Identification of three novel members of the calcium-dependent chloride channel (CaCC) family predominantly expressed in the digestive tract and trachea.

Three novel human sequences showing striking homology to the recently described bovine Ca2+/calmodulin kinase II-dependent epithelial chloride channel bCaCC have been identified in an expressed sequence tags database. Full-length clones were isolated using a 5' RACE approach. The encoded predicted proteins display 65% overall homology to bCaCC. Tissue expression patterns of the corresponding genes, designated as hCaCC-1, -2 and -3, appear to be highly restricted, with the first two genes primarily expressed in the digestive tract. Another original feature as compared to the CaCC family members is the fact that hCaCC-2 also shows expression in the brain. Taken together these findings demonstrate the existence of several CaCC-like genes in humans, some of which display distinct tissue specificity patterns within the CaCC subfamily of chloride channels.

Cloning and characterization of a novel human inwardly rectifying potassium channel predominantly expressed in small intestine.

A new member of the two transmembrane domain potassium (K+) channel family was identified and isolated from a human brain cDNA library. The cDNA clone contains an open reading frame which encodes a 360 amino acid sequence with a characteristic P domain flanked by two hydrophobic regions representing the membrane spanning segments. The closest homologue of this gene product is the inwardly rectifying potassium channel subunit, Kir1.2 (identity approximately 42%). Northern blot analysis of human tissues with a selective cDNA probe for this new K+ subunit showed a single major transcript of 3.4 kb predominantly expressed at high levels in small intestine, with lower levels in stomach, kidney and brain. The main regions of expression in the central nervous system were medulla, hippocampus and corpus callosum. cRNA-injected oocytes and transiently transfected HEK293 cells expressed a K+ conductance which displays an inward rectification. This conductance is blocked by cesium and barium but is insensitive to tolbutamide and diazoxide even upon co-transfection of this novel subunit with the plasmid encoding the sulfonylurea receptor SUR1. Taken together, these results demonstrate that we have isolated and characterized a novel K+ channel subunit belonging to the inwardly rectifying K+ (Kir) channel family to which, upon homology classification, we have given the nomenclature Kir7.1.

Human thrombopoietin structure-function relationships: identification of functionally important residues.

Thrombopoietin (TPO) is a haematopoietic growth factor responsible for megakaryocyte progenitor proliferation and differentiation. It belongs to the four-helix-bundle cytokine family and exerts its biological effects through binding to a specific receptor, c-Mpl. With the use of site-directed mutagenesis we have generated 20 TPO mutants. Each of the TPO mutants was produced in a eukaryotic expression system and the mutants' ability to induce the proliferation of factor-dependent c-Mpl-expressing megakaryoblastic M-O7e cells was compared with that of wild-type TPO. Among the mutations studied, 10 lead to a significant decrease in TPO bioactivity. Of these ten residues, three are located in helix A of the protein (Arg10, Lys14 and Arg17) and four in helix D (His133, Gln132, Lys138 and Phe141), indicating that in TPO, as in other cytokines, these two helices are important for functional cytokine/receptor interactions. Surprisingly, mutant Arg10-->Ala (R10A) lacked any proliferative activity, despite the fact that this mutation was recently reported to have no effect on TPO/c-Mpl binding in a TPO phage ELISA [Pearce, Potts, Presta, Bald, Fendly and Wells (1997) J. Biol. Chem. 272, 20595-20602]. The lack of M-O7e proliferation is probably due to an inability of R10A mutant to promote receptor dimerization and thus receptor activation. Moreover we found that the Arg10 and Arg17 residues of TPO seem to be specific determinants for TPO/c-Mpl recognition. We also demonstrate that the O-glycosylation site located at position 110 of TPO is not necessary for the bioactivity of the cytokine.

Pharmacological characterization of the cloned human 5-hydroxytryptamine transporter.

We performed an extensive pharmacological study of the 5-hydroxytryptamine (5-HT) transporter polypeptide cloned from human placenta. Transient expression of this 630 amino acid polypeptide in HeLa cells led to saturable 5-HT uptake activity (Km = 858 nM). This 5-HT uptake was blocked by selective 5-HT inhibitors, such as citalopram, litoxetine, sertraline, and indalpine, with Ki values in the low nanomolar range, and it exhibited a pharmacological profile similar to that found in rat brain. [3H]Citalopram binding to membrane preparations of the transfected cells occurred to a single class of high-affinity binding sites (Kd = 5.3 nM) and was potently inhibited by selective 5-HT uptake inhibitors. The pharmacological profile of [3H]citalopram binding to these transfected cells showed a good correlation with that of [3H]paroxetine binding to the rat cerebral cortical 5-HT transporter (r = 0.79). These data confirm that the full pharmacological characteristics of the 5-HT transport system are conferred by the expression of the 630 amino acid human placental 5-HT transporter polypeptide. [3H]Citalopram should, therefore, provide a useful probe for more insights at a molecular level into this cloned 5-HT transport system.

A Drosophila rotund transcript expressed during spermatogenesis and imaginal disc morphogenesis encodes a protein which is similar to human Rac GTPase-activating (racGAP) proteins.

The rotund (rn) locus of Drosophila melanogaster at cytogenetic position 84D3,4 has been isolated and cloned on the basis of the mutant phenotype: an absence of structures in the subdistal regions of the appendages. The shortened appendages are the consequence of a localized cell death in the imaginal discs, precursors of the adult appendages. Physical characterization of the rn locus has demonstrated that it is relatively large, occupying a minimum of 50 kb. There are two major transcripts of 1.7 kb (m1.7) and 5.3 kb (m5.3). We present here the sequence analysis of m1.7 and its putative product, rnprot1.7, and show that rnprot1.7 is similar to the product of the human n-chimaerin gene, which is expressed in brain and testes. Recently, the GAP activity of n-chimaerin was demonstrated and shown to be specific for the Rac subfamily of the Ras oncoproteins. The Rac proteins have been implicated in the regulation of secretory processes. In addition to being expressed in the imaginal discs, the m1.7 racGAP transcript was detected in developmentally specific germ line cells of the testes, the primary spermatocytes.

Two transcripts from the rotund region of Drosophila show similar positional specificities in imaginal disc tissues.

The rotund (rn) mutation in Drosophila is unique in that its phenotype is limited to the deletion of specific distal parts, though not the extremities, of all adult appendages. We have cloned the rn gene, located at cytogenetic position 84D3,4, by chromosomal walking. The functional rn unit, defined genetically by the localization of 13 noncomplementing rn alleles, covers approximately 50 kb of DNA. Despite different developmental profiles, two transcript size classes (1.7 and 5.3 kb) from this region show an indistinguishable pattern of spatial expression in the imaginal discs at the white pupa stage. There is a high correlation between the specificity of the mutant phenotype and the accumulation of transcripts in the presumptive distal regions of the cuticle-forming epithelial cells of the affected discs; it is, in fact, the first gene reported whose expression is localized with respect to the proximo distal-forming axis. For both transcripts, we have also found, uniquely in the wing disc, expression limited to the anterior region of the mesodermally derived epithelial cells, which contribute to the muscles of the thorax.