©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Functional Expression of a Novel Amiloride-sensitive Na Channel (*)

(Received for publication, September 11, 1995; and in revised form, September 25, 1995)

Rainer Waldmann Guy Champigny Frédéric Bassilana Nicolas Voilley Michel Lazdunski (§)

From the Institut de Pharmacologie Moléculaire et Cellulaire, Sophia Antipolis, 06560 Valbonne, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have isolated a cDNA for a novel human amiloride-sensitive Na channel isoform (called ) which is expressed mainly in brain, pancreas, testis, and ovary. When expressed in Xenopus oocytes, it generates an amiloride-sensitive Na channel with biophysical and pharmacological properties distinct from those of the epithelial Na channel, a multimeric assembly of alpha, beta, and subunits. The Na current produced by the new isoform is increased by two orders of magnitude after coexpression of the beta and subunit of the epithelial Na channel showing that can associate with other subunits and is part of a novel multisubunit ion channel.


INTRODUCTION

Amiloride-sensitive sodium channels (ASCs) (^1)are Na-permeable non-voltage-sensitive ion channels inhibited by the diuretic amiloride. They are abundant and well characterized in epithelial tissues such as kidney, colon, and lung (for review, see (1) ) where they control the rate and extent of Na reabsorption under the regulation of steroid hormones(2, 3, 4, 5) . The same ASCs also seem to play an important role in taste perception(6) . Different mammalian forms of ASCs with different biophysical (conductances, selectivity) and pharmacological properties (sensitivity to amiloride and derivatives) (for review, see (7) ) have been characterized recently in thyroid(8) , smooth muscle(9) , and vascular endothelial cells from brain(10) . In addition, amiloride-blockable nonselective ion channels are also important for mechanotransduction(11) . Molecular cloning of the highly Na-selective epithelial Na channel has demonstrated recently that it is made of at least three homologous subunits called alpha, beta, and (3, 12, 13, 14, 15) . Each of these subunits has homologies with the degenerins of the nematode Caenorhabditis elegans(16, 17, 18, 19) , which after certain mutations cause neurodegeneration. These degenerins are thought to be ion channels(17, 18, 22) .

It seems likely that the epithelial amiloride-sensitive Na channel is the first cloned member of a new family of ion channels which probably includes mammalian homologues of the C. elegans degenerins which might be involved in certain forms of neurodegeneration. This paper reports the molecular cloning and functional expression of a novel human isoform of an amiloride-sensitive Na channel expressed in brain, testis, ovary, and pancreas.


MATERIALS AND METHODS

PCR Amplifications Library Construction and Screening

cDNA was synthesized from human kidney poly(A) RNA (Clontech) using an oligo(dT) primer with a XhoI restriction site following the protocol supplied with the superscript reverse transcriptase (Life Technologies, Inc.), and a human kidney cDNA library in ZAP (Stratagene) was prepared following standard procedures. A fragment corresponding to nucleotides 42-265 of the expressed sequence tag (GenBank accession number T19320) was amplified by PCR, subcloned into Bluescript SK(-) (Stratagene), sequenced, and used to screen the library using standard techniques. Screening of 5 times 10^5 phages yielded one positive clone of 3.4 kb. For sequencing, deletions were prepared using the Erase-a-Base System (Promega) and sequenced on an Applied Biosystems automatic sequencer. The open reading frame and flanking sequences were sequenced on both strands.

Construction of an Oocyte Expression Vector

An oocyte expression vector (pBSK-SP6-globin) was constructed as follows. The noncoding sequences from Xenopus globin preceded by an SP6 promoter and flanking an EcoRI and XhoI site were amplified from the pEXO vector (3) by PCR using ATTTAGGTGACACTATAGAAGCTCAGA and a M13 reverse primer. The PCR product was digested with PstI and ligated into EcoRV/PstI-cut Bluescript SK(+) (Stratagene). The resulting vector was double-digested with ApaI and HindIII and blunt end-religated to remove the XhoI site in the Bluescript vector. In order to remove the long (1.2-kb) 5`-noncoding sequences of NaCh containing various start and stop codons, the cDNA was amplified using the Pwo polymerase (Boehringer) with a primer (CAGAATTCCTGCCCCCGCAATGGC) positioned on the first ATG codon (underlined) of the open reading frame and a primer complementary to the T7 promoter. The PCR product was digested with EcoRI and XhoI and ligated into EcoRI/XhoI-digested pBSK-SP6-globin vector.

Expression in Xenopus Oocytes

cRNA was prepared using SP6 or T7 RNA polymerase and the NotI-digested vector as template, and oocytes were injected with 5 to 15 pg of alphaNaCh or NaCh alone or together with 5-15 pg of one or several of the other subunits essentially as described(23) . Whole cell recordings were carried out essentially as described(23) . Cell-attached recordings were performed on oocytes clamped to 0 mV in high K medium. Pipettes contained (in mM): NaCl (or LiCl), 140; MgCl(2), 1; CaCl(2), 1; Hepes, 10, pH 7.4. Data were sampled at 1 kHz and filtered at 300 Hz for analysis (Biopatch software, Biologic). Single-channel conductances were calculated from i - V relationships from 0 mV to -100 mV.

Northern Blots

Human multi-tissue Northern blots containing about 2 µg of poly(A) RNA normalized for identical beta-actin expression in each lane were purchased from Clontech and hybridized with a random prime-labeled KspI fragment (bases 238-737) located just after the first transmembrane region. The blots were hybridized overnight at 65 °C in 5 times SSC, 10 times Denhardt's solution, 0.1% SDS, 100 µg/ml herring sperm DNA, washed with 0.1 times SSC, 0.1% SDS at 70 °C, and subsequently exposed to Kodak X-Omat AR film for 14 days at -70 °C. The apparent molecular weight of the NaCh RNA was calculated using the mobilities of a 0.24-9.5-kb RNA ladder (Life Technologies, Inc.).

All nucleic acid positions in the text refer to positions relative to the A in the ATG initiation codon of the nucleic acid sequence submitted to GenBank (accession number U38254).

All comparisons of sequences with data bases were done using the Blast network server at the NCBI (National Center for Biotechnology Information).


RESULTS AND DISCUSSION

In order to identify novel homologues of the epithelial Na channel (NaCh), the sequences of the cloned subunits (alphaNaCh, betaNaCh, NaCh) have been compared with the data base of expressed sequence tags. We found one good matching partial cDNA sequence (GenBank accession number T19320) in this data base. A fragment of this sequence was amplified by PCR from human kidney cDNA and used to screen a human kidney cDNA library. A positive clone of 3.4 kb was isolated and sequenced. It contains an open reading frame of 1914 bases preceded by stop codons in all three reading frames and codes for a protein of 638 amino acids (Fig. 1a).


Figure 1: Protein sequence of NaCh and comparison with alphaNaCh, betaNaCh, and NaCh. a, alignment of NaCh with human alphaNaCh, betaNaCh, and NaCh. Residues identical or similar to the corresponding amino acid in the subunit are printed white on black or black on gray background, respectively. The putative transmembrane regions for NaCh are labeled with bars. For MII, the hydrophobic region is longer than the some 20 amino acids required for an alpha helix to span the membrane. The sequence which was shown to participate in the formation of the ionic pore of alphaNaCh (22) is marked by a black bar, and flanking hydrophobic regions by gray bars. The sequence for alphaNaCh is from GenBank (accession number X76180), and those for betaNaCh and NaCh are from EMBL (accession numbers X877159 and X87160). The sequences were aligned using the GCG Pileup program. b, phylogenetic tree of the human NaCh subunits. The phylogenetic tree was established from the alignment shown in a using the Distances program (GCG) with Kimura substitution followed by the Growtree program (GCG) with the UPGMA option. c, identity between the cloned human NaCh subunits and the C. elegans degenerins mec10 and deg1. The sequences were aligned using the GCG Pileup program, and identities were calculated with the GCG Distances program without correction for multiple substitutions. The sequences for the degenerins deg1 and mec10 used are GenBank accession numbers L34414 and L25312, respectively.



The homology with the alpha, beta, and subunit (27-37% identity; Fig. 1c) is rather low and lies in the same range as observed between alphaNaCh, betaNaCh, and NaCh (29-36% identity; Fig. 1c). Nevertheless, the homology and phylogenetic analysis (Fig. 1b) places this new isoform, named NaCh, closer to the alpha subunit than to betaNaCh and NaCh. The NaCh is, as alphaNaCh, betaNaCh, and NaCh, about 20% identical with the degenerins mec10 and deg1 of C. elegans (Fig. 1c).

NaCh has a hydrophobicity profile similar to alphaNaCh, betaNaCh, and NaCh and to the degenerins with two hydrophobic regions (MI and MII, Fig. 1a) long enough to span the plasma membrane. Together with the sequence homologies, this suggests a transmembrane topology identical with that proposed for alphaNaCh (20) with intracellular amino and carboxyl termini and a large cysteine-rich extracellular loop between MI and MII.

When expressed alone in Xenopus oocytes, NaCh induced a small (38 ± 5 nA, n = 16) but very reproducible amiloride-sensitive Na current (Fig. 2) with macroscopic properties (pharmacology, selectivity) clearly distinct from those of alphaNaCh when expressed in the same conditions(13) .


Figure 2: Electrophysiology of NaCh. a, effect of amiloride on the whole cell current recorded at -70 mV on oocytes injected with NaCh. b, selectivity of NaCh. The bars represent the amiloride (100 µM)-sensitive current. c, dose response curves for amiloride and benzamil. Each point represents the mean of the values obtained from 5 oocytes. d, mean i - V relationship of the amiloride (100 µM)-sensitive Na current with 96 mM Na or Li in the external medium. Points are the mean values from 4 oocytes.



The first difference concerns the pharmacology. K(0.5) values (Fig. 2c) for the diuretic amiloride (2.6 µM) and for benzamil (0.27 µM) were about 30 times higher than those for alphaNaCh (K = 80 nM; K = 7 nM). The second difference was the ionic selectivity. The NaCh channel was more permeable for Na than for Li (I/I= 0.6) unlike the human alphaNaCh or rat alphaNaCh which have a higher permeability for Li than for Na (I/I 2) (Fig. 2b). NaCh was insensitive to ethylisopropylamiloride (Fig. 3b), a potent inhibitor of the Na/H exchanger(21) , at concentrations below 10 µM.


Figure 3: Electrophysiology of betaNaCh. a, amiloride (100 µM)-sensitive Na currents in oocytes injected with NaCh alone or together with human alpha, beta, and/or NaCh. b, dose-response curves of betaNaCh for amiloride, benzamil, and ethylisopropylamiloride (EIPA). c, cell-attached recordings on betaNaCh-injected oocytes at different membrane potentials with 140 mM Na in the pipette solution. d, mean i - V relationships with 140 mM Na or Li in the pipette solution.



Like alphaNaCh, NaCh is virtually impermeable for K. No amiloride-sensitive current could be detected when Na was substituted by K (Fig. 2b), and the i - V curve shows a positive reversal potential (+49 ± 7 mV, n = 5) (Fig. 2d).

Since the epithelial Na channel is known to be a multisubunit assembly and since alphaNaCh alone also induces only small currents when expressed in Xenopus oocytes without betaNaCh and NaCh(3, 12, 13, 14, 15) , we examined whether any of the other known human subunits (alpha, beta, or ) increases the NaCh current (Fig. 3a). Unlike alphaNaCh for which coexpression of just the subunit increases the current by one order of magnitude(3, 14) , none of the alpha, beta, or subunits alone altered or increased the NaCh current when coexpressed with NaCh. However, coexpression of both the beta and subunits with the NaCh increased the Na current by 50-fold (1.94 ± 0.4 µA, n = 7), an amplification that lies in the same range as that reported after coexpression of alphaNaCh with betaNaCh and NaCh (14) .

The macroscopic properties (pharmacology, ionic selectivity) of betaNaCh were indistinguishable from those of NaCh. Together with the fact that the macroscopic properties of alphaNaCh are also not altered by coexpression of betaNaCh and NaCh(3, 14) , this suggests either that alphaNaCh and NaCh are the pore-forming subunits or that low amounts of endogenous betaNaCh- and NaCh-like subunits are present in the oocyte and are responsible for the small currents observed after expression of alphaNaCh or NaCh alone.

The single-channel conductance (Fig. 3) for Na of betaNaCh was 11.6 ± 0.4 pS (n = 8). It was clearly different from that of alphabetaNaCh (4.8 ± 0.3 pS, n = 6). The betaNaCh conductance for Li (6.8 ± 0.5 pS, n = 4) was nearly identical with that of alphabetaNaCh (7.3 ± 0.2 pS, n = 4). The betaNaCh channel, like alphabetaNaCh, was highly selective for Naversus K (pNa/pK > 50). The gating of betaNaCh was slow ( = 3.3 ± 1.5 s, = 1.9 ± 0.7 s, n = 5; Fig. 3b), and the open probability did not show a marked voltage dependence (P(o) = 0.46 ± 0.05 at -20 mV and 0.49 ± 0.02 at -100 mV, n = 3).

It is particularly interesting that, despite their low homology (37% identity), both alphaNaCh and NaCh can associate with betaNaCh and NaCh to form a functional channel. Sequence comparisons between alphaNaCh and NaCh (Fig. 1) reveal some motifs that are well conserved and which are not found in betaNaCh and NaCh. Those ``common'' alpha and sequences, and particularly the sequence just before MI (Fig. 1a), are probably important elements for the functional association of alphaNaCh or NaCh with betaNaCh and NaCh.

The tissue distribution of NaCh mRNA was analyzed by Northern blot (Fig. 4). The highest expression levels of the 5.5-kDa mRNA were found in testis, ovary, pancreas, and brain. Those are tissues in which to our knowledge no amiloride-sensitive Na channels have been described yet. Small amounts of NaCh-mRNA can be detected in all other tissues examined except in spleen and small intestine. The dominant tissue distribution of NaCh is clearly nonepithelial, and, in kidney (the tissue we cloned NaCh from), there are only small amounts of NaCh-mRNA present. Therefore, it does not seem likely that the principal role of this new channel is to be searched in epithelia.


Figure 4: Tissue distribution. The tissue distribution of NaCh-mRNA was analyzed by Northern blot as described under ``Materials and Methods.''



The pharmacological and biophysical properties of NaCh do not really match those of any of the amiloride-sensitive Na channels described so far. Like alphaNaCh, the major subunit of the epithelial Na channel, NaCh, can associate with betaNaCh and NaCh to form a multisubunit ion channel. Whether this beta subunit combination is the one actually present in vivo or whether yet unknown subunits form a channel together with NaCh requires further investigation.

The presence of NaCh in brain is particularly interesting, because the C. elegans degenerins (16, 17, 18, 19) which are homologues to NaCh are expressed in neurons. A more detailed localization of NaCh especially in brain might clarify the physiological role of this new amiloride-sensitive Na channel.


FOOTNOTES

*
This work was supported by CNRS and the Association Française contre les Myopathies (AFM). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institut de Pharmacologie Moléculaire et Cellulaire, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France. Tel.: 33-93-95-77-00; Fax: 33-93-95-77-04; douy@unice.fr.

(^1)
The abbreviations used are: ASC, amiloride-sensitive Na channel; NaCh, epithelial amiloride-sensitive Na channel; alphaNaCh, betaNaCh, NaCh, and NaCh, epithelial Na channel alpha, beta, , and subunits, respectively; kb, kilobase(s); pS, picosiemens; GCG, Genetics Computer Group, Wisconsin package version 8.


ACKNOWLEDGEMENTS

We are very grateful to Drs. Jacques Barhanin, Marc Borsotto, and Eric Lingueglia for helpful discussions. We thank M. Bordes and F. Aguila for technical assistance and A. Douy for secretarial assistance. Thanks are due to Bristol-Myers Squibb Co. for an ``Unrestricted Award.''


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.