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.

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.

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)

Single-channel behavior of a purified epithelial Na+ channel subunit that binds amiloride

1992 ◽  
Vol 263 (5) ◽  
pp. C1111-C1117 ◽  
Author(s):  
S. Sariban-Sohraby ◽  
M. Abramow ◽  
R. S. Fisher
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Na Channel ◽  
Na Transport ◽  
Patch Clamp Technique ◽  
Transport Inhibitor ◽  
Open Time ◽  
The Mean ◽  
Epithelial Na Channel ◽  
Binding Subunit

The apical membrane of high electrical resistance epithelia, which is selectively permeable to Na+, plays an essential role in the maintenance of salt balance. Na+ entry from the apical fluid into the cells is mediated by amiloride-blockable Na(+)-specific channels. The channel protein, purified from both amphibian and mammalian sources, is composed of several subunits, only one of which the 150-kDa polypeptide, specifically binds the Na+ transport inhibitor amiloride. The goal of the present study was to investigate whether the isolated amiloride-binding subunit of the channel could conduct Na+. The patch-clamp technique was used to study the 150-kDa polypeptide incorporated into a lipid bilayer formed on the tip of a glass pipette. Unitary conductance jumps averaged 4.8 pS at 100 mM Na2HPO4. Open times ranged from 24 ms to several seconds. The channel spent most of the time in the closed state. Channel conductance and gating were independent of voltage between -60 and +100 mV. Amiloride (0.1 microM) decreased the mean open time of the channel by 98%. We conclude that the 150-kDa subunit of the amiloride-blockable Na+ channel conducts current and may be sufficient for the Na+ transport function of the whole channel.

Properties of veratridine-modified single Na+ channels in guinea pig ventricular myocytes

1993 ◽  
Vol 264 (2) ◽  
pp. H454-H463 ◽  
Author(s):  
A. Sunami ◽  
T. Sasano ◽  
A. Matsunaga ◽  
Z. Fan ◽  
T. Swanobori ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Voltage Dependence ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Simultaneous Occurrence ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
High Event ◽  
Event Times

Modification of single Na+ channels by the alkaloid neurotoxin veratridine was investigated in guinea pig ventricular myocytes using the cell-attached configuration of the patch-clamp technique. Pipette application of veratridine (50 microM) induced long-lasting openings with two different single-channel conductances of 7.6 and 3.0 pS, in addition to normal type of short openings with a single-channel conductance of 16 pS. The veratridine-modified high- and low-conductance channels appeared commonly, and they could coexist with the normal one in the same patch. The open-time distributions for the high- and low-conductance channels could be fitted by a single exponential. The mean open time for the high- and low-conductance events ranged between 19.1 ms at -120 mV and 86.0 ms at -10 mV and between 4.5 ms at -120 mV and 16.2 ms at -10 mV, respectively. The closed-time distributions for the two conductance channels consisted of at least two components, and their values and voltage dependence were similar. External Ca2+ block resulted in an apparent reduction of unitary current amplitudes with a similar voltage dependence and affinity for Ca2+ in the high- and low-conductance channels. However, the low-conductance channel was more resistant to tetrodotoxin than the high one. The probability of simultaneous occurrence of the high and low events was equal to the product of the probabilities of occurrence of the high event times that of the low event. Furthermore, we observed modified channel openings after a normal opening for the two conductance channels and a modified one turning into a normal one for the high-conductance channel. It is concluded that veratridine induces the two different types of modified Na+ channels in cardiac myocytes and these are correlated with normal openings.

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.

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)

Basolateral potassium channels in renal proximal tubule

1987 ◽  
Vol 253 (3) ◽  
pp. F476-F487 ◽  
Author(s):  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Basolateral Membrane ◽  
K Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
K Channels ◽  
Open Time ◽  
Excised Patches ◽  
The Mean ◽  
Inside Out

Potassium (K+) channels in the basolateral membrane of unperfused Necturus proximal tubules were studied in both cell-attached and excised patches, after removal of the tubule basement membrane by manual dissection without collagenase. Two different K+ channels were identified on the basis of their kinetics: a short open-time K+ channel, with a mean open time less than 1 ms, and a long open-time K+ channel with a mean open time greater than 20 ms. The short open-time channel occurred more frequently than the longer channel, especially in excised patches. For inside-out excised patches with Cl- replaced by gluconate, the current-voltage relation of the short open-time K+ channel was linear over +/- 60 mV, with a K+-Na+ selectivity of 12 +/- 2 (n = 12), as calculated from the reversal potential with oppositely directed Na+ and K+ gradients. With K-Ringer in the patch pipette and Na-Ringer in the bath, the conductance of the short open-time channel was 47 +/- 2 pS (n = 15) for cell-attached patches, 26 +/- 2 pS (n = 15) for patches excised (inside out) into Na-Ringer, and 36 +/- 6 pS (n = 3) for excised patches with K-Ringer on both sides. These different conductances can be partially explained by a dependence of single-channel conductance on the K+ concentration on the interior side of the membrane. In experiments with a constant K+ gradient across excised patches, large changes in Na+ at the interior side of the membrane produced no change in single-channel conductance, arguing against a direct block of the K+ channel by Na+. Finally, the activity of the short open-time channel was voltage gated, where the mean number of open channels decreased as a linear function of basolateral membrane depolarization for potentials between -60 and 0 mV. Depolarization from -60 to -40 mV decreased the mean number of open K+ channels by 28 +/- 8% (n = 6).

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)

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