loop between Na+ channel membrane segments M1 and M2 had no influence on the stimulatory effect of Ni2+, and neither did complete deletion of this segment. Next, we mutated His416 flanked by His411 and Cys417, a unique site for possible heavy metal ion chelation, and, with this quality, most proximal (∼100 amino acids upstream of the second putative amiloride binding site at the pore entrance), was found localized at M2. Replacing His416 with arginine, aspartate, tyrosine, and alanine clearly affected amiloride binding in all cases, as well as Na+ conductance, as expressed in the xENaC current-voltage relationship, especially with regard to aspartate and tyrosine. However, similarly to those obtained with the WYRFHY stretch, none of these mutations could either abolish the stimulating effect of Ni2+ or reverse it to an inhibitory type.

Genes and channels: patch/voltage-clamp analysis and single-cell RT-PCR

2000 ◽  
Vol 302 (3) ◽  
pp. 295-307 ◽  
Author(s):  
Nikolaus J. Sucher ◽  
David L. Deitcher ◽  
Deborah J. Baro ◽  
Ronald M. Harris Warrick ◽  
Elke Guenther
Keyword(s):  
Single Cell ◽  
Voltage Clamp ◽  
Rt Pcr ◽  
Voltage Clamp Analysis
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