scholarly journals Gating of IsK expressed in Xenopus oocytes depends on the amount of mRNA injected.

1994 ◽  
Vol 104 (1) ◽  
pp. 87-105 ◽  
Author(s):  
J Cui ◽  
R P Kline ◽  
P Pennefather ◽  
I S Cohen

IsK is a K+ channel of the delayed rectifier type widely distributed throughout both excitable and nonexcitable cells. Its structure is different from other cloned K+ channels and molecular details of its gating remain obscure. Here we show that the activation kinetics of IsK expressed in Xenopus oocytes depend upon the amount of its mRNA injected, with larger amounts resulting in slower activation kinetics with a longer initial delay during activation. Similar changes in activation kinetics occur with time after a single injection of IsK mRNA. We present two kinetic schemes which illustrate how our experimental results could arise. Both imply an interaction among individual channel proteins during IsK activation. The dependence of channel gating on mRNA concentration provides a novel mechanism for long term regulation of ion current kinetics.

1996 ◽  
Vol 108 (3) ◽  
pp. 207-219 ◽  
Author(s):  
J J Rosenthal ◽  
R G Vickery ◽  
W F Gilly

We have cloned the cDNA for a squid Kvl potassium channel (SqKv1A). SqKv1A mRNA is selectively expressed in giant fiber lobe (GFL) neurons, the somata of the giant axons. Western blots detect two forms of SqKv1A in both GFL neuron and giant axon samples. Functional properties of SqKv1A currents expressed in Xenopus oocytes are very similar to macroscopic currents in GFL neurons and giant axons. Macroscopic K currents in GFL neuron cell bodies, giant axons, and in Xenopus oocytes expressing SqKv1A, activate rapidly and inactivate incompletely over a time course of several hundred ms. Oocytes injected with SqKv1A cRNA express channels of two conductance classes, estimated to be 13 and 20 pS in an internal solution containing 470 mM K. SqKv1A is thus a good candidate for the "20 pS" K channel that accounts for the majority of rapidly activating K conductance in both GFL neuron cell bodies and the giant axon.


1998 ◽  
Vol 79 (5) ◽  
pp. 2345-2357 ◽  
Author(s):  
Kathryn G. Klemic ◽  
Dominique M. Durand ◽  
Stephen W. Jones

Klemic, Kathryn G., Dominique M. Durand, and Stephen W. Jones. Activation kinetics of the delayed rectifier potassium current of bullfrog sympathetic neurons. J. Neurophysiol. 79: 2345–2357, 1998. We examined the activation kinetics of the delayed rectifier K+ current of bullfrog sympathetic neurons, primarily using whole cell recording. On depolarization, currents activated with a sigmoid delay but did not show a Cole-Moore shift. The time course of activation differed systematically from an exponential raised to a power. At most voltages, a power of 2 gave the best overall fit but a power of 3 better described the initial delay. After the delay, the time course could be fitted by a single exponential. Time constants were 15–20 ms at 0 mV and decreased to a limiting τ = 7 ms at +50 to +100 mV. Tail currents were well fitted by single exponential functions and accelerated with hyperpolarization, from τ = 15–20 ms at 0 mV to τ = 2 ms at −110 mV ( e-fold for 40 mV). Eleven kinetic models were evaluated for their ability to describe the activation kinetics of the delayed rectifier. Hodgkin-Huxley–like models did not fit the data well. A linear model where voltage sensor movement is followed by a distinct channel opening step, allosteric models based on the Monod-Wyman–Changeux model, and an unconstrained C-C-C-O model could describe whole cell data from −100 to +40 mV. After including whole cell data at +60 and +80 mV, and a maximal p open of 0.8 from noise analysis of cell-attached patches, an allosteric model fit the data best, as the other models had difficulty describing qualitative features of the data. However, some more complex schemes (with additional free parameters) cannot be excluded. We propose the allosteric model as an empirical description of macroscopic ionic currents, and as a model worth considering in future studies on the molecular mechanism of potassium channel gating.


1997 ◽  
Vol 272 (3) ◽  
pp. H1309-H1314 ◽  
Author(s):  
A. Zou ◽  
M. E. Curran ◽  
M. T. Keating ◽  
M. C. Sanguinetti

HERG is a K+ channel with properties similar to the rapidly activating component (I(Kr)) of delayed rectifier K+ current, which is important for repolarization of human cardiac myocytes. In this study, we have characterized the single-channel properties of HERG expressed in Xenopus oocytes. Currents were measured in cell-attached patches with an extracellular K concentration of 120 mM. The single HERG channel conductance, determined at test potentials between -50 and -110 mV, was 12.1 +/- 0.6 pS. At positive test potentials (+40 to +80 mV), the probability of channel opening was low and slope conductance was 5.1 +/- 0.6 pS. The mean channel open times at -90 mV were 2.9 +/- 0.5 and 11.8 +/- 1.0 ms, and the mean channel closed times were 0.54 +/- 0.02 and 14.5 +/- 5.3 ms. Single HERG channels were blocked by MK-499, a class III antiarrhythmic agent that blocks I(Kr) in cardiac myocytes. The development of block was more rapid in inside-out patches than in cell-attached patches or in whole cell recordings, indicating that block occurs from the cytoplasmic side of the membrane. The single-channel properties of HERG are similar to I(Kr) channels of isolated cardiac myocytes, which provides further evidence that HERG proteins coassemble to form I(Kr) channels.


FEBS Letters ◽  
1996 ◽  
Vol 381 (1-2) ◽  
pp. 71-76 ◽  
Author(s):  
Tuvia Peretz ◽  
Gal Levin ◽  
Ofira Moran ◽  
William B. Thornhill ◽  
Dodo Chikvashvili ◽  
...  

1989 ◽  
Vol 257 (6) ◽  
pp. C1119-C1127 ◽  
Author(s):  
K. D. Gillis ◽  
W. M. Gee ◽  
A. Hammoud ◽  
M. L. McDaniel ◽  
L. C. Falke ◽  
...  

Intracellular ATP (ATPi)-sensitive K+ [K+(ATP)] channels are now a recognized site of action of clinically useful hypoglycemic and hyperglycemic sulfonamides. We have further examined the action of these agents on single K+ channels in rat pancreatic B-cells 1) Tolbutamide and glyburide, two hypoglycemic sulfonylureas which decrease K+(ATP) channel activity in the cell-attached patch, affect the kinetics of K+(ATP) channel in a manner similar to glucose. They shorten the duration of the “burst,” or cluster of open channel events, while lengthening the intervals between bursts. 2) The hyperglycemic vasodilator diazoxide increases mean K+(ATP) channel activity in the cell-attached patch as well as in the inside-out excised patch exposed to ATPi. It appears to lengthen channel bursts and shorten the intervals between them. Two structurally similar diuretics, hydrochlorothiazide and furosemide, which have mild hyperglycemic effects, do not increase K+(ATP) channel activity even at clinically toxic concentrations. 3) Neither the sulfonylureas nor diazoxide directly affect the activity of single delayed rectifier K+ channels or single calcium and voltage-activated K+ channels in normal B-cells.


1997 ◽  
Vol 273 (3) ◽  
pp. F404-F410 ◽  
Author(s):  
L. G. Palmer ◽  
H. Choe ◽  
G. Frindt

The biophysical properties of low-conductance secretory K (SK) channels in the apical membrane of the rat cortical collecting tubule were examined to compare these properties with those of the cloned renal K channels of the ROMK family expressed in oocytes. At room temperature, with the tubule superfused with 140 mM K and 110 mM cation in the pipette, the inward single-channel conductance of the SK channels was 36 +/- 1 pS for K, 41 +/- 2 pS for NH4, and 22 +/- 3 pS for Tl. The reversal potential was nearly the same for K and Tl in the pipette but was shifted by -60 mV for NH4. The kinetics of the channel when K was the permeant ion could be described by a single open state (mean open time, 24 ms) and two closed states (mean closed times, 1.6 and 65 ms). The kinetics of SK changed when Tl was the permeant ion (mean open times of 6.6 ms and no long closed state) and when NH4 was the permeant ion (mean open time of 3.0 ms and a more prevalent long closed state). Thus the gating kinetics of the channel depend strongly on the nature of the conducted ion. The properties of SK channels were quite similar to those of ROMK2 expressed in Xenopus oocytes and measured under similar conditions, suggesting that these channels are identical.


1977 ◽  
Vol 16 (01) ◽  
pp. 30-35 ◽  
Author(s):  
N. Agha ◽  
R. B. R. Persson

SummaryGelchromatography column scanning has been used to study the fractions of 99mTc-pertechnetate, 99mTcchelate and reduced hydrolyzed 99mTc in preparations of 99mTc-EDTA(Sn) and 99mTc-DTPA(Sn). The labelling yield of 99mTc-EDTA(Sn) chelate was as high as 90—95% when 100 μmol EDTA · H4 and 0.5 (Amol SnCl2 was incubated with 10 ml 99mTceluate for 30—60 min at room temperature. The study of the influence of the pH-value on the fraction of 99mTc-EDTA shows that pH 2.8—2.9 gave the best labelling yield. In a comparative study of the labelling kinetics of 99mTc-EDTA(Sn) and 99mTc- DTPA(Sn) at different temperatures (7, 22 and 37°C), no significant influence on the reduction step was found. The rate constant for complex formation, however, increased more rapidly with increased temperature for 99mTc-DTPA(Sn). At room temperature only a few minutes was required to achieve a high labelling yield with 99mTc-DTPA(Sn) whereas about 60 min was required for 99mTc-EDTA(Sn). Comparative biokinetic studies in rabbits showed that the maximum activity in kidneys is achieved after 12 min with 99mTc-EDTA(Sn) but already after 6 min with 99mTc-DTPA(Sn). The long-term disappearance of 99mTc-DTPA(Sn) from the kidneys is about five times faster than that for 99mTc-EDTA(Sn).


2016 ◽  
Vol 22 (2) ◽  
pp. 258-263 ◽  
Author(s):  
Gábor Steinbach ◽  
Radek Kaňa

AbstractPhotosynthesis research employs several biophysical methods, including the detection of fluorescence. Even though fluorescence is a key method to detect photosynthetic efficiency, it has not been applied/adapted to single-cell confocal microscopy measurements to examine photosynthetic microorganisms. Experiments with photosynthetic cells may require automation to perform a large number of measurements with different parameters, especially concerning light conditions. However, commercial microscopes support custom protocols (throughTime Controlleroffered by Olympus orExperiment Designeroffered by Zeiss) that are often unable to provide special set-ups and connection to external devices (e.g., for irradiation). Our new system combining an Arduino microcontroller with theCell⊕Findersoftware was developed for controlling Olympus FV1000 and FV1200 confocal microscopes and the attached hardware modules. Our software/hardware solution offers (1) a text file-based macro language to control the imaging functions of the microscope; (2) programmable control of several external hardware devices (light sources, thermal controllers, actuators) during imaging via the Arduino microcontroller; (3) theCell⊕Findersoftware with ergonomic user environment, a fast selection method for the biologically important cells and precise positioning feature that reduces unwanted bleaching of the cells by the scanning laser.Cell⊕Findercan be downloaded fromhttp://www.alga.cz/cellfinder. The system was applied to study changes in fluorescence intensity inSynechocystissp. PCC6803 cells under long-term illumination. Thus, we were able to describe the kinetics of phycobilisome decoupling. Microscopy data showed that phycobilisome decoupling appears slowly after long-term (>1 h) exposure to high light.


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