Bakhram K. Berdiev

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 α, β, γ-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 α,β,γ-rENaC. 2) α,β,γ-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 α,β,γ-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 α,β,γ-rENaC by ATP or PKA plus ATP. 5) Cytochalasin D, added after rundown of α,β,γ-rENaC activity following ATP or PKA plus ATP treatment, produced a second transient activation of α,β,γ-rENaC. 6) Gelsolin, a protein that stabilizes polymerization of actin filaments at certain lengths, evoked a sustained activation of α,β,γ-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 α,β,γ-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 α,β,γ-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.

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 α subunit alone and those formed by α, β, and γ 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 α-rENaC alone or α- in combination with β- and γ-rENaC, produced highly Na+-selective (PNa/PK = 10), amiloride-sensitive (Kiamil = 170 nM), and mechanosensitive cation channels in planar bilayers. α-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. α-, β-, γ-rENaC showed primarily the 13-pS level. α-rENaC and α,β,γ-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 α-, β-, and γ-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.

Identification of an Amiloride Binding Domain within the α-Subunit of the Epithelial Na+ Channel

Limited information is available regarding domains within the epithelial Na+ channel (ENaC) which participate in amiloride binding. We previously utilized the anti-amiloride antibody (BA7.1) as a surrogate amiloride receptor to delineate amino acid residues that contact amiloride, and identified a putative amiloride binding domain WYRFHY (residues 278–283) within the extracellular domain of αrENaC. Mutations were generated to examine the role of this sequence in amiloride binding. Functional analyses of wild type (wt) and mutant αrENaCs were performed by cRNA expression in Xenopus oocytes and by reconstitution into planar lipid bilayers. Wild type αrENaC was inhibited by amiloride with aK i of 169 nm. Deletion of the entire WYRFHY tract (αrENaC Δ278–283) resulted in a loss of sensitivity of the channel to submicromolar concentrations of amiloride (K i = 26.5 μm). Similar results were obtained when either αrENaC or αrENaC Δ278–283 were co-expressed with wt β- and γrENaC (K i values of 155 nm and 22.8 μm, respectively). Moreover, αrENaC H282D was insensitive to submicromolar concentrations of amiloride (K i = 6.52 μm), whereas αrENaC H282R was inhibited by amiloride with a K i of 29 nm. These mutations do not alter ENaC Na+:K+ selectivity nor single-channel conductance. These data suggest that residues within the tract WYRFHY participate in amiloride binding. Our results, in conjunction with recent studies demonstrating that mutations within the membrane-spanning domains of αrENaC and mutations preceding the second membrane-spanning domains of α-, β-, and γrENaC alters amiloride’s K i , suggest that selected regions of the extracellular loop of αrENaC may be in close proximity to residues within the channel pore.

Intracellular H+ regulates the α-subunit of ENaC, the epithelial Na+ channel

Protons regulate electrogenic sodium absorption in a variety of epithelia, including the cortical collecting duct, frog skin, and urinary bladder. Recently, three subunits (α, β, γ) coding for the epithelial sodium channel (ENaC) were cloned. However, it is not known whether pH regulates Na+ channels directly by interacting with one of the three ENaC subunits or indirectly by interacting with a regulatory protein. As a first step to identifying the molecular mechanisms of proton-mediated regulation of apical membrane Na+ permeability in epithelia, we examined the effect of pH on the biophysical properties of ENaC. To this end, we expressed various combinations of α-, β-, and γ-subunits of ENaC in Xenopusoocytes and studied ENaC currents by the two-electrode voltage-clamp and patch-clamp techniques. In addition, the effect of pH on the α-ENaC subunit was examined in planar lipid bilayers. We report that α,β,γ-ENaC currents were regulated by changes in intracellular pH (pHi) but not by changes in extracellular pH (pHo). Acidification reduced and alkalization increased channel activity by a voltage-independent mechanism. Moreover, a reduction of pHi reduced single-channel open probability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited α,β-ENaC, α,γ-ENaC, and α-ENaC currents. We conclude that pHi but not pHo regulates ENaC and that the α-ENaC subunit is regulated directly by pHi.

Mechanosensitivity of an epithelial Na+ channel in planar lipid bilayers: release from Ca2+ block.

A family of novel epithelial Na+ channels (ENaCs) have recently been cloned from several different tissues. Three homologous subunits (alpha, beta, gamma-ENaCs) from the core conductive unit of Na(+)-selective, amiloride-sensitive channels that are found in epithelia. We here report the results of a study assessing the regulation of alpha,beta,gamma-rENaC by Ca2+ in planar lipid bilayers. Buffering of the bilayer bathing solutions to [Ca2+] < 1 nM increased single-channel open probability by fivefold. Further investigation of this phenomenon revealed that Ca2+ ions produced a voltage-dependent block, affecting open probability but not the unitary conductance of ENaC. Imposing a hydrostatic pressure gradient across bilayers containing alpha,beta,gamma-rENaC markedly reduced the sensitivity of these channels to inhibition by [Ca2+]. Conversely, in the nominal absence of Ca2+, the channels lost their sensitivity to mechanical stimulation. These results suggest that the previously observed mechanical activation of ENaCs reflects a release of the channels from block by Ca2+.

Expression and regulation of normal and polymorphic epithelial sodium channel by human lymphocytes.

Gene expression, protein expression, and function of amiloride-sensitive sodium channels were examined in human lymphocytes from normal individuals and individuals with Liddles disease. Using reverse transcriptase polymerase chain reactions, expression of all three cloned epithelial sodium channel (ENaC) subunits was detected in lymphocytes. Polyclonal antibodies to bovine α-ENaC bound to the plasma membrane of normal and Liddles lymphocytes. A quantitative analysis of fluorescence-tagged ENaC antibodies indicated a 2.5-fold greater surface binding of the antibodies to Liddles lymphocytes compared with normal lymphocytes. The relative binding intensity increased significantly (25%;p < 0.001) for both normal and Liddles cells after treatment with 40 μm 8-CPT-cAMP. Amiloride-sensitive whole cell currents were recorded under basal and cAMP-treated conditions for both cell types. Liddles cells had a 4.5-fold larger inward sodium conductance compared with normal cells. A specific 25% increase in the inward sodium current was observed in normal cells in response to cAMP treatment. Outside-out patches from both cell types under both treatment conditions revealed no obvious differences in the single channel conductance. The P open was 4.2 ± 3.9% for patches from non-Liddles cells, and 27.7 ± 5.4% in patches from Liddles lymphocytes. Biochemical purification of a protein complex, using the same antibodies used for the immunohistochemistry, yielded a functional sodium channel complex that was inhibited by amiloride when reconstituted into lipid vesicles and incorporated into planar lipid bilayers. These four independent methodologies yielded findings consistent with the hypotheses that human lymphocytes express functional, regulatable ENaC and that the mutation responsible for Liddles disease induces excessive channel expression.

Point mutations in alpha bENaC regulate channel gating, ion selectivity, and sensitivity to amiloride

We have generated two site-directed mutants, K504E and K515E, in the alpha subunit of an amiloride-sensitive bovine epithelial Na+ channel, alpha bENaC. The region in which these mutations lie is in the large extracellular loop immediately before the second membrane-spanning domain (M2) of the protein. We have found that when membrane vesicles prepared from Xenopus oocytes expressing either K504E or K515E alpha bENaC are incorporated into planar lipid bilayers, the gating pattern, cation selectivity, and amiloride sensitivity of the resultant channel are all altered as compared to the wild-type protein. The mutated channels exhibit either a reduction or a complete lack of its characteristic burst-type behavior, significantly reduced Na+:K+ selectivity, and an approximately 10-fold decrease in the apparent inhibitory equilibrium dissociation constant (Ki) for amiloride. Single-channel conductance for Na+ was not affected by either mutation. On the other hand, both K504E and K515E alpha bENaC mutants were significantly more permeable to K+, as compared to wild type. These observations identify a lysine-rich region between amino acid residues 495 and 516 of alpha bENaC as being important to the regulation of fundamental channel properties.

A truncated CFTR protein rescues endogenous ΔF508-CFTR and corrects chloride transport in mice

Cystic fibrosis (CF) is most frequently associated with deletion of phenylalanine at position 508 (∆F508) in the CF transmembrane conductance regulator (CFTR) protein. The ∆F508‐CFTR mutant protein exhibits a folding defect that affects its processing and impairs chloride‐channel function. This study aimed to determine whether CFTR fragments approximately half the size of wild‐type CFTR and complementary to the portion of CFTR bearing the mutation can specifically rescue the processing of endogenous ∆F508‐CFTR in vivo. cDNA encoding CFTR fragments were delivered to human airway epithelial cells and mice harboring endogenous ∆F508‐CFTR. Delivery of small CFTR fragments, which do not act as chloride channels by themselves, rescue ∆F508‐CFTR Therefore, we can speculate that the presence of the CFTR fragment, which does not harbor a mutation, might facilitate intermolecular interactions. The rescue of CFTR was evident by the restoration of chloride transport in human CFBE41o‐ bronchial epithelial cells expressing ∆F508‐CFTR in vitro. More important, nasal administration of an adenovirus expressing a complementary CFTR fragment restored some degree of CFTR activity in the nasal airways of ∆F508 homozy‐gous mice in vivo. These findings identify complementary protein fragments as a viable in vivo approach for correcting disease‐causing misfolding of plasma membrane proteins.—Cormet‐Boyaka, E., Hong, J. S., Ber‐diev, B. K., Fortenberry, J. A., Rennolds, J., Clancy, J. P., Benos, D. J., Boyaka, P. N., Sorscher, E. J. A truncated CFTR protein rescues endogenous ∆F508‐CFTR and corrects chloride transport in mice. FASEB J. 23, 3743‐3751 (2009). www.fasebj.org

Purification and reconstitution of an outwardly rectified Cl-channel from tracheal epithelia

We reported the identification of three outwardly rectified Cl- channel (ORCC) candidate proteins (115, 85, and 52 kDa) from bovine tracheal epithelia. We have raised polyclonal antibodies against these isolated proteins. Incorporation into planar lipid bilayers of material partly purified from bovine tracheal apical membranes with one of these antibodies as a ligand (anti-p115) resulted in the incorporation of an ORCC identical in biophysical characteristics to one we previously described. We developed a new purification procedure to increase the yield and purity of this polypeptide. The purification scheme that gave the best results in terms of overall protein yield and purity was a combination of anion- and cation-exchange chromatography followed by immunopurification. By use of this purification scheme, 7 μg of the 115-kDa protein were purified from 20 mg of tracheal apical membrane proteins. Incorporation of this highly purified material into planar lipid bilayers revealed a DIDS-inhibitable channel with the following properties: linear conductance of 87 ± 9 pS in symmetrical Cl- solutions, halide selectivity sequence of I-> Cl- > Br-, and lack of sensitivity to protein kinase A, Ca2+, or dithiothreitol. Using anti-Gαiantibodies to precipitate Gαiprotein(s) from the partly purified preparations, we demonstrated that the loss of rectification of the ORCC was due to uncoupling of Gαi protein(s) from the ORCC protein and that the 115-kDa polypeptide is an ORCC.

Chapter 3 Subunit Stoichiometry of Heterooligomeric and Homooligomeric Epithelial Sodium Channels

Publisher Summary The chapter presents a study on subunit stoichiometry of hetero-oligomeric and homo-oligomeric epithelial sodium channels. Epithelial Na + channels (ENaCs) have a key role in the regulation of urinary Na + reabsorption, extracellular fluid volume homeostasis, and blood pressure, and are a major site of action of volume regulatory hormones. ENaCs are composed of three homologous subunits, termed α, β, and γ . One of the fundamental challenges pertaining to the structure of the Na + channel is the determination of its subunit stoichiometry. To determine the subunit stoichiometry of ENaC, a biophysical assay utilizing mutant subunits is used that display significant differences in sensitivity to channel blockers from the wild-type channel. The results suggest that ENaCs are a tetrameric channel, with a α 2 βγ stoichiometry. The α -subunit itself is sufficient to form functional channels. The results suggest that similar to heterooligomeric αβγ Na + channels, homooligomeric α -subunit Na + channels are also tetrameric in structure. The chapter presents an example of a biophysical approach, considering the situation in which an ion channel is formed by a single polypeptide “X,” it is assumed that the channel has been cloned, can be functionally expressed in Xenopus oocytes , and is inhibited by the compound “Y.”