Different homologous subunits of the amiloride-sensitive Na+ channel are differently regulated by aldosterone.

Long term regulation of the amiloride-sensitive Na+ channel activity by steroid hormones occurs via de novo protein synthesis. The messenger level of RCNaCh1, previously shown by expression cloning to be a component of this channel, was measured in colons from rats fed with a low sodium diet. After 1 week of this diet, the channel activity was increased in an all-or-none fashion, whereas the level of RCNaCh1 messenger remained constant. A cDNA coding for another subunit of the Na+ channel was obtained by polymerase chain reaction. The 650-amino acid protein, entitled RCNaCh2, is 58% homologous to RCNaCh1 and displays a similar structure. It had no intrinsic activity when expressed alone in Xenopus oocytes, but its co-expression with RCNaCh1 increased the channel activity 18 +/- 5-fold. The increase in messenger level for RCNaCh2 during the time course of the diet is likely to explain the positive regulation of the rat colon Na+ channel by steroids. Immunocytochemical localization of the RCNaCh1 subunit revealed an apical labeling in colon from sodium-depleted rats. No labeling was observed in colon from control animals. These results suggest that oligomerization is needed for the proper expression of RCNaCh1 at the cell surface.

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to the GenBankmIEMBL Data Bank with accession number(s) X78034.
The nucleotide sequence(s) reported in this paper has been submitted ll To whom correspondence should be addressed. Tel.: 33-93-95-77-00; translation and is thus known to involve de novo RNA and protein synthesis (reviewed in Ref. 3). The rat colon Na' channel has been cloned recently (4,5). Expression of the cloned protein (699 amino acids) in Xenopus oocytes induces a Na+ current, which is inhibited by low concentrations of amiloride and analogs such as phenamil. The present paper reports the molecular cloning of a second channel subunit, with structural homology to the first one, and analyzes the expression of the two subunits during steroid treatment.
EXPERIMENTAL. PROCEDURES Short Circuit Current-Male Wistar rats were fed with a standard and/or a low sodium diet (ICN). The colon was removed and its most distal part mounted in an Ussing chamber. Both sides were perfused with a solution containing 120 m M NaCI, 5.9 m M KCI, 15.5 m~ NaHCO,, 1.2 m M NaH,PO,, 1.2 m M MgCl,, 2.6 m M CaCI,, 11.5 m M glucose. After a stabilizing period, transepithelial potential difference (in mV) and short circuit current (in pA/cm2) were measured with a dual voltage/ current clamp system (DVC1000, World Precision Instruments), before and after addition of 20 PM amiloride to the luminal side.
Polymerase Chain Reaction+DNA was synthesized from mRNA purified from distal colon of a dexamethasone-treated rat using a cDNA synthesis kit (Pharmacia LKB Biotechnology Inc.). Two degenerate oligonucleotides that correspond to sequences conserved between rat colon Na' channel, human lung Na' channel, Mec4, MeclO, and Degl were used as primers for PCR a sense primer matching amino acids 331-338 of RCNaChl(5'-GGIARYTGYTWYACI"YAAYSA-3') and an antisense primer matching amino acids 419-426 of RCNaChl (5'-CCRTYYTY-IGTRCAITCICCRTA-3'). PCR was carried out for 30 s at 94 "C, 90 s at 46 "C and 90 s at 72 "C for 35 cycles. Amplified products were digested with BstXI and separated on an agarose gel. A fragment of about 280 bp resistant to BstXI digestion was reamplified by PCR and digested by HhaI, and a resistant 286-bp fragment was cloned into a pBluescript vector for sequencing.
cDNA Library Synthesis and Screening-A size-selected directional cDNA library was synthesized from the rat colon mRNA of a dexamethasone-treated rat as previously described (5). The Escherichia coli strain SURE was transformed with the cDNA library by electroporation. 215,000 clones were plated. Hybridization was carried out with the 286-bp cDNA probe described above.
Characterization of the Clone-The cDNA insert was sequenced in both directions on 2947 nucleotides by dideoxy sequencing (6) using a dye terminator kit and automatic sequencing (Applied Biosystems model 373A Sequencer). Deletions for sequencing were prepared with the Erase-A-Base system from Promega.
Expression in Oocytes-Experiments were carried out as described in Ref. 5. In vitro transcription was performed using a Stratagene kit. Capped cRNAs of RCNaChl and RCNaCh2 (at 100 nglpl) were injected into oocytes (50 nuegg). The oocytes were tested by zzNa+ flux measurements with and without amiloride (10 p~) and by electrophysiology.

RESULTS AND DISCUSSION
The previously cloned Na' channel subunit is homologous with three proteins identified in the nematode Caenorhabditis elegans, called Mec-4, Mec-10 and Deg-1 (4,5,8), which are all expressed in mechanosensory neurons and are involved in mechanosensitive function (9). Their expression in the same cell type has suggested that they might be associated within a multimeric structure to form an ion channel (8), as observed for ligand-gated ionic channels (10)(11)(12)(13). Multimeric associations of homologous subunits have also been reported for other channel types (14,15). Sequence alignments between the cloned rat colon Na' channel (5) and human lung Na' channel (16), on the one hand, and Mec-4, Deg-l(9) and Mec-10; on the other hand, have revealed several regions of significant identity. Two of them, corresponding to amino acids 331-338 (GNCYTFND) and amino acids 419-426 (YGDCTENG), were used to design degenerate oligonucleotides. PCR amplification of rat colon cDNA gave one band of 280 bp. Analysis by restriction enzyme digestion revealed that this PCR product actually contained M. Chalfie, personal communication.
two distinct products of identical size. The first product corresponded to the previously cloned subunit, and the second displayed 57% homology with the first. The screening of a rat colon cDNA library led to the isolation of a cDNA containing a 1950nucleotide open reading frame. It corresponds to a protein of 650 amino acids, with a predicted molecular mass of 74 kDa (Fig. 1). The overall homology between the two rat colon proteins is 58%, and there is a structural conservation of both the two hydrophobic domains and the cysteine-rich region (Fig. 1). The terminology that will be subsequently used is as follows. The first clone will now be called RCNaChl and the new one RCNaCh2. Fig. 2 shows the results obtained by co-expression of the two subunits in Xenopus oocytes. Injection of the RCNaCh2 alone did not increase "Na+ uptake (5 ng of RNA injectedoocyte), while a small but significant amiloride-sensitive uptake was measured after expression of RCNaChl alone, as previously described (5). A large potentiation of the amiloride-sensitive "Na+ uptake was observed when the two subunits were coexpressed (Fig. 2a). The stimulation factor was 18 * 5 (mean 2 S.D.; n = 4). Similar results were observed by measuring the amiloride-sensitive Na' current under voltage-clamped conditions (Fig. 2b). The current observed after expression of RC-NaChl alone was 24 2 6 nA, while the current observed after co-expression of the two subunits was 295 38 nA. This intensity roughly corresponds to the signal obtained by injection of total messenger RNAs from a steroid-treated colon. Clearly, in oocytes, RCNaCh2 potentiates the channel activity of RC-NaChl. Fig. 3a shows the time dependence of expression of the two rat colon subunits during a low sodium diet, which raises plasma aldosterone concentration and thus increases Na' channel activity (17). After a switch to a low sodium diet, there was a time-dependent increase of the amiloride-sensitive short circuit current, which reached a steady state after 2 weeks (Fig.  3a). A parallel rise in aldosterone levels was observed (not shown). Level of messenger RNA for RCNaChl was already high in the control and remained nearly constant (Fig. 3b). In 5 out of 11 independent experiments, an increase in RCNaChl RNAlevel was observed after steroid treatment. Because variations in RCNaChl RNA level existed between colons with similar short circuit currents, it is likely that other factors might also regulate its expression. The mRNA for RCNaCh2 was hardly detectable in control animals, and its level steadily increased as the amiloride-sensitive short circuit current became higher (Fig. 3b). The positive regulation of the rat colon Na' channel activity by aldosterone can therefore be explained by the large increase in transcription of RCNaCh2.

Regulation of Nu' Channel Subunits
An immunohistochemical localization of the RCNaChl was performed with polyclonal antibodies, in order to assess the effect of diet on the protein level (Fig. 4). These antibodies raised against peptides of RCNaChl have been characterized previously (7). Normal and sodium-depleted animals displayed a striking difference in protein expression. No signal was detected in colon from control rats (Fig. &), whereas an apical labeling was detected with sodium-depleted animals expressing high levels of Na' channel activity (Fig. 4, c and e). Similar results were obtained with two other polyclonal sera raised against two distinct epitopes of RCNaChl (not shown) and with anti-peptide antibodies purified by affinity chromatography on a peptide column (Fig. 4e). Moreover, the labeling was inhibited by a previous incubation with an excess of immunogenic peptide (Fig. 4f) and was absent after incubation with the preimmune serum (Fig. 4, b and d ) . Only the borders were labeled, and no labeling was observed in the crypts (Fig. 4, c and e). The increase in RCNaChl transcription hardly explained the differences between control and sodium-depleted colon. It is therefore likely that the expression of RCNaChl on the cell surface is dependent of the co-expression of other subunit(s), as previously demonstrated for the nicotinic receptor (18); in that case, p, -y, and 6 subunits stabilize the a subunit, as measured by the binding of a-bungarotoxin (19). A similar model might be built for the Na' channel, which would explain its regulation by aldosterone. In control animals, the RCNaChl subunit would be synthesized but would be rapidly degraded in the absence of RCNaCh2. When the animals are exposed to higher aldosterone concentrations, for instance after sodium depletion, at least one other component, RCNaCh2, would be expressed, and this expression would permit the processing of RCNaChl to the apical membrane. These two distinct regulatory mechanisms (transcription and stabilization of the subunits) would permit large variations of Na' reabsorption to occur for Na+ homeostasis. The present article shows that, in colon, RCNaCh2 is one limiting factor for Na+ channel expression, whereas RCNaChl messenger seems to be constitutively expressed. Oligomerization of the different subunits appears to be an early event during the sorting of the channel complex, since RCNaChl is