Feedback regulation of Na channels in rat CCT. III. Response to cAMP

1995 ◽  
Vol 268 (3) ◽  
pp. F480-F489 ◽  
Author(s):  
G. Frindt ◽  
R. B. Silver ◽  
E. E. Windhager ◽  
L. G. Palmer
Keyword(s):  
Open Channel ◽  
Single Channel ◽  
Feedback Inhibition ◽  
Feedback Regulation ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Channel Current ◽  
Fluorescence Measurements ◽  
Collecting Tubule ◽  
The Mean

The effects of exogenous adenosine 3',5'-cyclic monophosphate (cAMP) on apical Na channels in the rat cortical collecting tubule were studied using the patch-clamp technique and fura 2 fluorescence measurements of intracellular Ca2+ (Ca2+i). When the permeant analogue, 8-(4-chlorophenylthio)-cAMP (CPT-cAMP, 200 microM), was added to the superfusate during recording from cell-attached patches, both the mean number of open channels (NPo) and the single-channel current (i) decreased within 3 min. When the superfusate also contained amiloride (10 microM), there was no effect of CPT-cAMP on either NPo or i. When CPT-cAMP was added to the bath before formation of the patch, the density of conducting channels was increased from 10 +/- 2 to 37 +/- 6 per patch, as estimated by analysis of channel-induced noise. This suggests that cAMP increases open-channel density in the regions of the apical membrane outside the patch but not within the patch. Channels already active in the patch before stimulation with the nucleotide are subject to feedback inhibition secondary to increased Na entry into the cell. CPT-cAMP increased Ca2+i from 104 to 198 nM. This increase in Ca2+i was abolished by benzamil (0.5 microM) or by low extracellular Ca2+. The cAMP-dependent reduction in NPo was still observed in Ca(2+)-free medium, indicating that a rise in Ca2+i was not essential for the feedback response. The decrease in NPo was attenuated, however, when cAMP was added in the absence of Ca2+ and in the presence of ouabain (1 mM) in the superfusate.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Feedback regulation of Na channels in rat CCT. I. Effects of inhibition of Na pump

1993 ◽  
Vol 264 (3) ◽  
pp. F557-F564 ◽  
Author(s):  
R. B. Silver ◽  
G. Frindt ◽  
E. E. Windhager ◽  
L. G. Palmer
Keyword(s):  
Feedback Regulation ◽  
Peak Level ◽  
Na Channels ◽  
Channel Activity ◽  
Electrical Measurements ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Collecting Tubule ◽  
Intracellular Ca ◽  
The Mean

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (Po), and currents (i) were measured at 37 degrees C during continuous superfusion of the tubule. Addition of ouabain (1 mM) to the superfusate to increase cell Na resulted in a decrease in the mean number of open channels (NPo) to less than 20% of control values within 2 min. This effect was not reversible within 5 min after removal of ouabain. There was, in addition, a parallel decrease in i. The mechanism of inhibiton appeared to involve increased intracellular Ca (Cai). Cai was measured using the fluorescence of the Ca indicator fura-2 in principal cells of split tubules under conditions identical to those used for electrical measurements. Cai increased from a basal level (153 +/- 36 nM) to a peak level (588 +/- 53 nM) approximately 3 min after the addition of ouabain. When a Ca-free superfusate was used, ouabain did not increase Cai or decrease NPo, although the decrease in i was similar to that observed in Ca-containing solutions. Similar increases in Cai were elicited by the Ca ionophore ionomycin (5 microM) in the presence of 0.1 mM extracellular Ca. This maneuver also resulted in a decrease in NPo which was similar to that observed in the presence of ouabain. Ouabain had no observable effect on cell pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Feedback regulation of Na channels in rat CCT. II. Effects of inhibition of Na entry

1993 ◽  
Vol 264 (3) ◽  
pp. F565-F574 ◽  
Author(s):  
G. Frindt ◽  
R. B. Silver ◽  
E. E. Windhager ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Ion Homeostasis ◽  
Feedback Regulation ◽  
Feedback Mechanism ◽  
Acute Administration ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Single Channel Currents

Na channels in the apical membrane of the rat renal cortical collecting tubule were studied using the patch-clamp technique. Channel activity was monitored in cell-attached patches on tubules that were split open to expose the luminal surface. Channel number (N), open probability (Po), and single-channel currents (i) were measured at 37 degrees C during continuous superfusion of the tubule. Addition of amiloride (10 microM) or benzamil (0.5 microM) to the superfusate resulted in a twofold increase in the mean number of open channels (NPo) after 2 min. The effect closely paralleled an increase in i, presumably reflecting membrane hyperpolarization. The effects on both i and NPo reversed within 3 min after removal of amiloride. The increase in NPo was accounted for, at least in part, by an increase in Po. Several cellular events may contribute to this phenomenon. Channels could be activated directly by membrane hyperpolarization and by cell shrinkage, both of which are known to occur during acute administration of amiloride. In addition, benzamil elicited a 30% decrease in intracellular Ca compared with control levels as measured by fura-2 fluorescence. A comparable decrease observed after reducing extracellular Ca did not increase NPo. No changes in cell pH, measured with 2',7'-bis-(carboxyethyl)-5(6)-carboxyfluorescein fluorescence, were observed. The modulation of channel Po by the rate of Na entry into the cell will act as a feedback mechanism to maintain cellular ion homeostasis, and this may also serve to distribute Na reabsorption more evenly along the nephron.

scholarly journals Origins of open-channel noise in the large potassium channel of sarcoplasmic reticulum.

1994 ◽  
Vol 104 (5) ◽  
pp. 857-883 ◽  
Author(s):  
A H Hainsworth ◽  
R A Levis ◽  
R S Eisenberg
Keyword(s):  
Spectral Density ◽  
Power Spectral Density ◽  
Open Channel ◽  
Single Channel ◽  
Channel Noise ◽  
Mean Power ◽  
Channel Current ◽  
Power Spectral ◽  
The Mean ◽  
Aqueous Species

Open-channel noise was studied in the large potassium channel of the sarcoplasmic reticulum (SR). Inside-out patches were excised directly from the SR of split skeletal muscle fibers of lobster, with lobster relaxing ringer (LRR) in bath and pipette. The power spectrum of open-channel noise is very low and approximately flat in the 100 Hz-10 kHz frequency range. At 20 degrees C, with an applied voltage of 50 mV, the mean single-channel current (i) is 9 pA (mean single-channel conductance = 180 pS) and the mean power spectral density 1.1 x 10(-29) A2/Hz. The latter increases nonlinearly with (i), showing a progressively steeper dependence as (i) increases. At 20 mV, the mean power spectral density is almost independent of (i) and approximately 1.4 times that of the Johnson noise calculated for the equivalent ideal resistor with zero net current; at 70 mV it increases approximately in proportion to (i)2. The mean power spectral density has a weak temperature dependence, very similar to that of (i), and both are well described by a Q10 of 1.3 throughout the range 3-40 degrees C. Discrete ion transport events are thought to account for a significant fraction of the measured open-channel noise, probably approximately 30-50% at 50 mV. Brief interruptions of the single-channel current, due either to blockage of the open channel by an extrinsic aqueous species, or to intrinsic conformational changes in the channel molecule itself, were a possible additional source of open-channel noise. Experiments in modified bathing solutions indicate, however, that open-channel noise is not affected by any of the identified aqueous species present in LRR. In particular, magnesium ions, the species thought most likely to cause brief blockages, and calcium and hydrogen ions, have no detectable effect. This channel's openings exhibit many brief closings and substrates, due to intrinsic gating of the channel. Unresolved brief full closings are calculated to make a negligible contribution (< 1%) to the measured power spectral density. The only significant source of noise due to band width-limited missed events is brief, frequent 80% substrates (mean duration 20 microseconds, mean frequency 1,000 s-1) which account for a small part of the measured power spectral density (approximately 14%, at 50 mV, 20 degrees C). We conclude that a large fraction of the measured open-channel noise results from intrinsic conductance fluctuations, with a corner frequency higher than the resolution of our recordings, in the range 10(4)-10(7) Hz.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Kinetics of 9-aminoacridine block of single Na channels.

1984 ◽  
Vol 84 (3) ◽  
pp. 361-377 ◽  
Author(s):  
D Yamamoto ◽  
J Z Yeh
Keyword(s):  
Rate Constant ◽  
Voltage Dependence ◽  
Single Channel ◽  
Na Channel ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Voltage Dependent ◽  
The Mean ◽  
Kinetics Of

The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA). Following NBA treatment, the current flowing through individual Na channels was manifested by square-wave open events lasting from several to tens of milliseconds. When 9-AA was applied to the cytoplasmic face of Na channels at concentrations ranging from 30 to 100 microM, it caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single channel current. The histograms for the duration of blocked states and the histograms for the duration of open states could be fitted with a single-exponential function. The mean open time (tau o) became shorter as the drug concentration was increased, while the mean blocked time (tau b) was concentration independent. The association (blocking) rate constant, kappa, calculated from the slope of the curve relating the reciprocal mean open time to 9-AA concentration, showed little voltage dependence, the rate constant being on the order of 1 X 10(7) M-1s-1. The dissociation (unblocking) rate constant, l, calculated from the mean blocked time, was strongly voltage dependent, the mean rate constant being 214 s-1 at 0 mV and becoming larger as the membrane being hyperpolarized. The voltage dependence suggests that a first-order blocking site is located at least 63% of the way through the membrane field from the cytoplasmic surface. The equilibrium dissociation constant for 9-AA to block the Na channel, defined by the relation of l/kappa, was calculated to be 21 microM at 0 mV. Both tau -1o and tau -1b had a Q10 of 1.3, which suggests that binding reaction was diffusion controlled. The burst time in the presence of 9-AA, which is the sum of open times and blocked times, was longer than the lifetime of open channels in the absence of drug. All of the features of 9-AA block of single Na channels are compatible with the sequential model in which 9-AA molecules block open Na channels, and the blocked channels could not close until 9-AA molecules had left the blocking site in the channels.

Effects of cell Ca and pH on Na channels from rat cortical collecting tubule

1987 ◽  
Vol 253 (2) ◽  
pp. F333-F339 ◽  
Author(s):  
L. G. Palmer ◽  
G. Frindt
Keyword(s):  
Apical Membrane ◽  
Na Channels ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Collecting Tubule ◽  
Solution Bathing ◽  
The Mean ◽  
Inside Out ◽  
Cytoplasmic Side ◽  
Cortical Collecting Tubule

The patch-clamp technique was used to identify individual Na channels in the apical membrane of the rat cortical collecting tubule and to evaluate the effects of cytoplasmic Ca2+ and pH on channel activity. In excised, inside-out patches, the probability of a channels's being open (P0) increased with alkalinization of the solution bathing the cytoplasmic side of the patch. Estimates of P0 were 0.05 at pH 6.4, 0.19 at pH 6.9, and 0.41 at pH 7.4. Varying the free Ca2+ concentration of the solution bathing the cytoplasmic side of the patch had no measurable effect on P0. In cell-attached patches, addition of the Ca2+ ionophore ionomycin to the solution bathing the tubules to a final concentration of either 1 or 10 microM decreased channel activity measured as the mean number of open channels (no. open) = n X P0 where n is the number of channels in the membrane. (no. open) was significantly decreased at 3 min after addition of ionomycin and fell to less than 10% of control values after 10 min incubation. There was no fall in (no. open) either in time controls or in tubules exposed to ionomycin in the presence of low bath Ca2+ concentrations [no added Ca2+ with 1 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA)]. The results suggest that cytoplasmic pH can directly influence channel activity. Cytoplasmic Ca2+ does not interact directly with the channels, but increased cytoplasmic Ca2+ produces a fall in channel activity through an indirect process.

scholarly journals Slow currents through single sodium channels of the adult rat heart.

1985 ◽  
Vol 86 (1) ◽  
pp. 89-104 ◽  
Author(s):  
J B Patlak ◽  
M Ortiz
Keyword(s):  
Time Constant ◽  
Single Channel ◽  
Slow Component ◽  
Reversal Potential ◽  
Careful Examination ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Adult Rat ◽  
Ventricular Cells ◽  
The Mean

The currents through single Na+ channels from the sarcolemma of ventricular cells dissociated from adult rat hearts were studied using the patch-clamp technique. All patches had several Na+ channels; most had 5-10, while some had up to 50 channels. At 10 degrees C, the conductance of the channel was 9.8 pS. The mean current for sets of many identical pulses inactivated exponentially with a time constant of 1.7 +/- 0.6 ms at -40 mV. Careful examination of the mean currents revealed a small, slow component of inactivation at pulse potentials ranging from -60 to -30 mV. The time constant of the slow component was between 8 and 14 ms. The channels that caused the slow component had the same conductance and reversal potential as the fast Na+ currents and were blocked by tetrodotoxin. The slow currents appear to have been caused by repeated openings of one or more channels. The holding potential influenced the frequency with which such channel reopening occurred. The slow component was prominent during pulses from a holding potential of -100 mV, while it was very small during pulses from -140 mV. Ultraslow currents through the Na+ channel were observed occasionally in patches that had large numbers of channels. They consisted of bursts of 10 or more sequential openings of a single channel and lasted for up to 150 ms. We conclude that the single channel data cannot be explained by standard models, even those that have two inactivated states or two open states of the channel. Our results suggest that Na+ channels can function in several different "modes," each with a different inactivation rate.

Feedback regulation of Na channels in rat CCT. IV. Mediation by activation of protein kinase C

1996 ◽  
Vol 270 (2) ◽  
pp. F371-F376 ◽  
Author(s):  
G. Frindt ◽  
L. G. Palmer ◽  
E. E. Windhager
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Single Channel ◽  
Feedback Inhibition ◽  
Na Channel ◽  
Specific Cell ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Kinase C ◽  
Calmodulin Kinase

The hypothesis that feedback inhibition of the apical Na+ channels in the cortical collecting tubule (CCT) is mediated by activation of a Ca(2+)-dependent protein kinase was tested using the patch-clamp technique. Na+ channel activity was monitored in cell-attached patches in principal cells of split-open rat tubules. Mean number of open channels (NPo) and single-channel current (i) were measured at 37 degrees C during continuous tubule superfusion. Phorbol 12-myristate 13-acetate (PMA; 50 nM), an activator of protein kinase C (PKC), decreased NPo to 33% of the control value. Staurosporine (200 nM), an inhibitor of PKC and of Ca(2+)-calmodulin kinase II, practically abolished the effect of PMA. Ouabain (1 mM), reduced NPo to 29% of control values and decreased i. Ouabain did not downregulate the channels in tubules exposed to staurosporine, although it still reduced i. Incubation of the tubules with 10 microM KN-62, a specific cell membrane-permeable inhibitor of Ca(2+)-calmodulin kinase II, did not interfere with the ouabain-dependent downregulation of the channels. The results support the view that the downregulation caused by ouabain involves the Ca(2+)-dependent phosphorylation of the channel itself or of proteins regulating the channel.

scholarly journals Conductance and gating of epithelial Na channels from rat cortical collecting tubule. Effects of luminal Na and Li.

1988 ◽  
Vol 92 (1) ◽  
pp. 121-138 ◽  
Author(s):  
L G Palmer ◽  
G Frindt
Keyword(s):  
Patch Clamp ◽  
Single Channel ◽  
Partial Substitution ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Epithelial Na Channels ◽  
Collecting Tubule ◽  
Km Value ◽  
Series Of Experiments ◽  
Cortical Collecting Tubule

The behavior of individual Na channels in the apical membrane of the rat cortical collecting tubule (CCT) was studied at different concentrations of the permeant ions Na and Li. Tubules were opened to expose their luminal surfaces and bathed in K-gluconate medium to minimize tubule-to-tubule variation in cell membrane potential and intracellular Na concentration. The patch-clamp technique was used to resolve currents through individual channels. The patch-clamp pipette was filled with solutions containing variable concentrations of either NaCl or LiCl. In one series of experiments, the concentrations were changed without substitutions. In another series, the ionic strength and Cl concentration were maintained constant by partial substitution of Li with N-methyl-D-glucamine (NMDG). In cell-attached patches, both the single-channel conductance (g) and the single-channel current (i) saturated as functions of the Na or Li activity in the pipette. Without NMDG, the saturation of i was well described by Michaelis-Menten kinetics with an apparent Km of approximately 20 mM activity for Na and approximately 50 mM activity for Li. Km was independent of voltage for both ions. With substitution for Li by NMDG, the apparent Km value for Li transport through the channels increased. The values of the probability of a channel's being open (Po) varied from patch to patch, but no effect of pipette ion activity on Po could be demonstrated. A weak dependence of Po on membrane voltage was observed, with hyperpolarization increasing Po by an average of 2.3%/mV.

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.

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