Biomedical subjects

I I Ismailov

Publications and source records attributed to I I Ismailov.

At least 37 records · Page 2Linked to original sources

PubMed1996

Regulation of epithelial sodium channels by short actin filaments.

Cytoskeletal elements play an important role in the regulation of ion transport in epithelia. We have studied the effects of actin filaments of different length on the alpha, beta, gamma-rENaC (rat epithelial Na+ channel) in planar lipid bilayers. We found the following. 1) Short actin filaments caused a 2-fold decrease in unitary conductance and a 2-fold increase in open probability (Po) of alpha,beta,gamma-rENaC. 2) alpha,beta,gamma-rENaC could be transiently activated by protein kinase A (PKA) plus ATP in the presence, but not in the absence, of actin. 3) ATP in the presence of actin was also able to induce a transitory activation of alpha, beta,gamma-rENaC, although with a shortened time course and with a lower magnitude of change in Po. 4) DNase I, an agent known to prohibit elongation of actin filaments, prevented activation of alpha,beta,gamma-rENaC by ATP or PKA plus ATP. 5) Cytochalasin D, added after rundown of alpha,beta,gamma-rENaC activity following ATP or PKA plus ATP treatment, produced a second transient activation of alpha,beta,gamma-rENaC. 6) Gelsolin, a protein that stabilizes polymerization of actin filaments at certain lengths, evoked a sustained activation of alpha,beta,gamma-rENaC at actin/gelsolin ratios of <32:1, with a maximal effect at an actin/gelsolin ratio of 2:1. These results suggest that short actin filaments activate alpha, beta,gamma-rENaC. PKA-mediated phosphorylation augments activation of this channel by decreasing the rate of elongation of actin filaments. These results are consistent with the hypothesis that cloned alpha,beta,gamma-rENaCs form a core conduction unit of epithelial Na+ channels and that interaction of these channels with other associated proteins, such as short actin filaments, confers regulation to channel activity.

Actins↗

PubMed1996

Regulation of epithelial sodium channels by the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis airway epithelia exhibit enhanced Na+ reabsorption in parallel with diminished Cl- secretion. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) directly affects epithelial Na+ channel activity by co-incorporating into planar lipid bilayers immunopurified bovine tracheal CFTR and either heterologously expressed rat epithelial Na+ channel ( alpha,b eta,gamma-rENaC) or an immunopurified bovine renal Na+ channel protein complex. The single channel open probability (Po) of rENaC was decreased by 24% in the presence of CFTR. Protein kinase A (PKA) plus ATP activated CFTR, but did not have any effect on rENaC. CFTR also decreased the extent of elevation of the renal Na+ channel Po following PKA-mediated phosphorylation. Moreover, the presence of CFTR prohibited the inward rectification of the gating of this renal Na+ channel normally induced by PKA-mediated phosphorylation, thus down-regulating inward Na+ current. This interaction between CFTR and Na+ channels occurs independently of whether or not wild-type CFTR is conducting anions. However, the nonconductive CFTR mutant, G551D CFTR, cannot substitute for the wild-type molecule. Our results indicate that CFTR can directly down-regulate single Na+ channel activity, thus accounting, at least in part, for the observed differences in Na+ transport between normal and cystic fibrosis-affected airway epithelia.

Adenosine Triphosphate↗

PubMed1996

G-protein regulation of outwardly rectified epithelial chloride channels incorporated into planar bilayer membranes.

Experiments were designed to test if immunopurified outwardly rectified chloride channels (ORCCs) and the cystic fibrosis transmembrane conductance regulator (CFTR) incorporated into planar lipid bilayers are regulated by G-proteins. pertussis toxin (PTX) (100 ng/ml) + NAD (1 mM) + ATP (1 mM) treatment of ORCC and CFTR in bilayers resulted in a 2-fold increase in single channel open probability (Po) of ORCC but not of CFTR. Neither PTX, NAD, nor ATP alone affected the biophysical properties of either channel. Further, PTX conferred a linearity to the ORCC current-voltage curve, with a slope conductance of 80 +/- 3 picosiemens (pS) in the +/- 100 mV range of holding potentials. PKA-mediated phosphorylation of these PTX + NAD-treated channels further increased the Po of the linear 80-pS channels from 0.66 +/- 0.05 to >0.9, and revealed the presence of a small (16 +/- 2 pS) linear channel in the membrane. PTX treatment of a CFTR-immunodepleted protein preparation incorporated into bilayer membranes resulted in a similar increase in the Po of the larger conductance channel and restored PKA-sensitivity that was lost after CFTR immunodepletion. The addition of guanosine 5'-3-O-(thio)triphosphate (100 mum) to the cytoplasmic bathing solutions decreased the activity of the ORCC and increased its rectification at both negative and positive voltages. ADP-ribosylation of immunopurified material revealed the presence of a 41-kDa protein. These results demonstrate copurification of a channel-associated G-protein that is involved in the regulation of ORCC function.

Adenosine Diphosphate Ribose↗

PubMed1996

Triple-barrel organization of ENaC, a cloned epithelial Na+ channel.

A cloned rat epithelial Na+ channel (rENaC) was studied in planar lipid bilayers. Two forms of the channel were examined: channels produced by the alpha subunit alone and those formed by alpha, beta, and gamma subunits. The protein was derived from two sources: either from in vitro translation reaction followed by Sephadex column purification or from heterologous expression in Xenopus oocytes and isolation of plasma membranes. We found that either alpha-rENaC alone or alpha- in combination with beta- and gamma-rENaC, produced highly Na(+)-selective (PNa/PK = 10), amiloride-sensitive (Kamili = 170 nM), and mechanosensitive cation channels in planar bilayers. alpha-rENaC displayed a complicated gating mechanism: there was a nearly constitutively open 13-picosiemens (pS) state and a second 40-pS level that was achieved from the 13-pS level by a 26-pS transition. alpha-, beta-, gamma-rENaC showed primarily the 13-pS level. alpha-rENaC and alpha,beta,gamma-rENaC channels studied by patch clamp displayed the same gating pattern, albeit with > 2-fold lowered conductance levels, i.e. 6 and 18 pS, respectively. Upon treatment of either channel with the sulfhydryl reducing agent dithiothreitol, both channels fluctuated among three independent 13-pS sublevels. Bathing each channel with a high salt solution (1.5 M NaCl) produced stochastic openings of 19 and 38 pS in magnitude between all three conductance levels. Different combinations of alpha-, beta-, and gamma-rENaC in the reconstitution mixture did not produce channels of intermediate conductance levels. These findings suggest that functional ENaC is composed of three identical conducting elements and that their gating is concerted.

Animals↗

PubMed1996

Associated proteins and renal epithelial Na+ channel function.

The hypothesis that amiloride-sensitive Na+ channel complexes immunopurified from bovine renal papillary collecting tubules contain, as their core conduction component, an ENaC subunit, was tested by functional and immunological criteria. Disulfide bond reduction with dithiothreitol (DTT) of renal Na+ channels incorporated into planar lipid bilayers caused a reduction of single channel conductance from 40 pS to 13 pS, and uncoupled PKA regulation of this channel. The cation permeability sequence, as assessed from bi-ionic reversal potential measurements, and apparent amiloride equilibrium dissociation constant (K(amil)i) of the Na+ channels were unaltered by DTT treatment. Like ENaC, the DTT treated renal channel became mechanosensitive, and displayed a substantial decrease in K(amil)i following stretch (0.44 +/- 0.12 microM versus 6.9 +/- 1.0 microM). Moreover, stretch activation induced a loss in the channel's ability to discriminate between monovalent cations, and even allowed Ca2+ to permeate. Polyclonal antibodies generated against a fusion protein of alpha bENaC recognized a 70 kDa polypeptide component of the renal Na+ channel complex. These data suggest that ENaC is present in the immunopurified renal Na+ channel protein complex, and that PKA sensitivity is conferred by other associated proteins.

Amiloride↗

PubMed1996

Diversity and regulation of amiloride-sensitive Na+ channels.

Amiloride-sensitive Na+ channels play a vital role in many important physiological processes such as delineation of the final urine composition, sensory transduction, and whole-body Na+ homeostasis. These channels display a wide range of biophysical properties, and are regulated by cAMP-mediated second messenger systems. The first of these channels has recently been cloned. This cloned amiloride-sensitive Na+ channel is termined ENaC (Epithelial Na+ Channel) and, in heterologous cellular expression systems, displays a single channel conductance of 4 to 7 pS, a high PNa/PK (> 10), a high amiloride sensitivity (Ki(amil) = 150 nM), and relatively long open and closed times. ENaC may form the core conduction element of many of these functionally diverse forms of Na+ channel. The kinetic and regulatory differences between these channels may be due, in large measure, to unique polypeptides that associate with the core element, forming a functional channel unit.

Amiloride↗

PubMed1996

Protein kinase regulation of a cloned epithelial Na+ channel.

We examined the regulation of a cloned epithelial Na+ channel (alpha beta gamma-rENaC) by protein kinase A (PKA) and protein kinase C (PKC). Experiments were performed in Xenopus oocytes and in planar lipid bilayers. At a holding potential of -100 mV, amiloride-sensitive current averaged -1,279 +/- 111 nA (n = 7) in alpha beta gamma-rENaC-expressing oocytes. Currents in water-injected oocytes were essentially unresponsive to 10 microM amiloride. A 1-h stimulation of PKC with 100 nM of PMA inhibited whole-cell currents in Xenopus oocytes to 17.1 +/- 1.8, and 22.1 +/- 2.6% of control (n = 7), at holding potentials of -100 and +40 mV, respectively. Direct injection of purified PKC resulted in similar inhibition to that observed with PMA. Additionally, the inactive phorbol ester, phorbol-12-myristate-13-acetate, 4-O-methyl, was without effect on alpha beta gamma-rENaC currents. Pretreatment with the microtubule inhibitor colchicine (100 microM) did not modify the inhibitory effect of PMA; however, pretreatment with 20 microM cytochalasin B decreased the inhibitory action of PMA to < 20% of that previously observed. In vitro-synthesized alpha beta gamma-rENaC formed an amiloride-sensitive Na(+)-selective channel when incorporated into planar lipid bilayers. Addition of PKC, diacyl-glycerol, and Mg-ATP to the side opposite that which amiloride blocked, decreased the channel's open probability (Po) from 0.44 +/- 0.06 to 0.13 +/- 0.03 (n = 9). To study the effects of PKA on alpha beta gamma-rENaC expressed in Xenopus oocytes, cAMP levels were elevated with 10 microM forskolin and 1 mM isobutyl-methyl-xanthine. This cAMP-elevating cocktail did not cause any stimulation of alpha beta gamma-rENaC currents in either the inward or outward directions. This lack of activation was also observed in oocytes preinhibited with PMA and in oocytes pretreated with cytochalasin B and PMA. Neither alpha-rENaC nor alpha beta gamma-rENaC incorporated into planar lipid bilayers could be activated with PKA and Mg-ATP added to either side of the membrane, as Po remained at 0.63 +/- 0.06 (n = 7) and 0.45 +/- 0.05 (n = 9), respectively. We conclude that: alpha beta gamma-rENaC is inhibited by PKC, and that alpha beta gamma-rENaC is not activated by PKA.

Animals↗

PubMed1996

Liddle's disease: abnormal regulation of amiloride-sensitive Na+ channels by beta-subunit mutation.

Liddle's disease is an autosomal dominant genetic disorder characterized by severe low renin hypertension ("pseudoaldosteronism") that has been genetically linked to a locus on chromosome 16 encoding the beta-subunit of an amiloride-sensitive Na+ channel (ASSC) (15). Peripheral blood lymphocytes (PBL) express ASSC that are functionally indistinguishable from those expressed by Na(+)-reabsorbing renal epithelial cells (3, 5). The amiloride-sensitive Na+ conductance in PBL from affected and unaffected individuals from the original Liddle's pedigree was examined using whole cell patch clamp. Typically, the basal Na+ currents in cells from affected individuals were maximally activated. Basal Na+ currents in cells from unaffected individuals were minimal and could be maximally activated by superfusion with 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Affected cells could not be further stimulated with CPT-cAMP. Superfusion with a supermaximal concentration of amiloride (2 microM) inhibited both the cAMP-activated Na+ conductance in unaffected cells and the constitutively activated inward conductance in affected cells. Cytosolic addition of a peptide identical to the terminal 10 amino acids of the truncated beta-subunit normalized the cAMP-mediated but not the pertussis toxin-induced regulation of the mutant ASSC. The findings show that lymphocyte ASSC are constitutively activated in affected individuals, that a mutation of the beta-subunit alters ASSC responsiveness to specific regulatory effectors, and that the cellular mechanism responsible for the pathophysiology of Liddle's disease is abnormal regulation of Na+ channel activity. These findings have important diagnostic and therapeutic implications and provide a cellular phenotype for the diagnosis of pseudoaldosteronism.

Amiloride↗

PubMed1996

Peptide block of constitutively activated Na+ channels in Liddle's disease.

Hypertension is a multifactorial disorder that results in an increased risk of cardiovascular and end-stage renal disease. Liddle's disease represents a specific hypertensive disease and expresses itself in the human population as an autosomal dominant trait. Recent experimental evidence indicates that patients with Liddle's disease have constitutively active amiloride-sensitive Na+ channels and that these channels are phenotypically expressed in lymphocytes obtained from normal and affected members of the original Liddle's kindred. Linkage analysis indicates that this disease results from a deletion of the carboxy-terminal region of the beta-subunit of a recently cloned epithelial Na+ channel (ENaC). We report the successful immunopurification and reconstitution of both normal and constitutively active lymphocyte Na+ channels into planar lipid bilayers. These channels display all of the characteristics typical of renal Na+ channels, including sensitivity to protein kinase A phosphorylation. We demonstrate that gating of normal Na+ channels is removed by cytoplasmic trypsin digestion and that the constitutively active Liddle's Na+ channels are blocked by a beta- or gamma-ENaC carboxy-terminal peptide in a GTP-dependent fashion.

Amiloride↗

PubMed1995

Cloning of an epithelial chloride channel from bovine trachea.

We have isolated and cloned a novel epithelial Cl- channel protein from a bovine tracheal cDNA expression library using an antibody probe. The antibody (alpha p38) was raised against a 38-kDa component of a homopolymeric protein that behaves as a Ca2+/calmodulin kinase II-, DIDS-, and dithiothreitol (DTT)-sensitive, anion-selective channel when incorporated into planar lipid bilayers. The full-length cDNA is 3001 base pairs long and codes for a 903-amino acid protein. The clone does not show any significant homology to any other previously reported Cl- channel sequence. Northern analysis of bovine tracheal mRNA with a cDNA probe corresponding to the cloned sequence revealed a band at 3.1 kilobases, suggesting that close to the full-length sequence has been cloned. The full-length open reading frame (2712 base pairs) has been expressed in Xenopus oocytes and in mammalian COS-7 cells. In oocytes, expression of the clone was associated with the appearance of a novel DIDS-, and DTT-sensitive, anion-selective conductance that was outwardly rectified and exhibited a reversal potential close to 0 mV. Whole-cell patch clamp studies in COS-7 cells transfected with the clone identified an ionomycin-, and DTT-sensitive chloride conductance that was not apparent in mock-transfected or control cells. In vitro translation studies have shown that the primary transcript codes for a protein migrating at 140 kDa under reduced conditions, significantly larger than the polypeptide recognized by alpha p38. We therefore suggest that either the 140-kDa translated product is a prepro form of the 38-kDa subunit of the previously identified bovine tracheal anion channel and that the primary transcript is post-translationally cleaved to yield the final product, or that the cloned channel and the previously identified bovine tracheal anion channel protein share an epitope that is recognized by the alpha p38 antibody.

Amino Acid Sequence↗

PubMed1995

Interaction between cystic fibrosis transmembrane conductance regulator and outwardly rectified chloride channels.

We have previously described a protocol for the simultaneous isolation and reconstitution of a protein kinase A (PKA)-sensitive outwardly rectified chloride channel (ORCC) and the cystic fibrosis transmembrane conductance regulator (CFTR) from bovine tracheal epithelium. Immunoprecipitation of CFTR from this preparation prevented PKA activation of the ORCC, suggesting that CFTR regulated the ORCC and that this regulatory relationship was preserved throughout the purification procedure. We now report the purification of CFTR from bovine tracheal epithelia and the purification of a CFTR conduction mutant (G551D CFTR) from retrovirally transduced mouse L cells using a combination of alkali stripping, Triton-X extraction, and immunoaffinity chromatography. Immunopurified CFTR proteins were reconstituted in the absence and presence of ORCC. To test the hypothesis that only functional CFTR can support activation of ORCC by PKA and ATP, we used an inhibitory anti-CFTR505-511 peptide antibody or G551D CFTR. When anti-CFTR505-511 peptide antibodies were present prior to the addition of PKA and ATP, activation of both the ORCC and CFTR was prevented. If the antibody was added after activation of the ORCC and CFTR Cl- channels by PKA and ATP, only the CFTR Cl- channel was inhibited. When ORCC and G551D CFTR were co-incorporated into planar bilayers, only the ORCC was recorded and this channel could not be further activated by the addition of PKA and ATP. Thus, functional CFTR is required for activation of the ORCC by PKA and ATP. We also tested the hypothesis that PKA activation of ORCC was dependent on the extracellular presence of ATP. We added ATP on the presumed extracellular side of the lipid bilayer under conditions where it was not possible to activate the ORCC, i.e. in the presence of inhibitory anti-CFTR505-511 antibody or G551D CFTR. In both cases the ORCC regained PKA sensitivity. Moreover, the addition of hexokinase + glucose to the extracellular side prevented activation of the ORCCs by PKA and ATP in the presence of CFTR. These experiments confirm that both the presence of CFTR as well as the presence of ATP on the extracellular side is required for activation of the ORCC by PKA and ATP.

Adenosine Triphosphate↗

PubMed1995

Cystic fibrosis transmembrane conductance regulator is required for protein kinase A activation of an outwardly rectified anion channel purified from bovine tracheal epithelia.

Our laboratory has developed a protocol for the isolation of a 140-kDa protein that forms an anion-selective channel when reconstituted into planar lipid bilayers. Polyclonal antibodies have been raised against the 38-kDa component of this purified protein. This channel has a linear current-voltage relationship and is not activated by protein kinase A (PKA) plus ATP. Using the same antibody and a modified purification protocol (eliminating the ion exchange chromatography steps), we isolated and reconstituted two other anion channels from tracheal membrane vesicles. In vitro phosphorylation of these isolated proteins by PKA and ATP revealed four bands migrating at 52, 85, 120, and 174 kDa. Immunoprecipitation experiments with anti-CFTR antibodies indicate that the 174-kDa phosphoprotein was CFTR. Upon incorporation of these isolated proteins into planar bilayers, an anion channel that exhibited a marked outward rectification in symmetrical Cl- solutions with a slope conductance of 82 pS at depolarizing voltages was observed. PKA and ATP increased channel activity but only from one side of the bilayer. However, channel activity was unaffected by addition of ATP alone from either side of the membrane. DIDS (100 microM) applied to the opposite side of the bilayer to which PKA and ATP act, blocked channel activity. A linear anion-selective channel with a conductance of 16 pS could be also resolved after inhibition of the outwardly rectified anion channel by DIDS in the presence of PKA and ATP. This small conductance channel was inhibited by 300 microM diphenylamine-2-carboxylic acid. Immunodepletion of the 174-kDa phosphoprotein from the preparation prevented activation of the 82-pS outwardly rectified anion channel by PKA and ATP. However, the PKA-dependent in vitro phosphorylation of the 52-, 85-, and 120-kDa phosphoproteins was unaffected by the absence of CFTR. Our results suggest a direct regulatory relationship between an outwardly rectified anion channel and CFTR.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1995

Structure and function of amiloride-sensitive Na+ channels.

A new molecular biological epoch in amiloride-sensitive Na+ channel physiology has begun. With the application of these new techniques, undoubtedly a plethora of new information and new questions will be forthcoming. First and foremost, however, is the question of how many discrete amiloride-sensitive Na+ channels exist. This question is important not only for elucidating structure-function relationships, but also for developing strategies for pharmacological or, ultimately, genetic intervention in such diseases as obstructive nephropathy, Liddle's syndrome, or salt-sensitive hypertension where amiloride-sensitive Na+ channel dysfunction has been implicated [17, 62]. Epithelia Na+ channels purified from kidney are multimeric. However, it is not yet clear which subunits are regulatory and which participate directly as a part of the Na+ conducting core and what is the nature of the gate. The combination of electrophysiologic techniques such as patch clamp and the ability to study reconstituted channels in planar lipid bilayers along with molecular biology techniques to potentially manipulate the individual subunits should provide the answers to questions that have puzzled physiologists for decades. It seems clear that the robust versatility of the channel in responding to a wide range of differing and potentially synergistic regulatory inputs must be a function of its multimeric structure and relation to the cytoskeleton. Multiple mechanisms of regulation imply multiple regulatory sites. This hypothesis has been validated by the demonstration that enzymatic carboxyl methylation and phosphorylation have both individual and synergistic effects on the purified channel in planar lipid bilayers. Of the multiple mechanisms proposed for channel regulation, evidence is now available to support the ideas that channels may be activated (or inactivated) by direct modifications including phosphorylation and carboxyl methylation, by activation or association of regulatory proteins such as G proteins, and by recruitment from subapical membrane domains. The observation that channel gating is achieved primarily through regulation of open probability without alterations in conductance may simplify future understanding of the molecular events involved in gating once the regulatory sites have been identified. As more Na+ channels or Na+ channel subunits are cloned from different epithelia, it will become possible to piece together the puzzle of epithelial Na+ channels. It is interesting to observe that renal Na+ channel proteins contain a subunit which falls into the 70 kD range. This size protein is in the range reported for the aldosterone-induced proteins [12, 46, 153].(ABSTRACT TRUNCATED AT 400 WORDS)

Amiloride↗

PubMed1995

Biochemical status of renal epithelial Na+ channels determines apparent channel conductance, ion selectivity, and amiloride sensitivity.

Purified bovine renal papillary Na+ channels, when reconstituted into planar lipid bilayers, reside in three conductance states: a 40-pS main state, and two subconductive states (12-13 pS and 24-26 pS). The activity of these channels is regulated by phosphorylation and by G-proteins. Protein kinase A (PKA)-induced phosphorylation increased channel activity by increasing the open state time constants from 160 +/- 30 (main conductance), and 15 +/- 5 ms (both lower conductances), respectively, to 365 +/- 30 ms for all of them. PKA phosphorylation also altered the closed time of the channel from 250 +/- 30 ms to 200 +/- 35 ms, thus shifting the channel into a lower-conductance, long open time mode. PKA phosphorylation increased the PNa:PK of the channel from 7:1 to 20:1, and shifted the amiloride inhibition curve to the right (apparent K(i)amil from 0.7 to 20 microM). Pertussis toxin-induced ADP-ribosylation of either phosphorylated of either phosphorylated or nonphosphorylated channels decreased the PNa:PK to 2:1 and 4:1, respectively, and altered K(i)amil to 8 and 2 microM for phosphorylated and nonphosphorylated channels, respectively. GTP-gamma-S treatment of either phosphorylated or nonphosphorylated channels resulted in an increase of PNa:PK to 30:1 and 10:1, respectively, and produced a leftward shift in the amiloride dose-response curve, altering K(i)amil to 0.5 and 0.1 microM, respectively. These results suggest that amiloride-sensitive renal Na+ channel biophysical characteristics are not static, but depend upon the biochemical state of the channel protein and/or its associated G-protein.

Amiloride↗

PubMed1995

Effects of phosphorylation on ion channel function.

A fundamental property of ion channels is their ability to be modulated by intracellular second messenger systems acting via covalent modifications of the channel protein itself. One such important biochemical reaction is phosphorylation on serine, threonine, and tyrosine residues. Ion channels in the kidney are no exception. Moreover, many ion channels, including many amiloride-sensitive epithelial Na+ channels, are subject to modulation by a multiplicity of inputs. For example, renal Na+ channels are not gated by voltage in their unphosphorylated state. However, upon phosphorylation by PKA plus ATP, these channels become voltage-dependent as well as having their open probability increased. Phosphorylation by PKC inhibits channel activity regardless of whether the channel was previously phosphorylated by PKA. Likewise, Na+ channel ADP-ribosylation by PTX overrides the actions of cAMP-dependent phosphorylation. Consistent with this idea is the fact that the phosphorylation sites for PKA and PKC and the ADP-ribosylation sites occur on different polypeptides comprising the channel complex. Epithelial Na+ channel activity is also regulated by methylation, arachidonic acid metabolites, and by interactions with cytoskeletal components. An exciting new age in understanding renal Na+ channel function has begun. Canessa and collaborators [103, 104] and Lingueglia et al [105] have, for the first time, identified by expression cloning an amiloride-sensitive Na+ channel from rat distal colon. The messenger RNA encoding the subunits comprising this channel are expressed in the distal tubule and cortical collecting tubule of the kidney (Rossier, unpublished observations). In addition, our laboratory has successfully cloned a mammalian homologue of this same channel from bovine renal papillary collecting ducts [106].(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗

PubMed1995

Regulation by Na+ and Ca2+ of renal epithelial Na+ channels reconstituted into planar lipid bilayers.

Purified bovine renal epithelial Na+ channels when reconstituted into planar lipid bilayers displayed a specific orientation when the membrane was clamped to -40 mV (cis-side) during incorporation. The trans-facing portion of the channel was extracellular (i.e., amiloride-sensitive), whereas the cis-facing side was intracellular (i.e., protein kinase A-sensitive). Single channels had a main state unitary conductance of 40 pS and displayed two subconductive states each of 12-13 pS, or one of 12-13 pS and the second of 24-26 pS. Elevation of the [Na+] gradient from the trans-side increased single-channel open probability (Po) only when the cis-side was bathed with a solution containing low [Na+] (< 30 mM) and 10-100 microM [Ca2+]. Under these conditions, Po saturated with increasing [Na+]trans. Buffering of the cis compartment [Ca2+] to nearly zero (< 1 nM) with 10 mM EGTA increased the initial level of channel activity (Po = 0.12 +/- 0.02 vs 0.02 +/- 0.01 in control), but markedly reduced the influence of both cis- and trans-[Na+] on Po. Elevating [Ca2+]cis at constant [Na+] resulted in inhibition of channel activity with an apparent [KiCa2+] of 10-100 microM. Protein kinase C-induced phosphorylation shifted the dependence of channel Po on [Ca2+]cis to 1-3 microM at stationary [Na+]. The direct modulation of single-channel Po by Na+ and Ca2+ demonstrates that the gating of amiloride-sensitive Na2+ channels is indeed dependent upon the specific ionic environment surrounding the channels.

Animals↗

PubMed1995

A cloned renal epithelial Na+ channel protein displays stretch activation in planar lipid bilayers.

We have previously cloned a bovine renal epithelial channel homologue (alpha-bENaC) belonging to the epithelial Na+ channel (ENaC) family. With the use of a rabbit nuclease-treated in vitro translation system, mRNA coding for alpha-bENaC was translated and the polypeptide products were reconstituted into liposomes. On incorporation into planar lipid bilayers, in vitro-translated alpha-bENaC protein 1) displayed voltage-independent Na+ channel activity with a single-channel conductance of 40 pS, 2) was mechanosensitive in that the single-channel open probability was maximally activated with a hydrostatic pressure gradient of 0.26 mmHg across the bilayer, 3) was blocked by low concentrations of amiloride [apparent inhibitory constant of amiloride (K(i)amil approximately 150 nM], and 4) was cation selective with a Li+:Na+:K+ permselectivity of 2:1:0.14 under nonstretched conditions. These pharmacological and selectivity characteristics were altered to a lower amiloride affinity (K(i)amil > 25 microM) and a lack of monovalent cation selectivity in the presence of a hydrostatic pressure gradient. This observation of stretch activation (SA) of alpha-bENaC was confirmed in dual electrode recordings of heterologously expressed alpha-bENaC whole cell currents in Xenopus oocytes swelled by the injection of 15 nl of a 100 mM KCl solution. We conclude that alpha-bENaC, and by analogy other ENaCs, represent a novel family of cloned SA channels.

Amiloride↗

PubMed1995

Immunopurification and functional reconstitution of a Na+ channel complex from rat lymphocytes.

Patch-clamp experiments have demonstrated an amiloride-sensitive Na+ conductance in human B lymphoid cells. We measured whole cell currents in rat lymphocytes and observed a similar Na(+)-specific inward conductance. The presence of 400 microM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate in the bath significantly increased the inward current, and this adenosine 3',5'-cyclic monophosphate activation was abolished by 2 microM amiloride. We immunopurified a protein complex from rat lymphocyte membranes using an anti-bovine kidney Na+ channel antibody. The complex consisted of five distinct polypeptides with apparent M(r) values of 110,000, 92,000, 59,000, 48,000, and 42,000. This putative channel complex was incorporated into planar lipid bilayers, where we observed single Na+ channel activity that was blocked by amiloride in a concentration-dependent manner. The addition of protein kinase A and ATP to the "intracellular" solution elicited a twofold increase in channel activity. Reverse transcription-polymerase chain reaction analysis was used to determine if the rat lymphocytes express the message for the recently cloned Na+ channel of the rat colon (rENaC). Primers for the alpha-subunit of rENaC identified no message in the lymphocyte RNA, while primers for the beta-subunit of the clone produced low levels of the expected product. Thus it appears that a rENaC-like beta-subunit may be an essential component of the lymphocyte Na+ channel that was isolated. At the same time, this channel is different from those recently cloned in that it does not include an alpha-subunit homologous to that of rENaC.

Animals↗