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Objective Prior to the discovery of CLCNKB-T481S there were no variants or clinical disorders associated with gain-of-function defects in thick ascending limb (TAL) of the kidney channels or transporters. CLCNKB-T481S is a novel gain-of-function variant that has been associated with essential hypertension. This finding has not been replicated until our current study. In this study we re-examine CLCNKB-T481S using a large homogenous population from Ghana, and coupled genetic analyses with the functional characterization of this polymorphism using a mammalian expression system. Methods We genotyped CLCNKB-T481S in four ethnically-defined control populations and a homogenous cohort of normotensive and hypertensive Ghanaians. Functional analysis was performed by whole-cell patch-clamp recording of tsA201 cells (a cell line derived from the human renal cell line, HEK-293) transiently transfected with ClC-Kb and barttin. Results CLCNKB-T481S was found more commonly in the African and Caucasian-Americans when compared to the Asian and Hispanic American populations having minor allele frequencies of 0.20, 0.15 and 0.06 and 0.01 respectively. Additionally, CLCNKB-T481S was significantly associated with hypertension in Ghanaian males. In stratified logistic regression analysis with Ghanaian males we observed a significant odds ratio of 3.29 (1.17 - 9.20 95% CI, p=0.024) in the recessive model (TT v AT&TT). Unlike previous results obtained in Xenopus oocytes, co-expression of CLCNKB-T481S with the obligatory accessory subunit barttin in tsA201 cells did not generate larger currents than co-expression of the wild type allele. Conclusions We conclude that CLCNKB-T481S is associated with essential hypertension in males within the Ghanaian population; however further studies are needed to understand its gender and ethnic segregation as well as to identify cellular factors that account for the divergent functional expression of ClC-Kb-T481S plus barttin in Xenopus oocytes and mammalian cells.

Chloride channels belonging to the ClC family are ubiquitous and participate in a wide variety of physiological and pathophysiological processes. To define sequence segments in ClC channels that contribute to the formation of their ion conduction pathway, we employed a combination of site-directed mutagenesis, heterologous expression, patch clamp recordings, and chemical modification of the human muscle ClC isoform, hClC-1. We demonstrate that a highly conserved 8-amino acid motif (P3) located in the linker between transmembrane domains D2 and D3 contributes to the formation of a wide pore vestibule facing the cell interior. Similar to a previously defined pore region (P1 region), this segment functionally interacts with the corresponding segment of the contralateral subunit. The use of cysteine-specific reagents of different size revealed marked differences in the diameter of pore-forming regions implying that ClC channels exhibit a pore architecture quite similar to that of certain cation channels, in which a narrow constriction containing major structural determinants of ion selectivity is neighbored by wide vestibules on both sides of the membrane. The ClC family of voltage-gated ClϪ channels represents the largest known gene family coding for anion channels (1-3). At least nine human isoforms (ClC-1 to ClC-7, ClC-Ka, and ClCKb) are expressed in various tissues and play important roles in the function of various organs. Mutations in the genes coding for three ClC isoforms cause inherited human diseases. CLCN1 represents the genetic locus for myotonia congenita (4, 5), a muscle disease characterized by stiffness upon sudden forceful movement. Mutations in CLCN5 cause Dent's disease, an inherited renal disorder associated with hypercalciuria, nephrolithiasis, and low molecular weight proteinuria (6). Genetic alterations of CLCNKB are responsible for type III Bartter's syndrome, a salt-wasting renal tubular disorder causing hypovolemia, hyponatremia, and hypotension (7).Heterologous expression of many cloned ClC isoforms have revealed highly anion-selective ion channels, and it is therefore reasonable to predict that evolutionarily conserved structures confer anion selectivity to their ion conduction pathways. Elucidating the molecular mechanisms responsible for ion selectivity and conduction in ClC channels is key to understanding the function and dysfunction of this important family of ion channels. Moreover, primary sequence information about the ionic pore represents the first step for a rationally designing compounds to block or open these ion channels that play important roles in human diseases.We recently probed the ion conduction pathway of human ClC-1 in the presence of several different permeant anions (8), and we demonstrated an unusual ion selectivity mechanism that depends upon differential ion binding to discriminate among anions (8, 9). Human ClC-1 (hClC-1) 1 exhibits at least two functionally distinct ion binding sites within the pore (8, 10), both preferring large and polyatomic anions over chl...

We genotyped 326 “frequently medicated” individuals of European-descent in Vanderbilt’s biorepository linked to de-identified electronic medical records, BioVU, on the ADME Core Panel to assess quality and performance of the assay. We compared quality control metrics and determined the extent of direct and indirect marker overlap between the ADME Core Panel and the Illumina Omni1-Quad. We found the quality of the ADME Core Panel data to be high, with exceptions in select copy number variants (CNVs) and markers in certain genes (notably CYP2D6). Most of the common variants on the ADME panel are genotyped by the Omni1, but absent rare variants and CNVs could not be accurately tagged by single markers. Finally, our frequently medicated study population did not convincingly differ in allele frequency from reference populations, suggesting that heterogeneous clinical samples (with respect to medications) follow similar allele frequency distributions in pharmacogenetics genes as their appropriate reference populations.