Apical Na+ conductance in maturing rabbit principal cell

1996 ◽  
Vol 270 (3) ◽  
pp. F391-F397 ◽  
Author(s):  
L. M. Satlin ◽  
L. G. Palmer
Keyword(s):  
Principal Cell ◽  
Na Channel ◽  
Postnatal Life ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Pipette Solution ◽  
Principal Cells ◽  
Inward Currents ◽  
The Mean

Net Na+ absorption in microperfused rabbit cortical collecting ducts (CCDs) is low during the 1st wk of postnatal life, increasing substantially thereafter [L. M. Satlin. Am. J. Physiol. 266 (Renal Fluid Electrolyte Physiol. 35): F57-F65, 1994]. To establish whether the low rate of Na+ absorption observed immediately after birth is due to a low apical Na+ permeability of the neonatal principal cell, we used the patch-clamp technique in split-open CCDs isolated from maturing rabbits to estimate conductance, number (N), and open probability (Po) of apical Na+ channels in principal cells. With LiCl in the pipette and a NaCl or potassium gluconate solution, warmed to 37 degrees C, in the bath, inward currents with a conductance of approximately 11 pS (n = 23) were observed in 17% of cell-attached patches at 1 wk, 41% of patches at 2 wk, and 43% of patches at 5 wk. The mean N per patch in the 1st wk (0.22 +/- 0.09; n = 36) was significantly less than that observed in the 2nd (1.38 +/- 0.39; n = 34) and 5th (1.24 +/- 0.37; n = 21) wk of life. Po, studied at positive pipette voltages, was significantly lower in the 1st wk (0.085 +/- 0.035; n = 5) than in the 2nd wk (0.345 +/- 0.063; n = 9) and 5th wk (0.291 +/- 0.058; n = 4). To confirm that the 11-pS channel represented the amiloride-sensitive apical Na+ channel, cell-attached patches in CCDs isolated from 2-wk-old rabbits were studied with 0.5 microM amiloride added to the LiCl pipette solution. Amiloride led to > 90% reductions in mean open and closed times of the 11-pS conductance, consistent with blockade of the channel. These data indicate that N and Po of apical amiloride-sensitive Na+ channels in principal cells increase significantly after birth.

Apical K+ conductance in maturing rabbit principal cell

1997 ◽  
Vol 272 (3) ◽  
pp. F397-F404 ◽  
Author(s):  
L. M. Satlin ◽  
L. G. Palmer
Keyword(s):  
Reversal Potential ◽  
Principal Cell ◽  
K Channel ◽  
Postnatal Life ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
K Channels ◽  
Inward Currents ◽  
K Secretion

Net K+ secretion is not detected in cortical collecting ducts (CCDs) isolated from newborn rabbits and perfused in vitro. To establish whether a low apical K+ permeability of the neonatal principal cell limits K+ secretion early in life, we used the patch-clamp technique in split-open CCDs isolated from maturing rabbits to study the properties and density of conducting K+ channels in principal cells. With KCl in the pipette and a NaCl solution warmed to 37 degrees C in the bath, inward currents with a conductance of approximately 42 pS were observed in 0% (0 out of 13 or 0/13), 10% (2/21), 18% (5/28), 29% (4/14), and 56% (10/18) of cell-attached patches obtained in 1-, 2-, 3-, 4-, and 5-wk-old animals, respectively. The conductance and reversal potential of this channel led us to suspect that it represented the low-conductance K+ channel previously described in the rat CCD by L. G. Palmer, L. Antonian, and G. Frindt (J. Gen. Physiol. 104: 693-710, 1994). The mean number of open channels per patch (NPo) increased progressively (P < 0.05) after birth, from 0 at 1 wk, to 0.06 +/- 0.04 at 2 wk, to 0.40 +/- 0.18 at 3 wk, to 0.74 +/- 0.41 at 4 wk, and to 1.06 +/- 0.28 at 5 wk. The increase in NPo appeared to be due primarily to a developmental increase in N, which is the number of channels; open probability, Po, remained constant at approximately 0.5 for all channels identified after the 2nd wk of life. The increase in number of conducting K+ channels during postnatal life is likely to contribute to the maturational increase in net K+ secretion in the CCDs.

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)

Insulin activates single amiloride-blockable Na channels in a distal nephron cell line (A6)

1992 ◽  
Vol 263 (3) ◽  
pp. F392-F400 ◽  
Author(s):  
Y. Marunaka ◽  
N. Hagiwara ◽  
H. Tohda
Keyword(s):  
Cell Line ◽  
Single Channel ◽  
Apical Membrane ◽  
Na Channel ◽  
Na Channels ◽  
Distal Nephron ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Patch Clamp Technique ◽  
Open Probability

Using the patch-clamp technique, we studied the effect of insulin on an amiloride-blockable Na channel in the apical membrane of a distal nephron cell line (A6) cultured on permeable collagen films for 10-14 days. NPo (N, number of channels per patch membrane; Po, average value of open probability of individual channels in the patch) under baseline conditions was 0.88 +/- 0.12 (SE)(n = 17). After making cell-attached patches on the apical membrane which contained Na channels, insulin (1 mU/ml) was applied to the serosal bath. While maintaining the cell-attached patch, NPo significantly increased to 1.48 +/- 0.19 (n = 17; P less than 0.001) after 5-10 min of insulin application. The open probability of Na channels was 0.39 +/- 0.01 (n = 38) under baseline condition, and increased to 0.66 +/- 0.03 (n = 38, P less than 0.001) after addition of insulin. The baseline single-channel conductance was 4pS, and neither the single-channel conductance nor the current-voltage relationship was significantly changed by insulin. These results indicate that insulin increases Na absorption in the distal nephron by increasing the open probability of the amiloride-blockable Na channel.

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.

Regulation of Na channels in the rat cortical collecting tubule: effects of cAMP and methyl donors

1996 ◽  
Vol 271 (5) ◽  
pp. F1086-F1092 ◽  
Author(s):  
G. Frindt ◽  
L. G. Palmer
Keyword(s):  
Na Channel ◽  
Kinetic Properties ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Whole Cell ◽  
Methyl Donors ◽  
Membrane Rupture ◽  
Collecting Tubule ◽  
Cortical Collecting Tubule

The whole cell patch-clamp technique was used to investigate the interactions of the amiloride-sensitive Na channel of the rat cortical collecting tubule (CCT) with adenosine 3',5'-cyclic monophosphate (cAMP) and with methyl donors. The amiloride-sensitive whole cell current (INa) was measured in principal cells of dissected, split-open tubules from rats maintained either on a control diet or on a low-Na diet to increase endogenous aldosterone secretion. With Na-depleted animals, INa was highest immediately after rupture of the membrane patch and averaged 325 pA at a membrane potential of -60 mV. INa declined over 15 min to approximately 35% of the initial value. With 8-(4-chlorophenylthio)-cAMP in the pipette, INa increased within 5 min of membrane rupture and was maintained for 15 min at levels three- to fourfold higher than the corresponding control values. With Na-replete animals, INa was undetectable (< 10 pA) without cAMP. With cAMP in the pipette, INa averaged 40 pA. In cell-attached patches on tubules from Na-replete rats exposed to cAMP, single Na channels were observed with conductive and kinetic properties similar to those from Na-depleted rats but at lower density. Inclusion of the methyl donor S-adenosyl methionine to the pipette solution did not increase INa in CCTs from Na-replete rats, either in the presence or absence of cAMP. The methylation inhibitor S-adenosyl homocysteine did not affect INa in CCT from Na-depleted animals.

scholarly journals Properties of single cardiac Na channels at 35 degrees C.

1994 ◽  
Vol 104 (5) ◽  
pp. 801-820 ◽  
Author(s):  
K Benndorf
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Band Width ◽  
Na Channel ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Total Noise ◽  
Na Currents ◽  
Transition Points

Single Na channel currents were recorded in cell-attached patches of mouse ventricular myocytes with an improved patch clamp technique. Using patch pipettes with a pore diameter in the range of 200 nm, seals with a resistance of up to 4 T omega were obtained. Under those conditions, total noise could be reduced to levels as low as 0.590 pA rms at 20 kHz band width. At this band width, properties of single-channel Na currents were studied at 35 degrees C. Six out of a total of 23 patches with teraohm seals contained channel activity and five of these patches contained one and only one active channel. Amplitude histograms excluding transition points showed heterogenous distributions of levels. In one patch, part of the openings was approximately Gaussian distributed at different potentials yielding a slope conductance of 27 pS. The respective peak open probability at -10 mV was 0.26. The mean open time was determined at voltages between -60 and -10 mV by evaluation of the distribution of the event-related gaps in the center of the baseline noise to be approximately 40 microseconds at -60 mV and 50-74 microseconds between -50 and -10 mV. It is concluded that single cardiac Na channels open at 35 degrees C frequently with multiple levels and with open times in the range of several tens of microseconds.

scholarly journals Two modes of gating during late Na+ channel currents in frog sartorius muscle.

1986 ◽  
Vol 87 (2) ◽  
pp. 305-326 ◽  
Author(s):  
J B Patlak ◽  
M Ortiz
Keyword(s):  
Reversal Potential ◽  
Current Amplitude ◽  
Na Channel ◽  
Sartorius Muscle ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Na Currents ◽  
Inward Currents ◽  
Channel Currents

Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods.

Nitric oxide-induced hyperpolarization stimulates low-conductance Na+ channel of rat CCD

1997 ◽  
Vol 272 (4) ◽  
pp. F498-F504 ◽  
Author(s):  
M. Lu ◽  
G. Giebisch ◽  
W. Wang
Keyword(s):  
Nitric Oxide ◽  
Collecting Duct ◽  
No Synthase ◽  
Na Channel ◽  
Na Channels ◽  
Cortical Collecting Duct ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
No Donor ◽  
K Channels

We used the patch-clamp technique in the split-open cortical collecting duct (CCD) to investigate the effect of nitric oxide (NO) on the low-conductance (6-pS) Na+ channel that can be blocked by 1 microM amiloride. We confirmed that the number of Na+ channels increased significantly in CCDs of rats on a low-Na+ diet (17). Application of 100 microM N(G)-nitro-L-arginine methyl ester (L-NAME), an agent that blocks endogenous NO synthase, reduced NPo [the product of channel number (N) and open probability (Po)] to 45% of the control value. The effect of L-NAME was specific, since addition of D-NAME, which does not inhibit NO synthase, did not change the activity of the Na+ channel. That the effect of L-NAME results from inhibition of NO synthase is further confirmed by experiments in which addition of an exogenous NO donor, either 10 microM S-nitroso-N-acetyl penicillamine or sodium nitroprusside (SNP), restored the Na+ channel activity when it had been blocked by L-NAME. The action of NO involves a guanosine 3',5'-cyclic monophosphate (cGMP)-dependent pathway, since 100 microM 8-bromo-cGMP (8-BrcGMP) mimicked the effect of SNAP on K+ channels. However, 100 microM 8-BrcGMP did not alter the activity of Na+ channels in inside-out patches, suggesting an indirect action. Because the Na+ channel is activated by hyperpolarization (19) and NO stimulates basolateral K+ channels (16), we tested whether hyperpolarization mediated the effect of NO. In perforated whole cell recordings, addition of L-NAME depolarized the cell membrane from -73 to 51 mV, and application of 10 microM SNP repolarized the membrane to -68 mV. Furthermore, the L-NAME-induced decrease in NPo was effectively restored by 25 mV hyperpolarization of the patch membranes, and addition of 2 mM Ba2+ also abolished the effect of L-NAME. We concluded that the stimulatory effect of NO on the Na+ channel is an indirect effect mediated by a NO-induced increase of basolateral K+ conductance.

scholarly journals Regulation of apical K and Na channels and Na/K pumps in rat cortical collecting tubule by dietary K.

1994 ◽  
Vol 104 (4) ◽  
pp. 693-710 ◽  
Author(s):  
L G Palmer ◽  
L Antonian ◽  
G Frindt
Keyword(s):  
K Channel ◽  
Na Channel ◽  
Sk Channels ◽  
Na Channels ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
High K ◽  
Collecting Tubule ◽  
Cortical Collecting Tubule

The patch-clamp technique was used to study the properties and the density of conducting K and Na channels in the apical membrane of rat cortical collecting tubule. The predominant K channel observed in cell-attached patches (SK channels) had an outward single-channel conductance (with LiCl in the pipette) of 10 pS. The inward conductance (with KCl in the pipette) was 42 pS. The channel had a high open probability that increased with depolarization. Kinetic analysis indicated the presence of a single open state and two closed states. Increasing K intake by maintaining animals on a high K diet for 12-16 d increased the number of SK channels per patch by threefold (0.7-2.0/patch) over control levels. In addition, conducting Na-selective channels, which were not observed in control animals, were seen at low density (0.5/patch). These channels had properties similar to those observed when the animals were on a low Na diet, except that the mean open probability (0.84) was higher. In other experiments, the whole-cell patch clamp technique was used to measure Na channel activity (as amiloride-sensitive current, INa) and Na pump activity (as ouabain-sensitive current, Ipump). In animals on a high K diet, INa was greater than in controls but much less than in rats on a low Na diet. Ipump was greater after K loading than in controls or Na-depleted animals. These K diet-dependent effects were not accompanied by a significant increase in plasma aldosterone concentrations. To further investigate the relationship between K channel activity and mineralocorticoids, rats were maintained on a low Na diet to increase endogenous aldosterone secretion. Under these conditions, no increase in SK channel density was observed, although there was a large increase in the number of Na channels (to 2.7/patch). Aldosterone was also administered exogenously through osmotic minipumps. As with the low Na diet, there was no change in the density of conducting SK channels, although Na channel activity was induced. These results suggest that SK channels, Na channels and Na/K pumps are regulated during changes in K intake by factors other than aldosterone.

Amiloride-sensitive sodium channels in rabbit cortical collecting tubule primary cultures

1991 ◽  
Vol 261 (6) ◽  
pp. F933-F944 ◽  
Author(s):  
B. N. Ling ◽  
C. F. Hinton ◽  
D. C. Eaton
Keyword(s):  
High Selectivity ◽  
Primary Cultures ◽  
Principal Cell ◽  
Na Channel ◽  
Open Probability ◽  
Inward Current ◽  
Collecting Tubule ◽  
Apical Membranes ◽  
Rabbit Cortical Collecting Tubule ◽  
Cortical Collecting Tubule

Patch-clamp methodology was applied to principal cell apical membranes of rabbit cortical collecting tubule (CCT) primary cultures grown on collagen supports in the presence of aldosterone (1.5 microM). The most frequently observed channel had a unit conductance of 3-5 pS, nonlinear current-voltage (I-V) relationship, Na permeability (PNa)-to-K permeability (PK) ratio greater than 19:1, and inward current at all applied potentials (Vapp) less than +80 mV (n = 41). Less frequently, an 8- to 10-pS channel with a linear I-V curve, PNa/PK less than 5:1, and inward current at Vapp less than +40 mV was also observed (n = 7). Luminal amiloride (0.75 microM) decreased the open probability (Po) for both of these channels. Mean open time for the high-selectivity Na+ channel was 2.1 +/- 0.5 s and for the low-selectivity Na+ channel was 50 +/- 12 ms. In primary cultures grown without aldosterone the high-selectivity Na+ channel was rarely observed (1 of 32 patches). Lastly, a 26- to 35-pS channel, nonselective for Na+ over K+, was not activated by cytoplasmic Ca2+ or voltage nor inhibited by amiloride (n = 17). We conclude that under specific growth conditions, namely permeable transporting supports and chronic mineralocorticoid hormone exposure, principal cell apical membranes of rabbit CCT primary cultures contain 1) both high-selectivity and low-selectivity, amiloride-inhibitable Na+ channels and 2) amiloride-insensitive, nonselective cation channels.

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