Maintenance of a stable internal environment within complex organisms requires specialized cells that sense changes in the extracellular concentration of specific ions (such as Ca2+). Although the molecular nature of such ion sensors is unknown, parathyroid cells possess a cell surface Ca(2+)-sensing mechanism that also recognizes trivalent and polyvalent cations (such as neomycin) and couples by changes in phosphoinositide turnover and cytosolic Ca2+ to regulation of parathyroid hormone secretion. The latter restores normocalcaemia by acting on kidney and bone. We now report the cloning of complementary DNA encoding an extracellular Ca(2+)-sensing receptor from bovine parathyroid with pharmacological and functional properties nearly identical to those of the native receptor. The novel approximately 120K receptor shares limited similarity with the metabotropic glutamate receptors and features a large extracellular domain, containing clusters of acidic amino-acid residues possibly involved in calcium binding, coupled to a seven-membrane-spanning domain like those in the G-protein-coupled receptor superfamily.

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Individuals from different populations vary considerably in their susceptibility to immune-related diseases. To understand how genetic variation and natural selection contribute to these differences, we tested for the effects of African versus European ancestry on the transcriptional response of primary macrophages to live bacterial pathogens. A total of 9.3% of macrophage-expressed genes show ancestry-associated differences in the gene regulatory response to infection, and African ancestry specifically predicts a stronger inflammatory response and reduced intracellular bacterial growth. A large proportion of these differences are under genetic control: for 804 genes, more than 75% of ancestry effects on the immune response can be explained by a single cis- or trans-acting expression quantitative trait locus (eQTL). Finally, we show that genetic effects on the immune response are strongly enriched for recent, population-specific signatures of adaptation. Together, our results demonstrate how historical selective events continue to shape human phenotypic diversity today, including for traits that are key to controlling infection.

Nearly 30 mutations have been identified to date in the coding region of the extracellular calcium-sensing receptor (CaR) that are associated with inherited human hypo-and hypercalcemic disorders. To understand the mechanisms by which the mutations alter the function of the receptor may help to discern the structurefunction relationships in terms of ligand-binding and G protein coupling. In the present studies, we transiently expressed eight known CaR mutations in HEK293 cells. The effects of the mutations on extracellular calciumand gadolinium-elicited increases in the cytosolic calcium concentration were then examined. Seven inactivating mutations, which cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism, show a reduced functional activity of the receptor because they may 1) reduce its affinity for agonists; 2) prevent conversion of the receptor from a putatively immature, high mannose form into the fully glycosylated and biologically active form of the CaR, in addition to lowering its affinity for agonists; or 3) fail to couple the receptor to and/or activate its respective G protein(s). Conversely, one activating mutation, which causes a form of autosomal dominant hypocalcemia, appears to increase the affinity of the receptor for its agonists.The recently cloned extracellular Ca 2ϩ (Ca 2ϩ o )-sensing receptor (CaR) 1 (1) has provided key insights into the pathogenesis of inherited human hypo-and hypercalcemic disorders (2, 3). The receptor, BoPCaR (bovine parathyroid Ca 2ϩ -sensing receptor) was first isolated from bovine parathyroid using expression cloning in Xenopus laevis oocytes and shows pharmacological properties nearly identical to those of the native receptor in its responses to extracellular divalent cations (i.e. Ca o ), and polyamines (e.g. neomycin) (1). Subsequently, cDNAs encoding the human homolog of the same receptor have been cloned from human parathyroid (4) and kidney (5), using a homology-based strategy. The human and bovine receptors share a high degree of homology at the amino acid level (93% identity). Stimulation of the CaR by agonists activates phospholipase C, with resultant increases in inositol phosphates and the cytosolic calcium concentration (Ca 2ϩ

Mutations in the serine-threonine kinases WNK1 and WNK4 [with no lysine (K) at a key catalytic residue] cause pseudohypoaldosteronism type II (PHAII), a Mendelian disease featuring hypertension, hyperkalemia, hyperchloremia, and metabolic acidosis. Both kinases are expressed in the distal nephron, although the regulators and targets of WNK signaling cascades are unknown. The Cl ؊ dependence of PHAII phenotypes, their sensitivity to thiazide diuretics, and the observation that they constitute a ''mirror image'' of the phenotypes resulting from loss of function mutations in the thiazide-sensitive Na-Cl cotransporter (NCCT) suggest that PHAII may result from increased NCCT activity due to altered WNK signaling. To address this possibility, we measured NCCTmediated Na ؉ influx and membrane expression in the presence of wild-type and mutant WNK4 by heterologous expression in Xenopus oocytes. Wild-type WNK4 inhibits NCCT-mediated Na-influx by reducing membrane expression of the cotransporter ( 22 Nainflux reduced 50%, P < 1 ؋ 10 ؊9 , surface expression reduced 75%, P < 1 ؋ 10 ؊14 in the presence of WNK4). This inhibition depends on WNK4 kinase activity, because missense mutations that abrogate kinase function prevent this effect. PHAII-causing missense mutations, which are remote from the kinase domain, also prevent inhibition of NCCT activity, providing insight into the pathophysiology of the disorder. The specificity of this effect is indicated by the finding that WNK4 and the carboxyl terminus of NCCT coimmunoprecipitate when expressed in HEK 293T cells. Together, these findings demonstrate that WNK4 negatively regulates surface expression of NCCT and implicate loss of this regulation in the molecular pathogenesis of an inherited form of hypertension.protein serine-threonine kinases ͉ hypertension ͉ thiazide-sensitive Na-Cl cotransporter ͉ ion transport ͉ medical genetics H ypertension is the most common disease in industrialized societies, affecting Ͼ20% of the adult population and contributing to morbidity and mortality from stroke, myocardial infarction, renal failure, and congestive heart failure (1). Its pathogenesis is largely unknown, resulting in empiric pharmacologic therapy. In recent years, genetic approaches investigating rare Mendelian forms of high and low blood pressure have provided fundamental insight into mechanisms that contribute to blood pressure variation (2). These have demonstrated the causal role of inherited variation in renal salt homeostasis in blood pressure variation, with mutations in many genes known to play a role in mediating or regulating renal salt reabsorption resulting in altered blood pressure.Pseudohypoaldosteronism type II (PHAII; Online Mendelian Inheritance in Man database no. 145260) is an autosomal dominant disease featuring hypertension with hyperkalemia despite normal glomerular filtration rate; renal tubular acidosis is a variable associated finding. The clinical features of this disease are chloride dependent and are also corrected with thiazide diuretics, specific a...

In the nervous system, the intracellular chloride concentration ([Cl(-)](i)) determines the strength and polarity of gamma-aminobutyric acid (GABA)-mediated neurotransmission. [Cl(-)](i) is determined, in part, by the activities of the SLC12 cation-chloride cotransporters (CCCs). These transporters include the Na-K-2Cl cotransporter NKCC1, which mediates chloride influx, and various K-Cl cotransporters--such as KCC2 and KCC3-that extrude chloride. A precise balance between NKCC1 and KCC2 activity is necessary for inhibitory GABAergic signaling in the adult CNS, and for excitatory GABAergic signaling in the developing CNS and the adult PNS. Altered chloride homeostasis, resulting from mutation or dysfunction of NKCC1 and/or KCC2, causes neuronal hypoexcitability or hyperexcitability; such derangements have been implicated in the pathogenesis of seizures and neuropathic pain. [Cl(-)](i) is also regulated to maintain normal cell volume. Dysfunction of NKCC1 or of swelling-activated K-Cl cotransporters has been implicated in the damaging secondary effects of cerebral edema after ischemic and traumatic brain injury, as well as in swelling-related neurodegeneration. CCCs represent attractive therapeutic targets in neurological disorders the pathogenesis of which involves deranged cellular chloride homoestasis.

Defects in the human Ca(2+)-sensing receptor gene have recently been shown to cause familial hypocalciuric hypercalcaemia and neonatal severe hyperparathyroidism. We now demonstrate that a missense mutation (Glu128Ala) in this gene causes familial hypocalcaemia in affected members of one family. Xenopus oocytes expressing the mutant receptor exhibit a larger increase in inositol 1,4,5-triphosphate in response to Ca2+ than oocytes expressing the wild-type receptor. We conclude that this extracellular domain mutation increases the receptor's activity at low Ca2+ concentrations, causing hypocalcaemia in patients heterozygous for such a mutation.

K(+) channels are widely distributed in both plant and animal cells where they serve many distinct functions. K(+) channels set the membrane potential, generate electrical signals in excitable cells, and regulate cell volume and cell movement. In renal tubule epithelial cells, K(+) channels are not only involved in basic functions such as the generation of the cell-negative potential and the control of cell volume, but also play a uniquely important role in K(+) secretion. Moreover, K(+) channels participate in the regulation of vascular tone in the glomerular circulation, and they are involved in the mechanisms mediating tubuloglomerular feedback. Significant progress has been made in defining the properties of renal K(+) channels, including their location within tubule cells, their biophysical properties, regulation, and molecular structure. Such progress has been made possible by the application of single-channel analysis and the successful cloning of K(+) channels of renal origin.