Single-channel analysis of a K channel at basolateral membrane of rabbit proximal convoluted tubule

1988 ◽  
Vol 254 (1) ◽  
pp. F105-F113 ◽  
Author(s):  
L. Parent ◽  
J. Cardinal ◽  
R. Sauve
Keyword(s):  
Single Channel ◽  
Basolateral Membrane ◽  
Proximal Convoluted Tubule ◽  
Time Interval ◽  
Distribution Analysis ◽  
Patch Clamp Technique ◽  
Bathing Medium ◽  
Patch Pipette ◽  
K Concentration ◽  
Interval Distribution

The basolateral membrane of the rabbit proximal convoluted tubule (PCT) is known to be largely permeable to K ions. The patch-clamp technique was used to investigate the molecular basis of this K permeability. At room temperature and with a high-K solution (127 mM) in both the bathing medium and the patch pipette, current jumps associated with an inward-rectifying channel could be detected in every active cell-attached experiment. When the K concentration in the pipette was changed from 200 to 5 mM KCl (NaCl replacement), the single-channel conductance for inward currents changed from 54 to 10 pS. The observed shift in the zero current potential measured as a function of the patch pipette K concentration could be fitted using the Goldman-Hodgkin-Katz equation with a permeability ratio PNa/PK = 0.06. The channel was found to be moderately voltage dependent (e-fold per 56 mV depolarization). For instance, the open-channel probability (Po) increased from 0.06 to 0.16 following a membrane depolarization from -50 to +50 mV. A time interval distribution analysis showed for the open state a dominant single time constant of 14 and 10 ms at 50 and -50 mV, respectively. Two time constants equal to 1 (flickering) and 26 ms at +50 mV and to 0.6 and 300 ms at -50 mV were obtained for the closed-state interval distribution. Based on this analysis, it was concluded that the decrease of Po at negative potentials was due more to the appearance of prolonged silent periods than from a change in the channel mean open time.

scholarly journals Functional characterization of a voltage-gated anion channel from teleost fish intestinal epithelium

1988 ◽  
Vol 136 (1) ◽  
pp. 383-403 ◽  
Author(s):  
C. A. Loretz ◽  
C. R. Fourtner
Keyword(s):  
Single Channel ◽  
Basolateral Membrane ◽  
Functional Characterization ◽  
Anion Channel ◽  
Intestinal Epithelial ◽  
Channel Conductance ◽  
Patch Clamp Technique ◽  
Voltage Gated

An anion channel was isolated, using patch-clamp technique, from the basolateral membrane of goby intestinal epithelial cells. Single-channel conductance varied over a range from 20 to 90 pS. The channel was voltage-gated over the physiological range of cell membrane potential with depolarization increasing the proportion of time in the open state. There was no Ca2+ sensitivity. The selectivity sequence was SO4(2-) greater than Cl- greater than Mes-. The channel may function in vivo as one of several avenues of basolateral membrane Cl- exit with the voltage-gating property serving to match basolateral Cl- exit to apical entry.

Ionic conductive properties of rabbit proximal straight tubule basolateral membrane

1990 ◽  
Vol 258 (4) ◽  
pp. F940-F950 ◽  
Author(s):  
P. A. Welling ◽  
R. G. O'Neil
Keyword(s):  
Basolateral Membrane ◽  
Electrical Potential ◽  
Disulfonic Acid ◽  
Electrical Potential Difference ◽  
Bicarbonate Buffer ◽  
Bathing Medium ◽  
Straight Tubule ◽  
Proximal Straight Tubule ◽  
K Concentration ◽  
Conductive Properties

The ionic conductive properties of the nonperfused rabbit proximal straight tubule (S2) basolateral membrane were assessed by microelectrode techniques. The response of the basolateral membrane electrical potential difference, Vbl, to rapid changes in the peritubular bath concentration of K, HCO3, Na, and Cl were monitored with microelectrodes. The control steady-state Vbl averaged -41 mV (cell negative). An increase in peritubular bathing medium K concentration from 5 to 40 mM resulted in an instantaneous and sustained depolarization of +14.6 mV (27% of delta EK). Addition of barium (2 mM) depolarized the Vbl by +15.8 mV and abolished the Vbl response to the high-K medium. In other studies, reduction of peritubular bicarbonate at constant pH from 25 to 2.5 mM instantaneously and transiently depolarized Vbl by +15.8 mV (26% of delta EHCO3). In these same tubules reduction of peritubular Na from 126 to 2.2 mM resulted in an instantaneous and paradoxical depolarization of Vbl of +21.5 mV. Both depolarization transients resulting from reduction of Na and HCO3 were simultaneously inhibited by the addition of 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 0.5 mM), consistent with the presence of a SITS-sensitive Na-HCO3-coupled conductive pathway. In the absence of the bicarbonate buffer, reduction of Na resulted in a small sustained hyperpolarization of -5.8 mV (5% of delta ENa). Reduction of peritubular Cl from 120 to 4 mM resulted in an instantaneous and sustained depolarization of Vbl of +5.3 mV (6% of ECl) and was not affected by the addition of bumetanide (0.1 mM). It is concluded that the basolateral membrane of the nonperfused proximal straight tubule is characterized by a major barium-sensitive K conductance and a SITS-sensitive Na-coupled HCO3 conductance that carries net negative charge. These pathways are paralleled by relatively minor, but important, Na-conductive and Cl-conductive pathways.

Basolateral membrane potassium channels in rabbit cortical thick ascending limb

1992 ◽  
Vol 263 (2) ◽  
pp. F262-F267 ◽  
Author(s):  
A. M. Hurst ◽  
M. Duplain ◽  
J. Y. Lapointe
Keyword(s):  
Patch Clamp ◽  
Single Channel ◽  
Basolateral Membrane ◽  
Fold Increase ◽  
Thick Ascending Limb ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Collagenase Treatment ◽  
Voltage Dependent ◽  
Selective Channel

The nature of K exit across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) was investigated using the patch clamp technique. The basolateral membrane was exposed by mild collagenase treatment (0.1 U/ml), and a K-selective inwardly rectifying channel was identified. In cell-attached patches (140 mM K pipette) the inward conductance was 35.0 +/- 1.3 pS (n = 9) compared with an outward conductance of 7.0 +/- 0.9 pS (n = 5), and the current reversed at a pipette potential of -63.5 +/- 3.1 mV (n = 9). The channel is strongly voltage dependent, showing an e-fold increase in open probability per 18-mV depolarization. Barium blocked the channel, reducing both mean open probability and single-channel current amplitude; however, the channel was not Ca sensitive. On excision the channel exhibited rundown, which could not be prevented by 0.1 mM ATP or ATP plus 20 U/ml catalytic subunit of protein kinase A. A few excised patch recordings were possible, which confirmed the presence of a highly K-selective channel with a K-to-Na permeability ratio of 100. In conclusion, 1) it is possible to obtain patch clamp recordings from the rabbit CTAL basolateral membrane using a very mild collagenase treatment, and 2) the exit of K across the basolateral membrane is mediated at least in part by the presence of voltage-sensitive K channels.

A large conductance K(+)-selective channel of guinea pig villus enterocytes is Ca2+ independent

1992 ◽  
Vol 262 (2) ◽  
pp. G369-G374 ◽  
Author(s):  
G. M. Mintenig ◽  
A. S. Monaghan ◽  
F. V. Sepulveda
Keyword(s):  
Guinea Pig ◽  
Single Channel ◽  
K Channel ◽  
Constant Field ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Current Voltage ◽  
Selective Channel ◽  
K Concentration ◽  
Current Voltage Relationship

The presence of K(+)-selective channels has been probed in enterocytes isolated from guinea pig small intestinal villi by the patch-clamp technique. A channel with a single-channel conductance of approximately 130 pS was observed in excised inside-out patches bathed in symmetrical K+. A change in the K+ concentration in the intracellular aspect of the membrane altered the current-voltage relationship as expected from the constant-field equation when it is assumed that K+ is the only permeant ion. A change in Cl- concentration was without effect. Neither the activity of the channel nor its conductance was altered by addition of ATP or Ba2+ to the intracellular side of the patches. Changes in the free Ca2+ concentration were also without effect. The channel's open probability showed no voltage dependence and appeared only occasionally active in cell-attached patches where it had a linear current-voltage relation. The K+ channel described, which cannot be readily classified in any of the known classes of K+ channels, might provide an exit pathway for K+ recycling in guinea pig villus enterocytes.

Potassium channels in Necturus proximal tubule

1987 ◽  
Vol 253 (3) ◽  
pp. F488-F494 ◽  
Author(s):  
K. Kawahara ◽  
M. Hunter ◽  
G. Giebisch
Keyword(s):  
Potassium Channels ◽  
Proximal Tubule ◽  
Basolateral Membrane ◽  
K Channel ◽  
Selectivity Ratio ◽  
Patch Clamp Technique ◽  
Open Time ◽  
K Concentration ◽  
Inside Out ◽  
Necturus Proximal Tubule

Potassium channels from the apical and basolateral membranes of Necturus proximal tubule were studied using the patch-clamp technique. The conductance of the basolateral channel was dependent on the pipette K+ concentration (apparent Km, 65.5 mM K+; maximum channel conductance, 49.8 pS). The permeability ratio (PK+/PNa+) was approximately 10:1. The fractional open time increased with hyperpolarization, whereas mean open times did not change. Ba2+ (0.1 mM pipette concentration) blocked the channel and reduced the mean open time. The apical K+ channel was activated with depolarization and had a slope conductance of 60 pS in the inside-out configuration (100 mM KCl in the pipette and 2.5 mM KCl in the bath). The K+-Na+ selectivity ratio was 32:1. These different channel types will allow independent control of the apical and basolateral membrane K+ conductances.

Slowly activating voltage-dependent K+ conductance is apical pathway for K+ secretion in vestibular dark cells

1994 ◽  
Vol 267 (3) ◽  
pp. C857-C864 ◽  
Author(s):  
D. C. Marcus ◽  
Z. Shen
Keyword(s):  
Patch Clamp ◽  
Single Channel ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Equilibrium Potential ◽  
Patch Clamp Technique ◽  
Dark Cell ◽  
K Secretion ◽  
K Concentration

Dark cell epithelium secretes K+ into the lumen of the vestibular labyrinth by a previously unidentified apical transport mechanism. Previous single-channel patch-clamp studies demonstrated nonselective cation channels and maxi-K+ channels in the apical membrane, but in too low a density to account for transepithelial K+ transport. In this report, we demonstrated with the cell-attached macro-patch-clamp technique an outward apical membrane current at 0-mV pipette voltage, which was stimulated by elevating bath K+ concentration from 3.6 to 25 mM and inhibited by 10 microM bumetanide, similar to their known effects on transepithelial short-circuit current and K+ secretion. Furthermore, the patch current was activated over several seconds by a sustained depolarization and deactivated over several hundred milliseconds by a hyperpolarization. Current-voltage relationships from tail currents were obtained with either NaCl or KCl in the pipette. Depolarization from -40 to +40 mV led to an increased conductance by a factor of 7.3 +/- 1.7 (n = 7) and 19.2 +/- 7.6 (n = 6) for NaCl and KCl, respectively, and to a reversal voltage near the presumed equilibrium potential for K+. The results demonstrate that dark cell K+ secretion occurs via K(+)-selective channels with characteristics similar to those associated with the IsK protein.

Low-conductance K channels in apical membrane of rat cortical collecting tubule

1989 ◽  
Vol 256 (1) ◽  
pp. F143-F151 ◽  
Author(s):  
G. Frindt ◽  
L. G. Palmer
Keyword(s):  
Patch Clamp ◽  
Single Channel ◽  
Apical Membrane ◽  
Ringer Solution ◽  
Patch Clamp Technique ◽  
Outward Currents ◽  
Collecting Tubule ◽  
Inward Currents ◽  
K Concentration ◽  
Cortical Collecting Tubule

Low-conductance, K-selective channels were identified in the apical membrane of the rat cortical collecting tubule (CCT) by use of the patch-clamp technique. Isolated, split tubules were bathed in K gluconate medium to depolarize the cell while keeping the intracellular K concentration high. With the patch-clamp pipette containing predominantly either Na+ or Li+ but no K, outward currents were observed through channels that had a single-channel conductance (g) of 9 pS and a probability of being open (Po) of greater than 0.9, independent of the voltage (+/- 40 mV) applied to the pipette (Vp). Similarly, only outward currents were observed when the patch was excised into high-K solution, implying a high selectivity of the channel for K+. When 1 mM BaCl2 was added to the pipette, Po decreased to 0.36 at Vp = 0; however, g was not changed but the channels flickered rapidly between open and blocked states; Po decreased as Vp was made positive, and increased as Vp was made negative. With the pipette filled with KCl + 1 mM Ba, the channels conducted K+ in both directions. The inward currents (at positive Vp were larger than the outward currents (at negative Vp) and g near Vp = 0 increased to 25 pS. When the pipette was filled with RbCl + 1 mM Ba the inward and outward currents were similar in magnitude, suggesting that the channels can conduct Rb, although not as well as K. With the tubules bathed in NaCl Ringer solution and the pipette containing KCl, inward currents were observed that could be attributed to the same pathway for K.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression by extracellular K+ of N-methyl-D-aspartate responses in cultured rat hippocampal neurons

1990 ◽  
Vol 64 (5) ◽  
pp. 1361-1367 ◽  
Author(s):  
S. Ozawa ◽  
M. Iino ◽  
K. Tsuzuki
Keyword(s):  
Hippocampal Neurons ◽  
Single Channel ◽  
Reversal Potential ◽  
External Solution ◽  
Dependent Manner ◽  
Induced Current ◽  
Patch Clamp Technique ◽  
Experimental Conditions ◽  
K Concentration ◽  
External K

1. The effects of increasing K+ concentration in Mg2(+)-free extracellular solution on N-methyl-D-aspartate (NMDA)-induced current were studied in cultured rat hippocampal neurons with the use of the whole-cell and outside-out configurations of the patch-clamp technique. 2. When the K+ concentration in the external solution was increased by replacement of Na+ with isomolar K+, the amplitude of the NMDA-induced current decreased in a concentration-dependent manner. The effect of K+ was almost saturated at 100 mM, when the NMDA response was reduced to 12% of that in K(+)-free, 150 mM Na+ solution. Increasing the external K+ concentration did not affect either the kainate- or quisqualate-induced current in these experimental conditions. 3. Increase in the external K+ concentration reduced the NMDA-induced current almost equally over the whole range of membrane potential tested (-60-30 mV). The reversal potential of the NMDA-induced current was not significantly shifted by the replacement of Na+ with K+. 4. A rise in the external K+ concentration to 100 mM did not reduce the single-channel conductance of the NMDA channel, whereas it reduced the mean open time to about two-thirds of that in the control external solution. 5. The suppressed activation of the NMDA receptor channel in high-K+ environments may have a functional significance to alleviate entry of toxic Ca2+ into neurons of the CNS in pathological conditions such as hypoxia and ischemia.

Oscillations of Glial Membrane Potential in a Localized Region of the Blood-Brain Barrier of an Insect

1990 ◽  
Vol 148 (1) ◽  
pp. 335-351
Author(s):  
P. K. SCHOFIELD
Keyword(s):  
Blood Brain Barrier ◽  
Periplaneta Americana ◽  
Basolateral Membrane ◽  
American Cockroach ◽  
Brain Barrier ◽  
Bathing Medium ◽  
Perineurial Cell ◽  
Blood Brain ◽  
High K ◽  
K Concentration

In an abdominal ganglion of the American cockroach, Periplaneta americana, electrophysiological characteristics of the perineurium, the glial layer that forms the insect blood-brain barrier, were investigated by microelectrode recording. The potential across the barrier was positive relative to the bathing medium. Its value increased when the external K concentration was raised, as to be expected from a depolarization of the perineurial cell membrane facing the saline, the basolateral membrane. A negative ‘all-or-nothing’ transient, having an amplitude of some 30mV and a half-amplitude duration of about 1.5 min, was also induced, either during the K elevation or after. Given sufficient K exposure, a series of these transients occurred, with a periodicity of 3–15 min, indicating that a cellular oscillator had been activated. The delay between application of high K and the appearance of the first transient was variable, and couldbe as long as 60 min for a threefold increase in K level. The transients could persist upon return of normal saline, for atleast 85min, with little change in amplitude. By recording transperineurial potential simultaneously with recording from superficial cells, one of which was identified as a perineurial cell by peroxidase injection, the transients were found to begenerated by a depolarization of the membrane adjacent to the underlying nerve cells, the adglial membrane. Analysis using a simple electrical model yielded values for the resting electromotive force (e.m.f.) generated by the membranes, −50mV for the basolateral, −71 mV for the adglial, and indicated that the paracellular pathway had a resistance 4.3 times larger than the transcellular resistance. These results reveal a cellular oscillator, apparently of cytosolic type, in the perineurial glia of an insect ganglion, and demonstrate physiological differences between the perineurium of the ganglion and that of other regions of the insect nervous system.

scholarly journals Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch.

1996 ◽  
Vol 107 (1) ◽  
pp. 35-45 ◽  
Author(s):  
L G Palmer ◽  
G Frindt
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Apical Membrane ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Collecting Tubule ◽  
Excised Patches ◽  
The Mean ◽  
Cortical Collecting Tubule

The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed in cell-attached and inside-out excised patches from split-open tubules using the patch-clamp technique. In patches containing a single channel the open probability (Po) was variable, ranging from 0.05 to 0.9. The average Po was 0.5. However, the individual values were not distributed normally, but were mainly < or = 0.25 or > or = 0.75. Mean open times and mean closed times were correlated directly and inversely, respectively, with Po. In patches where a sufficient number of events could be recorded, two time constants were required to describe the open-time and closed-time distributions. In most patches in which basal Po was < 0.3 the channels could be activated by hyperpolarization of the apical membrane. In five such patches containing a single channel hyperpolarization by 40 mV increased Po by 10-fold, from 0.055 +/- 0.023 to 0.58 +/- 0.07. This change reflected an increase in the mean open time of the channels from 52 +/- 17 to 494 +/- 175 ms and a decrease in the mean closed time from 1,940 +/- 350 to 336 +/- 100 ms. These responses, however, could not be described by a simple voltage dependence of the opening and closing rates. In many cases significant delays in both the activation by hyperpolarization and deactivation by depolarization were observed. These delays ranged from several seconds to several tens of seconds. Similar effects of voltage were seen in cell-attached and excised patches, arguing against a voltage-dependent chemical modification of the channel, such as a phosphorylation. Rather, the channels appeared to switch between gating modes. These switches could be spontaneous but were strongly influenced by changes in membrane voltage. Voltage dependence of channel gating was also observed under whole-cell clamp conditions. To see if mechanical perturbations could also influence channel kinetics or gating mode, negative pressures of 10-60 mm Hg were applied to the patch pipette. In most cases (15 out of 22), this maneuver had no significant effect on channel behavior. In 6 out of 22 patches, however, there was a rapid and reversible increase in Po when the pressure was applied. In one patch, there was a reversible decrease. While no consistent effects of pressure could be documented, membrane deformation could contribute to the variation in Po under some conditions.

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