AG340 Sigma-AldrichEpithelial Sodium Channel γ, control peptide for AB3534P

The pathophysiological basis of Liddle's syndrome, a rare autosomal dominant form of arterial hypertension, has been found to rest on missense mutations or truncations of the beta- and gamma-subunits of the epithelial sodium channel. The hypothesis has been advanced that molecular variants of these genes might also contribute to the common polygenic forms of hypertension. We tested this hypothesis by performing a cosegregation study in a reciprocal cross between the stroke-prone spontaneously hypertensive rat (SHRSPHD) and a Wistar-Kyoto rat (WKY-1HD) reference strain. We carried out genetic mapping and chromosomal assignment of the alpha-, beta-, and gamma-subunits of the epithelial sodium channel using both linkage analysis and fluorescent in situ hybridization techniques. We demonstrate that in the rat, the beta- and gamma-subunits, as in humans, are in close linkage; they map to rat chromosome 1 and cosegregate with systolic pressure after dietary NaCl (logarithm of the odds [LOD] score, 3.7), although the peak LOD score of 5.0 for this quantitative trait locus was detected 4.4 cM away from the beta-/gamma-subunit locus. The alpha-subunit was mapped to chromosome 4 and exhibited no linkage to blood pressure phenotype. Comparative analysis of the complete coding sequences of all three subunits in the SHRSPHD and WKY-1HD strains revealed no biologically relevant mutations. Furthermore, Northern blot comparison of mRNA levels for all three subunits in the kidney showed no differences between SHRSPHD and WKY-1HD. Our results fail to support a material contribution of the epithelial sodium channel genes to blood pressure regulation in this model of polygenic hypertension.

The amiloride-sensitive epithelial sodium channel (ENaC) complex is made up of at least three different subunits alpha, beta, and gamma, which are developmentally regulated, selectively expressed, and variously up-regulated by steroid hormones. To understand mechanisms involved in regulation of the gamma subunit, we have determined the structure of the human gammaENaC gene. By 5' rapid amplification of cDNA ends, primer extension analysis, and nuclease protection assay, we identified transcription start sites in human brain, kidney, and lung. A human genomic library was screened and overlapping cosmid clones that span approximately 50 kilobases and contain the hgammaENaC gene were identified. The 5'-untranslated region is 141 bases long, and the translation start codon is contained within the second exon. The human gene spans at least 35 kilobases. The 5' end of the gene including portions of 5' flanking genomic DNA and the first intron are G + C rich and contain several CpG dinucleotides, consistent with a CpG island. The 5' flanking region contains no CCAAT or TATA-like elements but does contain two GC boxes as well as several putative transcription factor binding sites including AP-2, Sp1, CRE, PEA-3, and NF-IL6. This is the first description of the structural organization and the 5' flanking region of a member of the epithelial sodium channel complex.

Amiloride-sensitive Na+ channels are an important component of the Na+ reabsorption pathway in a number of epithelia. Here we report the cloning and characterization of cDNAs encoding two subunits of the human kidney epithelial Na+ channel (beta- and gamma-hENaC). Their predicted amino acid sequences were highly homologous (83-85% identical) to the corresponding subunits reported from rat colon (beta- and gamma-rENaC). Both beta- and gamma-hENaC mapped to human chromosome 16. Northern blot analysis showed high expression of beta- and gamma-hENaC in kidney and lung and differential expression of the three subunits in other tissues. Coexpression of beta- and gamma-hENaC with alpha-hENaC in Xenopus oocytes produced Na+ channels with high selectivity for Na+ and high sensitivity to amiloride. In addition, human subunits were able to substitute for the corresponding rat subunits in forming functional Na+ channels, suggesting conservation of function and suggesting that differences in sequence do not disrupt interactions between subunits. These results suggest that human alpha-, beta-, and gamma-ENaC together form Na+ channels with properties that are similar to those observed in epithelia, and will allow further investigation into the role that these channels may play in human disease.

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.

The epithelial amiloride-sensitive sodium channel constitutes the rate limiting step for sodium reabsorbtion by the epithelial lining the distal part of the kidney tubule, the urinary bladder and the distal colon. Reabsorbtion of sodium through this channel, which is regulated by hormones such as aldosterone and vasopressin, is one of the essential mechanisms involved in the regulation of sodium balance, blood volume and blood pressure. Here we isolate a DNA from epithelial cells of rat distal colon and identify it by functional expression of an amiloride-sensitive sodium current in Xenopus oocyte. The deduced polypeptide (698 amino acids) has at least two putative transmembrane segments. Expression of this protein in Xenopus oocytes reconstitutes the functional properties of the highly selective amiloride-sensitive, epithelial sodium channel. The gene encoding this rat sodium channel subunit shares significant sequence similarity with mec-4 and deg-1, members of a family of Caenorhabditis elegans genes involved in sensory touch transduction and, when mutated, neuronal degeneration. We propose that the gene products of these three genes are members of a gene family coding for cation channels.