Malia M. Collins

The ClC-3 Cl-/H+ Antiporter Becomes Uncoupled at Low Extracellular pH

Adenovirus expressing ClC-3 (Ad-ClC-3) induces Cl−/H+ antiport current (IClC-3) in HEK293 cells. The outward rectification and time dependence of IClC-3 closely resemble an endogenous HEK293 cell acid-activated Cl− current (IClacid) seen at extracellular pH ≤ 5.5. IClacid was present in smooth muscle cells from wild-type but not ClC-3 null mice. We therefore sought to determine whether these currents were related. IClacid was larger in cells expressing Ad-ClC-3. Protons shifted the reversal potential (Erev) of IClC-3 between pH 8.2 and 6.2, but not pH 6.2 and 5.2, suggesting that Cl− and H+ transport become uncoupled at low pH. At pH 4.0 Erev was completely Cl− dependent (55.8 ± 2.3 mV/decade). Several findings linked ClC-3 with native IClacid; 1) RNA interference directed at ClC-3 message reduced native IClacid; 2) removal of the extracellular “fast gate” (E224A) produced large currents that were pH-insensitive; and 3) wild-type IClC-3 and IClacid were both inhibited by (2-sulfonatoethyl)methanethiosulfonate (MTSES; 10–500 μm)-induced alkanethiolation at exposed cysteine residues. However, a ClC-3 mutant lacking four extracellular cysteine residues (C103_P130del) was completely resistant to MTSES. C103_P130del currents were still acid-activated, but could be distinguished from wild-type IClC-3 and from native IClacid by a much slower response to low pH. Thus, ClC-3 currents are activated by protons and ClC-3 protein may account for native IClacid. Low pH uncouples Cl−/H+ transport so that at pH 4.0 ClC-3 behaves as an anion-selective channel. These findings have important implications for the biology of Cl−/H+ antiporters and perhaps for pH regulation in highly acidic intracellular compartments.

Linkage of congenital isolated adrenocorticotropic hormone deficiency to the corticotropin releasing hormone locus using simple sequence repeat polymorphisms

Genetic screening techniques using simple sequence repeat polymorphisms were applied to investigate the molecular nature of congenital isolated adrenocorticotropic hormone (ACTH) deficiency. We hypothesize that this rare cause of hypocortisolism shared by a brother and sister with two unaffected sibs and unaffected parents is inherited as an autosomal recessive single gene mutation. Genes involved in the hypothalamic-pituitary axis controlling cortisol sufficiency were investigated for a causal role in this disorder. Southern blotting showed no detectable mutations of the gene encoding pro-opiomelanocortin (POMC), the ACTH precursor. Other candidate genes subsequently considered were those encoding neuroendocrine convertase-1, and neuroendocrine convertase-2 (NEC-1, NEC-2), and corticotropin releasing hormone (CRH). Tests for linkage were performed using polymorphic di- and tetranucleotide simple sequence repeat markers flanking the reported map locations for POMC, NEC-1, NEC-2, and CRH. The chromosomal haplotypes determined by the markers flanking the loci for POMC, NEC-1, and NEC-2 were not compatible with linkage. However, 22 individual markers defining the chromosomal haplotypes flanking CRH were compatible with linkage of the disorder to the immediate area of this gene on chromosome 8. Based on these data, we hypothesize that the ACTH deficiency in this family is due to an abnormality of CRH gene structure or expression. These results illustrate the useful application of high density genetic maps constructed with simple sequence repeat markers for inclusion/exclusion studies of candidate genes in even very small nuclear families segregating for unusual phenotypes.

Constitutive human steroid 21-hydroxylase promoter gene and pseudogene activity in steroidogenic and nonsteroidogenic cells with the luciferase gene as a reporter

This study was directed toward initial comparison and characterization of the activities of the human steroid 21-hydroxylase gene (CYP21) and pseudogene (CYP21P) promoters. DNA fragments containing the promoter regions of CYP21 and CYP21P were amplified and cloned into promoterless luciferase reporter plasmids either containing or lacking an enhancer element. Cells of the nonsteroidogenic COS-1 cell line, and the steroidogenic Y-1 cell line were transiently transfected with these recombinant plasmids and a beta-galactosidase cotransfection control plasmid. Cellular lysates were analyzed for luciferase and beta-galactosidase activities. In the nonsteroidogenic system, transfectants with either the CYP21 or CYP21P upstream sequence in enhancer containing plasmids showed a 2.3 fold increase (p < .001) in light production over controls. In the steroidogenic Y-1 cell system, these same CYp21 and CYP21P transfectants showed a 14.3 (+/- 0.8) and 5.2 (+/- 0.6) fold increase in luciferase activity respectively (p < .001) Transfections with recombinant reporter plasmids lacking an enhancer produced light emission which was not significantly different than controls. These observations indicate that 1.) one or more of the 35 nucleotide differences between the CYP21 and CYP21P upstream regions alters a DNA recognition site important for transcriptional activation of this gene in steroidogenic cells, 2.) the steroidogenic milieu has a stimulatory effect on both CYP21 and CYP21P promoter activities, and 3.) based on the minimal promoter activity observed in either cell type transfected with constructs lacking an enhancer element, both of these promoter sequences are enhancer dependent under constitutive conditions in both steroidogenic and nonsteroidogenic cells.

The human complement C4B/steroid 21-hydroxylase (CYP21) and complement C4A/21-hydroxylase pseudogene (CYP21P) intergenic sequences: Comparison and identification of possible regulatory elements

We determined the 1.8 kb intergenic sequences between the human complement C4B gene and the active steroid 21-hydroxylase gene in two subjects, and between the C4A gene and the steroid 21-hydroxylase pseudogene in one subject. Comparison of these sequences with each other and with published homologues revealed no differences which were unique to either intergenic region. Sequence analysis revealed two copies of an AGGTCA motif in all sequences. This motif is common to steroidogenic enzyme gene promoters and to the response elements for nuclear hormone receptors. Similarities with human enhancers were also found.