Renal sodium channels: regulation and single channel properties.
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Aldosterone↗
Biomedical subjects
D C Eaton
Publications and source records attributed to D C Eaton.
Role of growth factors in mesangial cell ion channel regulation.
Our single channel work has characterized two ion channels capable of depolarizing mesangial cells and activating classic, voltage-activated Ca2+ channels in response to growth-stimulatory peptides (such as Ang II, ET and insulin): (1) Ca(2+)-dependent, 4 pS Cl- channel promoting Cl- efflux; and (2) Ca(2+)-dependent, 27 pS nonselective cation channels promoting cation influx. We have also characterized a third channel which provides an alternative, receptor-operated pathway for Ca2+ entry in response to the growth factor, PDGF: (3) Ca(2+)-permeable, 1 pS cation channel. Consistent with our model of mesangial cell signal transduction (Fig. 1), these three mesangial cell ion channels are activated by binding of growth factors to membrane receptors (Fig. 8). Defective channel regulation, such as occurs in early diabetes mellitus, would promote mesangial cell relaxation and pathogenic glomerular hyperfiltration. Glomerular hyperfiltration and hypertension have been proposed to be major pathogenic factors in renal disease progression [4, 29, 38, 39]. Compensatory renal growth factor responses initially provide adaptive changes in glomerular hemodynamics after loss of functional renal mass. However, chronic stimulation of these mesangial cell ion channels by renal growth factors would promote sustained extracellular Ca2+ entry, resulting in mesangial cell contraction and growth, and progressive decreases in Kf and GFR. Eventually, this process leads to irreversible renal damage due to the development of glomerulosclerosis and interstitial fibrosis.
Angiotensin II↗
Extracellular glucose reduces the responsiveness of mesangial cell ion channels to angiotensin II.
Abnormal cellular ion homeostasis is a well-recognized component of diabetic glomerular disease. In cultured rat glomerular mesangial cells, we have previously shown that insulin regulates Ca(2+)-dependent activation of 4-pS Cl- channels and 27-pS nonselective cation channels (NSCC) by angiotensin II (ANG II). To assess whether extracellular glucose also affects mesangial ion channels, we applied patch-clamp techniques to cells incubated in constant insulin (100 mU/ml) and either "normal" (5 mM) or "high" (30 mM) glucose for 1 wk. In normal glucose, 100 nM ANG II increased Cl- and NSCC activity by > 16-fold and > 60-fold, respectivley. Direct release of intracellular Ca2+ ([Ca2+]i) stores (0.25 microM thapsigargin) mimicked ANG II-induced channel stimulation. In high glucose, Cl- and NSCC stimulation by ANG II was attenuated (< 7-fold), whereas channel activation by thapsigargin was unaffected. Protein kinase C (PKC) inhibition (30-min exposure to 0.5 microM calphostin) or downregulation (24-h exposure to 0.1 microM 4 beta-phorbol 12-myristate 13-acetate), but not aldose reductase inhibition (0.5 mM sorbinil), restored channel responsiveness to ANG II despite high glucose. Channel responsiveness was also restored if mesangial cells were coincubated in both high glucose and 500 microM myo-inositol. Acute exposure to a synthetic diacylglycerol (100 microM 1-oleoyl-2-acetyl glycerol) reestablished channel unresponsiveness to ANG II. We conclude the following in rat mesangial cell cultures: 1) Activation of Ca(2+)-dependent Cl- and NSCCs by ANG II is reduced by high extracellular glucose.(ABSTRACT TRUNCATED AT 250 WORDS)
Aldehyde Reductase↗
The effect of racemic ketamine on the large conductance Ca(+2)-activated potassium (BK) channels in GH3 cells.
Recently, inhalation anesthetics have been reported to block BK channels in adrenal chromaffin cells. To determine if BK block was characteristic only of inhalation anesthetics or was also a property of other general anesthetics we examined the effects of ketamine, an intravenous general anesthetic which is structurally different than inhalation anesthetics. Cell-attached and excised patch single channel and standard whole cell recording techniques were used to examine the effect of racemic ketamine on the BK channel activity in GH3 cells. When solutions containing 150 mM KCl are used in both the pipette and bath, the BK channels are characterized as a voltage-dependent channel with a unit conductance of 150-300 pS. Racemic ketamine (at clinically relevant concentrations; 2-500 microM) selectively blocked BK channels in a dose-dependent, reversible manner as evidenced by decreases in NPo (number of channels x open probability). This decrease was due to both a decrease in mean open time and an increase in the mean closed time but without a decrease in single-channel current amplitude. Ketamine shifts the Po vs voltage curve to higher potentials without a change in the slope of the voltage dependence. Ketamine also shifts the Po vs [Ca+2] relationship to higher Ca+2 concentrations. The IC50 for the single-channel block by ketamine is 20.3 +/- 15.9 microM. In an effort to confirm that the effect of ketamine was predominantly due to a block of the BK channels, standard whole cell techniques were utilized.(ABSTRACT TRUNCATED AT 250 WORDS)
Analysis of Variance↗
Postnatal changes in the G-proteins, cyclic nucleotides and adenylyl cyclase activity in rabbit heart cells.
We have studied the postnatal changes in the levels of isoforms of stimulatory (Gs) and inhibitory (Gi) G-proteins, cAMP and cGMP in washed particulate membranes (WPM) from whole ventricles as well as from isolated ventricular myocytes and have also measured adenylyl cyclase (AC) activity in WPM prepared from isolated myocytes of adult (AD) and newborn (NB) rabbit heart. Immunoblot analysis for the levels of Gi alpha 1, G alpha 2, Gi alpha 3 and Gs alpha subunits showed that Gi alpha 2 and Gi alpha 3 were higher in WPM from whole ventricles of NB compared to AD. This ratio was much higher in WPM from isolated ventricular myocytes since Gi alpha 2 and Gi alpha 3 were either absent or present in extremely low immunodetectable levels in WPM from AD ventricular myocytes. Gi alpha 1 levels were not different for AD compared to NB WPM, whether prepared from whole ventricle or from isolated myocytes. Two forms of Gs alpha, a small form (Gs alpha-S) and a large form (Gs alpha-L), were immunodetected at 43 and 48 kDa, respectively. The Gs alpha-S form was higher in AD WPM and the Gs alpha-L form was higher in NB WPM while the total Gs alpha(L+S) was not different. The Gs alpha results for WPM from isolated myocytes were not different from the results for WPM from whole ventricles. Basal levels of cAMP were 80% higher in NB compared to AD whole ventricles and were 200% higher in NB compared to AD isolated myocytes. Levels of cGMP were 4-5 fold higher in NB than in AD myocytes and ventricular tissue. Basal AC activity was higher in NB than in AD WPM from isolated myocytes and was enhanced by Gpp(NH)p pretreatment in AD but not in NB WPM. The isoproterenol-induced increase in AC activity was higher in AD compared to NB WPM and was completely abolished by Gpp(NH)p pretreatment in NB but not in AD WPM. Forskolin caused a greater increase in AC activity in NB than in AD WPM. The post-natal decrease in the levels of Gi alpha 2 and Gi alpha 3, particularly in isolated ventricular myocytes, may help to explain the smaller effects of isoproterenol and greater muscarinic influence on ICa, as we previously showed, and the smaller effect of isoproterenol on AC activity in NB compared to AD WPM.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenylyl Cyclases↗
Ca(2+)-permeable channel associated with platelet-derived growth factor receptor in mesangial cells.
We used patch-clamp methods to study the effect of platelet-derived growth factor (PDGF) on Ca2+ entry in cultured rat glomerular mesangial cells. In cell-attached patches, application of 50 ng/ml PDGF-BB inside, but not outside, the pipette frequently induced channel openings. The unitary conductance was 0.67 +/- 0.09 pS (n = 8) with 110 mM Mn2+ and 1.03 +/- 0.19 pS (n = 11) with 110 mM Ca2+ as the charge carrier. Number of channels times open probability was 0.515 +/- 0.144 (n = 14) with intrapipette PDGF and 0.037 +/- 0.022 (n = 12) without. Channel kinetics were only slightly voltage dependent. There was no effect of replacing chloride with gluconate in excised inside-out patches, showing that the channel was cation selective. The permeability (P) ratio for PMn/PNa was 1.65 and for PCa/PNa was 1.24. With the use of amphotericin B " perforated" whole cell patches, PDGF induced a small inward current (-16.1 +/- 4.33 pA; n = 11, membrane potential = -70 mV) consistent with 3,000-4,000 channels/cell. In summary, we have described a very-low-conductance Ca(2+)-permeable channel in rat mesangial cells with the following properties. 1) Activation by PDGF-BB occurs only when applied in close proximity to the channel. 2) Once activated, open probability is only slightly voltage dependent. 3) Under normal circumstances, the channel would probably appear to be cation nonselective, but with a permeability to divalent more than monovalent cations. 4) This PDGF-induced channel could provide a ligand-gated pathway for Ca2+ entry into mesangial cells that does not require membrane depolarization.
Animals↗
Effects of prostaglandin E2 on amiloride-blockable Na+ channels in a distal nephron cell line (A6).
We studied the mechanisms by which prostaglandin E2 (PGE2) regulates amiloride-blockable 4-pS Na+ channels in A6 distal nephron cells. With each apical cell-attached patch acting as its own control, acute (3-6 min) basolateral, but not apical, exposure to 1 microM PGE2 inhibited Na+ channel activity by decreasing the open probability (Po). This PGE2-induced inhibition was attenuated by 30 min pretreatment with the protein kinase C (PKC) antagonists 1 microM staurosporine or 100 microM D-sphingosine but was insensitive to pertussis toxin (PTX). Furthermore, the time course for channel inhibition by acute PGE2 correlated with a transient increase in intracellular inositol 1,4,5-trisphosphate (IP3) levels. In contrast, after chronic (10-50 min) exposure of A6 cells to 1 microM basolateral PGE2, channel activity was stimulated compared with controls. This stimulation was due to an increase in the number of apical Na+ channels, similar to the effect of maneuvers that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels in A6 cells (22). Indeed, chronic exposure to basolateral PGE2 correlated with a sustained increase in cAMP levels. In conclusion, 1) the regulation of apical 4-pS highly selective Na+ channel activity by basolateral PGE2 is a complicated biphasic process, which includes inhibition by acute PGE2 and stimulation by chronic PGE2 exposure; 2) acute PGE2 promotes a transient generation of IP3 which activates Ca(2+)-dependent PKC and promotes a decrease in Po; 3) chronic PGE2 promotes a sustained generation of cAMP that leads to an increase in channel density; and 4) both the acute and chronic effects of PGE2 on Na+ channels are PTX-insensitive processes.
Adenylyl Cyclases↗
Ketamine inhibition of large conductance Ca(2+)-activated K+ channels is modulated by intracellular Ca2+.
The present investigation was conducted to study the relationship between intracellular Ca2+ and inhibition of large conductance Ca(2+)-activated K+ (BK) currents by ketamine using excised patches from GH3 cells. Five ketamine concentrations were studied in the presence of six Ca2+ concentrations. The half-maximal inhibition for BK channel block by ketamine was increased from 4.1 +/- 0.7 microM at 0.1 microM intracellular Ca2+ to 230 +/- 74 microM at 100 microM intracellular Ca2+. Open probability (Po), Ca2+ concentration, and ketamine concentration data were best described by a competitive inhibition model. The inhibition constant for ketamine was 20.5 +/- 5.2 microM, and the Michaelis-Menten constant (Km) value for Ca2+ was 3.58 +/- 0.49 microM, which was not different from Km for Ca2+ in the absence of ketamine (3.33 +/- 0.37 microM). Taken alone, these data would suggest that Ca2+ and ketamine were competing for the same site on the channel protein. However, examination of open and closed interval data from patches containing only one channel show that ketamine primarily produces a decrease in the frequency of long-lived open events, suggesting that the effect of ketamine on BK channels may not be by a direct effect on channel proteins.
Calcium↗
Prostaglandin E2 activates clusters of apical Cl- channels in principal cells via a cyclic adenosine monophosphate-dependent pathway.
UNLABELLED: We examined cell-attached patches on principal cells of primary cultured, rabbit cortical collecting tubules. Under basal conditions, apical 9-pS Cl(-)-selective channels were observed in 9% of patches (11/126), and number of channels times open probability (NP0) was 0.56 +/- 0.21. The channel had a linear current-voltage relationship, reversal potential (Erev) near resting membrane potential, a P0 (0.30-0.70) that was independent of voltage, and complicated kinetics (i.e., bursting) at hyperpolarized potentials. NP0 and channel frequency were increased after 30 min of basolateral exposure to 0.5 microM PGE2 (18/56), 10 microM forskolin (23/36), or 0.5 mM dibutyryl cyclic adenosine monophosphate (cAMP) (25/41). Increases in NP0 appeared to be mediated primarily through an increase in the number of observed channels per patch (N), not changes in P0. After these cAMP-increasing maneuvers, N was inconsistent with a uniform distribution of channels in the apical membrane (P < 0.001), but rather the channels appeared to be clustered in pairs. Apical 0.5 microM PGE2 (12/91), apical or basolateral 0.5 microM PGF2 alpha (8/110), or 0.25 microM thapsigargin (releaser of intracellular Ca2+ stores) (7/73) did not increase NP0 or channel frequency. CONCLUSIONS: (a) 9-pS Cl- channels provide a conductive pathway for apical membrane Cl- transport across principal cells. (b) Channel activation by basolateral PGE2 is mediated via a cAMP-, but not a Ca(2+)-dependent mechanism. (c) Apical channels are clustered in pairs. (d) With its low baseline frequency and Erev near resting membrane potential, this channel would not contribute significantly to transcellular Cl- flux under basal conditions. (e) However, cAMP-producing agonists (i.e., PGE2, arginine vasopressin) would increase apical Cl- transport with the direction determined by the apical membrane potential.
Animals↗
Cyclosporin A inhibits apical secretory K+ channels in rabbit cortical collecting tubule principal cells.
UNLABELLED: We used the cell-attached patch clamp configuration to examine the effect of basolateral cyclosporin A (CsA) exposure on low conductance K+ channels found in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) primary cultures. Baseline K+ channel activity, measured as mean NPo (number of channels x open probability), was 2.7 +/- 1.1 (N = 29). NPo fell by 69% (0.84 +/- 0.32; N = 32) in cultures pretreated with 500 ng/ml CsA for 30 minutes prior to patching. Chelation of intracellular [Ca2+]i (10 mM BAPTA/AM; N = 8) or removal of extracellular Ca2+ (N = 9), but not prevention of [Ca2+]i store release (10 microM TMB-8; N = 7), abolished CsA-induced inhibition. This suggested that CsA effects were mediated by an initial rise in [Ca2+]i via Ca2+ influx. Either 25 nM AVP (N = 10) or 0.25 microM thapsigargin (N = 8) (causing IP3-dependent and -independent release of [Ca2+]i stores, respectively) augmented, while 25 pM (N = 6) or 250 pM AVP (N = 8) reversed CSA-induced channel inhibition. Apical membrane protein kinase C (PKC) activation with 0.1 microM phorbol ester, PMA (N = 8) or 10 microM synthetic diacylglycerol, OAG (N = 7), mimicked (mean NPo = 0.99 +/- 0.40) the inhibitory effect of CsA. Apical PKC inhibition by prolonged apical exposure to PMA (N = 10) or 100 microM D-sphingosine (N = 6) blocked CsA's effect. Cyclic AMP increasing maneuvers, 10 microM forskolin (N = 5) or 0.5 mM db-cAMP (N = 8), stimulated basal K+ channel activity in the absence of CsA. IN CONCLUSION: (1) basolateral exposure to CsA inhibits the activity of apical membrane 13 pS channels responsible for physiologic K+ secretion in rabbit CCT principal cells. (2) The inhibition is mediated by changes in intracellular Ca2+ and activation of apical PKC. (3) Pharmacologic AVP (nM) augments CsA-induced inhibition by releasing intracellular Ca2+ stores; more physiologic AVP (pM) attenuates channel inhibition, probably through cAMP generation. (4) Inhibition of apical secretory K+ channels by CsA likely contributes to decreased kaliuresis and clinical hyperkalemia observed in patients on CsA therapy.
Animals↗
Tyrosine kinase regulates epithelial sodium transport in A6 cells.
Insulin increases epithelial Na+ reabsorption, and many of its actions involve tyrosine kinase. We used tyrosine kinase inhibitors to examine the role of tyrosine kinase in the action of insulin. Pretreatment of Na+ transporting cells with tyrosine kinase inhibitors attenuates the subsequent action of insulin, suggesting that the action of insulin on epithelial Na+ transport involves tyrosine kinase activity. In addition to their effect on insulin-induced Na+ transport, the tyrosine kinase inhibitors also significantly reduce Na+ transport in Na(+)-transporting epithelial cells, suggesting that there is a significant tonic tyrosine kinase activity that modulates epithelial Na+ transport. Using patch-clamp methods, we found that one inhibitor, genistein, reduces the number of active Na+ channels in cell-attached patches without significantly affecting the open probability of any remaining channels. The effects of the tyrosine kinase inhibitors are not due to inhibition of protein kinase A (PKA), since H89, a PKA inhibitor, does not affect Na+ transport of control cells (as the tyrosine kinase inhibitors do), and the tyrosine kinase inhibitor, genistein or tyrphostin 23, does not alter the stimulation of ion transport by 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, a membrane-permeable adenosine 3',5'-cyclic monophosphate analogue (as H89 does).
Animals↗
G protein activation inhibits amiloride-blockable highly selective sodium channels in A6 cells.
Single-channel methods were used to examine the regulation of amiloride-blockable highly selective sodium channels by guanine nucleotide-binding proteins (G proteins). A6 cells (a renal cell line derived from Xenopus laevis kidney) were cultured on permeable collagen films, and patch recordings were made from the apical membranes of confluent cells. The predominant channel in the apical membranes is a highly selective, 4-pS, amiloride-blockable sodium channel (the Na(+)-to-K+ permeability ratio is > 30). In inside-out patches, application to the cytosolic surface of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), which deactivates G proteins, increased sodium channel activity. GDP beta S produced a sevenfold increase in channel activity. In contrast, GTP and guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) decreased sodium channel activity to about one-twentieth of the untreated value. The effect of GTP (but not GTP gamma S) was reversible. In cell-attached patches, a 3- to 4-h exposure of the apical membrane to pertussis toxin (PTX) increased the mean open time of sodium channels approximately 2.7 times and the open probability approximately 1.6-fold, but pretreatment of apical membranes with cholera toxin (250 ng/ml) for 3-4 h had no effect on open probability or mean open time. These results imply that a PTX-sensitive G protein regulates amiloride-blockable highly selective sodium channels in the apical membranes of A6 cells and that the G protein in a GTP-bound, activated state inhibits sodium channel activity.
Amiloride↗
Regulation of mesangial cell ion channels by insulin and angiotensin II. Possible role in diabetic glomerular hyperfiltration.
We used patch clamp methodology to investigate how glomerular mesangial cells (GMC) depolarize, thus stimulating voltage-dependent Ca2+ channels and GMC contraction. In rat GMC cultures grown in 100 mU/ml insulin, 12% of cell-attached patches contained a Ca(2+)-dependent, 4-picosiemens Cl- channel. Basal NPo (number of channels times open probability) was < 0.1 at resting membrane potential. Acute application of 1-100 nM angiotensin II (AII) or 0.25 microM thapsigargin (to release [Ca2+]i stores) increased NPo. In GMC grown without insulin, Cl- channels were rare (4%) and unresponsive to AII or thapsigargin in cell-attached patches, and less sensitive to [Ca2+]i in excised patches. GMC also contained 27-pS nonselective cation channels (NSCC) stimulated by AII, thapsigargin, or [Ca2+]i, but again only when insulin was present. In GMC grown without insulin, 15 min of insulin exposure increased NPo (insulin > or = 100 microU/ml) and restored AII and [Ca2+]i responsiveness (insulin > or = 1 microU/ml) to both Cl- and NSCC. GMC AII receptor binding studies showed a Bmax (binding sites) of 2.44 +/- 0.58 fmol/mg protein and a Kd (binding dissociation constant) of 3.02 +/- 2.01 nM in the absence of insulin. Bmax increased by 86% and Kd was unchanged after chronic (days) insulin exposure. In contrast, neither Kd nor Bmax was significantly affected by acute (15-min) exposure. Therefore, we concluded that: (a) rat GMC cultures contain Ca(2+)-dependent Cl- and NSCC, both stimulated by AII. (b) Cl- efflux and cation influx, respectively, would promote GMC depolarization, leading to voltage-dependent Ca2+ channel activation and GMC contraction. (c) Responsiveness of Cl- and NSCC to AII is dependent on insulin exposure; AII receptor density increases with chronic, but not acute insulin, and channel sensitivity to [Ca2+]i increases with both acute and chronic insulin. (d) Decreased GMC contractility may contribute to the glomerular hyperfiltration seen in insulinopenic or insulin-resistant diabetic patients.
Angiotensin II↗
Regulation of renal epithelial sodium channels.
The high selectivity, low conductance, amiloride-blockable, sodium channel of the mammalian distal nephron (i.e. cortical collecting tubule) is the site of discretionary regulation which allows maintainance of total body sodium balance. In order to understand the physiological events that participate in this regulation, we have used the patch-clamp technique which allows us to measure individual Na+ channel currents and permits access to the cytosolic side of the channel-protein as well as its associated regulatory components. Most of our experiments have utilized the A6 amphibian renal cell line, which when grown on permeable supports is an excellent model for the mammalian distal nephron. Different mechanisms have been examined: (1) regulation by hormonal factors such as Anti-Diuretic Hormone (ADH) and aldosterone, (2) regulation by G-proteins, (3) modulation by protein kinase C (PK-C), and (4) modulation by products of arachidonic acid metabolism. Consistent with noise analysis of tight epithelial tissues, ADH treatment increased the number of active channels in apical membrane patches of A6 cells, without any apparent change in the open probability (Po) of the individual channels. Agents that increased intracellular cAMP mimicked the effects of ADH. In contrast, aldosterone was found to act through a dramatic increase in Po rather than through changes in channel density. Inhibition of methylation by deazaadenosine antagonizes the stimulatory effect of aldosterone. In excised inside-out patches GTP gamma S inhibits channel activity, whereas GDP beta S or pertussis toxin stimulates activity suggesting regulatory control by G-proteins. PK-C has been shown to contribute to 'feed-back inhibition' of apical Na+ conductance in tight epithelia.(ABSTRACT TRUNCATED AT 250 WORDS)
Aldosterone↗
Role of sodium in mediator release from human basophils.
We studied the effect of extracellular sodium concentration on histamine release (HR) from human basophils initiated by immunologic and nonimmunologic stimuli. We found that lowering extracellular sodium markedly enhances HR induced by an immunologic stimulus from these cells. In buffer in which sodium had been replaced with univalent ions of strong bases, enhancement of HR increased as extracellular sodium decreased. Enhancement was the result of increased duration of release. When sucrose was used for replacement of sodium, we also observed that enhancement of HR increased as extracellular sodium decreased, but there was some lessening of enhancement at [Na+]e between 5 and 10 mmol/L. Ouabain, which is an inhibitor of the Na+/K+ adenosine triphosphatase, and bumetanide and furosemide, which are inhibitors of Cl(-)-dependent Na(+)-K+ cotransport, caused small increases in enhancement of HR by sodium-deficient buffers; 4,4'-diisothiocyanostilbene-2-2'-disulfonic acid, an anion transport inhibitor, caused some inhibition of enhancement of HR. Analogues of amiloride, such as 5-(N-N-hexamethylene) amiloride (HMA) and 5-(N-4-chlorobenzyl)-2'-4'dimethylbenzamil (CBDMB), inhibit Na+/H+ exchange, Na+/Ca++ exchange, and Na+ channels. Interestingly, at higher doses, HMA and CBDMB caused marked enhancement of HR in both normal and sodium-deficient buffers. These results suggest that several cellular regulatory mechanisms potentially are important for normal basophil secretion. The most likely are pH regulatory mechanisms that include Na+/H+ exchange and anion exchangers that transport alkaline equivalents. Our findings enhancement of basophil HR by HMA and CBDMB is particularly noteworthy in light of the recent interest in use of amiloride by inhalation for therapy of lung disease in patients with cystic fibrosis.
Basophils↗
Aldosterone alters the open probability of amiloride-blockable sodium channels in A6 epithelia.
We used patch-clamp methods to examine the effects of depletion and readdition of aldosterone on single, highly selective, amiloride-blockable sodium channels in the A6 cell line. Single-channel characteristics changed little before 24 h of continuous aldosterone depletion, although there was some reduction in short-circuit current. Thereafter, apical sodium permeability, measured as product of channel number per patch and individual channel open probability (NPo), was reduced between five- and sevenfold, primarily due to a large decrease in channel mean open time. With about the same time course, short-circuit current also decreased approximately fivefold. Readdition of aldosterone to depleted cells produced an increase in NPo within 2 h, primarily through an increase in mean open time. After readdition, channel number per patch increased twofold compared with cells not hormone deprived, with a return to control levels between 24 and 48 h after continuous exposure. The increase in short-circuit current followed a similar time course. The primary effect of aldosterone appears to be modulation of the open time of channels continuously present in the apical membrane, rather than promotion of the appearance or disappearance of channels from the membrane. In particular, it cannot be demonstrated statistically that aldosterone removal reduces the number of channels per patch, and there may actually be up to a twofold increase after a long period of aldosterone depletion.
Aldosterone↗
Chemical modification of Ca(2+)-activated potassium channels of GH3 anterior pituitary cells.
The effects of amino group specific reagents were examined on single, large-conductance, Ca(2+)-activated, K+ channels in excised membrane patches from GH3 cells. The reagents used include trinitrobenzene sulfonic acid, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and its 4-acetamido derivative, and sulfophenyl-isothiocyanate. These reagents react covalently with peptide terminal amino groups and the epsilon amino groups of lysine residues, thereby removing positive charge. Internal application of 0.1-1.0 mM reagent to inside-out patches irreversibly increases channel open probability. Single-channel conductance and voltage sensitivity are not affected by modification. Analysis of channel openings and closures shows that the increase in open probability is predominantly due to the loss of long-duration closures of the channel; however, the lengths of long-duration openings are increased. After the modification in the presence of Ca2+ was performed, the channel open probability remains large, regardless of the internal Ca2+ concentration. Transitions among several open and closed states of the modified channel are present in the absence of Ca2+, suggesting that many state transitions are not directly dependent on Ca2+ binding or dissociation.
Acetamides↗
Eicosanoids modulate apical Ca(2+)-dependent K+ channels in cultured rabbit principal cells.
Patch clamp technology was utilized to study the effects of apical phospholipase A2 (PLA2) metabolites on "maxi K" channels in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) primary cultures (B. N. Ling, C. F. Hinton, and D. C. Eaton. Kidney Int. 40: 441-452, 1991). At resting membrane potential, this channel is quiescent in the cell-attached configuration. Apical application of the PLA2 agonist melittin (1 microgram/ml) for 10 min increased single-channel open probability (Po) from 0.0004 +/- 0.0010 to 0.11 +/- 0.05. Similarly, apical exposure to 50 microM arachidonic acid (AA) or 0.5 microM prostaglandin (PG) E2, but not 0.5 microM PGF2 alpha, also increased channel activity. Conversely, 10 microM of the PLA2 antagonist quinacrine applied apically decreased Po. Removal of apical bath Ca2+ did not prevent melittin-, AA-, or PGE2-induced channel activation. We then examined the role of maxi K channels and eicosanoids in principal cell volume regulation. Within seconds of reducing basolateral bath tonicity (285 to 214 mosmol/kgH2O), NPo (i.e., no. of channels x Po) initially increased approximately 200%, followed by a delayed but prolonged activation phase that was attenuated by removal of apical bath Ca2+. Pretreatment with 10 microM quinacrine, 100 microM indomethacin (cyclooxygenase inhibitor), or 0.25 microM thapsigargin (to deplete intracellular Ca2+ stores) abolished the initial phase of swelling-induced channel activation.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗