Aims/hypothesis We sought to determine the mRNA transcriptome of all major human pancreatic endocrine and exocrine cell subtypes, including human alpha, beta, duct and acinar cells. In addition, we identified the cell type-specific distribution of transcription factors, signalling ligands and their receptors. Methods Islet samples from healthy human donors were enzymatically dispersed to single cells and labelled with cell type-specific surface-reactive antibodies. Live endocrine and exocrine cell subpopulations were isolated by FACS and gene expression analyses were performed using microarray analysis and quantitative RT-PCR. Computational tools were used to evaluate receptor–ligand representation in these populations. Results Analysis of the transcriptomes of alpha, beta, large duct, small duct and acinar cells revealed previously unrecognised gene expression patterns in these cell types, including transcriptional regulators HOPX and HDAC9 in the human beta cell population. The abundance of some regulatory proteins was different from that reported in mouse tissue. For example, v-maf musculoaponeurotic fibrosarcoma oncogene homologue B (avian) (MAFB) was detected at equal levels in adult human alpha and beta cells, but is absent from adult mouse beta cells. Analysis of ligand–receptor interactions suggested that EPH receptor–ephrin communication between exocrine and endocrine cells contributes to pancreatic function. Conclusions/interpretation This is the first comprehensive analysis of the transcriptomes of human exocrine and endocrine pancreatic cell types—including beta cells—and provides a useful resource for diabetes research. In addition, paracrine signalling pathways within the pancreas are shown. These results will help guide efforts to specify human beta cell fate by embryonic stem cell or induced pluripotent stem cell differentiation or genetic reprogramming.

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 ␣rENaC. Mutations were generated to examine the role of this sequence in amiloride binding. Functional analyses of wild type (wt) and mutant ␣rENaCs were performed by cRNA expression in Xenopus oocytes and by reconstitution into planar lipid bilayers. Wild type ␣rENaC was inhibited by amiloride with a K i of 169 nM. Deletion of the entire WYRFHY tract (␣rENaC ⌬278 -283) resulted in a loss of sensitivity of the channel to submicromolar concentrations of amiloride (K i ‫؍‬ 26.5 M). Similar results were obtained when either ␣rENaC or ␣rENaC ⌬278 -283 were co-expressed with wt ␤-and ␥rENaC (K i values of 155 nM and 22.8 M, respectively). Moreover, ␣rENaC H282D was insensitive to submicromolar concentrations of amiloride (K i ‫؍‬ 6.52 M), whereas ␣rENaC 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 ␣rENaC and mutations preceding the second membrane-spanning domains of ␣-, ␤-, and ␥rENaC alters amiloride's K i , suggest that selected regions of the extracellular loop of ␣rENaC may be in close proximity to residues within the channel pore.The diuretic amiloride is a prototypic inhibitor of epithelial Na ϩ channels (ENaCs) 1 (1), although amiloride and its various derivatives inhibit many Na ϩ -selective transport proteins. Several laboratories have recently identified domains within the epithelial Na ϩ channel and the Na ϩ /H ϩ exchanger that appear to participate in amiloride binding. Residues within the second membrane-spanning domain of ␣rENaC may interact with amiloride, as mutations of a serine residue at position 589 result in a large decrease of the apparent K i for amiloride and the amiloride analog benzamil, as well as alter cation selectivity (2). Selected mutations of residues within a hydrophobic region, termed H2 (3), immediately preceding the second membrane-spanning domains of the ␣-, ␤-, and ␥-subunits of rENaC (i.e. Trp-␣582, Ser-␣583, Gly-␤525, Gly-␥537) and the ␣-subunit of bovine ENaC (Lys-504, Lys-515) affect the K i for amiloride, and several of these mutations affect single-channel conductance (4, 5). Snyder and co-workers have identified splice variants of ␣rENaC in which the C-terminal 199 or 216 amino acid residues, including the second membrane-spanning domain, are truncated (6). These splice variants are not functional when expressed in Xenopus oocytes, but retain amiloride and phenamil binding activity, s...

Mutations in a Cl- channel (cystic fibrosis transmembrane conductance regulator or CFTR) are responsible for the cystic fibrosis (CF) phenotype. Increased Na+ transport rates are observed in CF airway epithelium, and recent studies suggest that this is due to an increase in Na+ channel open probability (Po). The Xenopus renal epithelial cell line, A6, expresses both cAMP-activated 8-picosiemen (pS) Cl- channels and amiloride-sensitive 4-pS Na+ channels, and provides a model system for examining the interactions of CFTR and epithelial Na+ channels. A6 cells express CFTR mRNA, as demonstrated by reverse transcriptase-polymerase chain reaction and partial sequence analysis. A phosphorothioate antisense oligonucleotide, complementary to the 5' end of the open reading frame of Xenopus CFTR, was used to inhibit functional expression of CFTR in A6 cells. Parallel studies utilized the corresponding sense oligonucleotide as a control. CFTR protein expression was markedly reduced in cells incubated with the antisense oligonucleotide. Incubation of A6 cells with the antisense oligonucleotide led to inhibition of forskolin-activated amiloride-insensitive short circuit current (Isc). After a 30-min exposure to 10 microM forskolin, 8-pS Cl- channel activity was detected in only 1 of 31 (3%) cell-attached patches on cells treated with antisense oligonucleotide, compared to 5 of 19 (26%) patches from control cells. A shift in the single-channel current-voltage relationship derived from antisense-treated cells was also consistent with a reduction in Cl- reabsorption. Both amiloride-sensitive Isc and Na+ channel Po were significantly increased in antisense-treated, forskolin-stimulated A6 cells, when compared with forskolin-stimulated controls. These data suggest that the regulation of Na+ channels by CFTR is not limited to respiratory epithelia and to epithelial cells in culture overexpressing CFTR and epithelial Na+ channels.

.-One of the defining characteristics of the epithelial sodium channel (ENaC) is its block by the diuretic amiloride. This study investigates the role of the extracellular loop of the ␣-subunit of ENaC in amiloride binding and stabilization. Mutations were generated in a region of the extracellular loop, residues 278-283. Deletion of this region, WYRFHY, resulted in a loss of amiloride binding to the channel. Channels formed from wild-type ␣-subunits or ␣-subunits containing point mutations in this region were examined and compared at the single-channel level. The open probabilities (P o) of wild-type channels were distributed into two populations: one with a high P o and one with a low P o. The mean open times of all the mutant channels were shorter than the mean open time of the wild-type (high-P o) channel. Besides mutations Y279A and H282D, which had amiloride binding affinities similar to that of wild-type ␣-ENaC, all other mutations in this region caused changes in the amiloride binding affinity of the channels compared with the wild-type channel. These data provide new insight into the relative position of the extracellular loop with respect to the pore of ENaC and its role in amiloride binding and channel gating. open probability; extracellular loop; channel pore; sodium channel; single-channel recording EPITHELIAL SODIUM CHANNELS (ENaC) play a critical role in the control of blood pressure and regulation of total body sodium balance. Although the original work in which the channel components were cloned suggested that functional channels consist of three subunits, ␣, ␤, and ␥ (5), under appropriate circumstances, ␣-subunits alone can form sodium-permeable channels (12,13,17). It has been proposed that some of the diversity in conductance, gating, and selectivity of functional ENaC may be due to different subunit combinations. The expression of ␣-subunits alone, rather than the expression of all three subunits together, depends on the environmental conditions to which renal epithelial cells (A6) (7) or alveolar type II cells (14) are exposed. However, regardless of which channel type is expressed, one of the defining characteristics of all these channels is their block by the diuretic amiloride, a substituted pyrazinoylguanidine. The tertiary structure of all the subunits is similar: each subunit is predicted to span the membrane twice, to have a large extracellular loop, and to have short intracellular NH 2 and COOH termini (2, 6). Because amiloride blocks the channel from the extracellular surface of the channel protein, it is possible that one or more of the extracellular loops could play a role in amiloride's interaction with and block of the channel by stabilizing amiloride in the channel pore.About 70% of each ENaC subunit is extracellular. Structure-function studies of the extracellular loop have focused primarily on the region immediately preceding and including the second transmembrane (M2) domain of ␣-ENaC. These data have implicated these regions in binding to amiloride and also play a role in...

We previously raised an antibody (RA6.3) by an antiidiotypic approach which was designed to be directed against an amiloride binding domain on the epithelial Na ؉ channel (ENaC). This antibody mimicked amiloride in that it inhibited transepithelial Na ؉ transport across A6 cell monolayers. RA6.3 recognized a 72-kDa polypeptide in A6 epithelia treated with tunicamycin, consistent with the size of nonglycosylated Xenopus laevis ␣ENaC. RA6.3 specifically recognized an amiloride binding domain within the ␣-subunit of mouse and bovine ENaC. The deduced amino acid sequence of RA6.3 was used to generate a three-dimensional model structure of the antibody. The combining site of RA6.3 was epitope mapped using a novel computer-based strategy. Organic residues that potentially interact with the RA6.3 combining site were identified by data base screening using the program LUDI. Selected residues docked to the antibody in a manner corresponding to the ordered linear array of amino acid residues within an amiloride binding domain on the ␣-subunit of ENaC. A synthetic peptide spanning this domain inhibited the binding of RA6.3 to ␣ENaC. This analysis provided a novel approach to develop models of antibody-antigen interaction as well as a molecular perspective of RA6.3 binding to an amiloride binding domain within ␣ENaC.Epithelial Na ϩ channels (ENaCs) 1 are expressed in a variety of tissues, including the distal nephron of the kidney, airway and alveolar epithelia in the lung, surface cells in the distal colon, urinary bladder epithelia, skin, and ducts within salivary and sweat glands (1). These transporters facilitate the movement of Na ϩ across the apical (or luminal) plasma membrane and have a critical role in extracellular fluid volume homeostasis, control of blood pressure, fetal lung maturation, and maintenance of airway fluids (1). ENaCs consist of at least three homologous subunits, termed ␣-, ␤-, and ␥ENaC, and are thought to form a tetrameric complex consisting of 2␣-, 1␤-, and 1␥-subunits (2-5), although one group has suggested a subunit stoichiometry of 3␣-, 3␤-, and 3␥-subunits (6). cDNAs encoding these Na ϩ channel subunits have been isolated and characterized from a variety of species. Each ENaC subunit has two predicted membrane-spanning domains. The amino-and carboxyl-terminal regions of the ENaCs are cytoplasmic, and each subunit has a large ectodomain (7-9). Domains of largely hydrophobic residues are located immediately following the putative first membrane-spanning domains and immediately preceding the second membrane-spanning domains of ␣-, ␤-and ␥rENaC (8, 10, 11). The hydrophobic domain immediately preceding the second membrane-spanning region of ENaC (referred to as the H2 domain) may insert in the membrane and form part of the channel pore (3,7,12).The diuretic drug amiloride is a prototypic inhibitor of epithelial Na ϩ channels. Several sites have been identified within the epithelial Na ϩ channel which participate in amiloride binding. Mutagenesis studies suggest that residues preceding the second ...