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Inheritable familial hypocalciuric hypercalcemia (FHH) and neonatal severe hyperparathyroidism (NSHPT) are caused by mutations in the Ca2+ receptor gene. Two nonconservative missense mutations occur in the extracellular N-terminal domain, while the third occurs in the final intracellular loop. The mutations lead to defects in the Ca2+-sensing receptor which is responsible for 'setting' extracellular Ca2+ levels, as exhibited in FHH and NSHPT afflicted individuals.

Specialized cells which change the extracellular concentration of specific ions maintain the stable internal environment within the body of a complex multicellular organism. Parathyroid cells have a cell surface calcium ion concentration-sensing mechanism which also recognizes the polyvalent cations such as neomycin. The changes in phosphoinositide turnover and cytosolic calcium ion concentration are monitored by the same cells. The calcium ion regulation restores the normocalcaemia by acting on the kidney and bone cells.

Membrane transport processes and cell volume regulation can be analyzed through laser light scattering, which enables rapid tracing of cell volume variations and also helps to maintain natural cell morphology. Solute and gas composition and the regulation of solution temperature is possible by this technique. Epithelial monolayer cell cultures can be investigated for their basolateral and apical membrane transport processes.

ROMK1 is a potassium (K+) ion channel polypeptide protein whose amino acid sequence and unusual structure have been inferred from the cloning of complementary DNA from mammalian kidney. ROMK1 conserves an amino acid sequence homologous to the H5 region of the voltage-gated K+ channel. Moreover, ROMK1 consists of two potential cell membrane-spanning helices and an adenosine triphosphate-binding domain.

The effects of intracellular pH on cellular NH4+/K+ transport pathways in the renal medullary thick ascending limb of Henle (MTAL) were studied using fluorescence and electrophysiological methods. Results show that the intrinsic cellular buffering power of MTAL cells is 29.7 plus or minus 2.4 millimoles per pH unit at pH values between 7.0 and 7.6, but below a pH of 7.0 the intrinsic buffering power increases linearly to nearly 50 millimoles per pH unit at pH 6.5.

The Gitelman's and Bartter's syndromes are disorders resulting from mutations in the sodium chloride cotransporters. These mutations lead to the loss of function of the cotransporters that mediate specific pathways in the kidneys. Using molecular cloning techniques, it has been shown that mutations in the gene coding for the thiazide-sensitive Na-Cl cotransporter causes Gitelman's syndrome, while mutations in the diuretic-sensitive Na-K-2Cl cotransporter gene causes Bartter's syndrome.

A study was conducted on the calcium-sensing receptor CaSR agonists (calcimetics) and antagonists (calcilytics), which have been identified and current and potential therapies for a variety of disorders are provided. Results show that calcimimetics can become a major therapy for hyperparathyroidism and calcilytics may prove useful in the treatment of osteoporosis.

Plata, Consuelo, Patricia Meade, Amy Hall, Rick C. Welch, Norma Vazquez, Steven C. Hebert, and Gerardo Gamba. Alternatively spliced isoform of apical [Na.sup.+]-[K.sup.+]-[Cl.sup.-]cotransporter gene encodes a furosemide-sensitive [Na.sup.+][Cl.sup.-]cotransporter. Am J Physiol Renal Physiol 280: F574-F582, 2001.--In the absence of vasopressin, medullary thick ascending limb cells express a [K.sup.+]-independent, furosemide-sensitive [Na.sup.+]-[Cl.sup.-] cotransporter that is inhibited by hypertonicity. The murine renal specific [Na.sup.+]-[K.sup.+]-2 [Cl.sup.-] cotransporter gene (SLC12A1) gives rise to six alternatively spliced isoforms. Three feature a long COOH-terminal domain that encodes the butmetanide-sensitive [Na.sup.+]-[K.sup.+]-2 [Cl.sup.-] cotransporter (BSC1-9/NKCC2), and three with a short COOH-terminal domain, known as mBSC1-A4, B4, or F4 (19). Here we have determined the functional characteristics of mBSC1-A4, as expressed in Xenopus laevis oocytes. When incubated at normal oocyte osmolarity (~200 mosmol/ kg[H.sub.2]O), mBSC1-4-injected oocytes do not express significant [Na.sup.+] uptake over [H.sub.2]O-injected controls, and immunohistochemical analysis shows that the majority of mBSC1-4 protein is in the oocyte cytoplasm and not at the plasma membrane. In contrast, when mBSC1-4 oocytes are exposed to hypotonicity (~100 mosmol/kg[H.sub.2]O), a significant increase in [Na.sup.+] uptake but not in [sup.86][Rb.sup.+] uptake is observed. The increased [Na.sup.+] uptake is [Cl.sup.-] dependent, furosemide sensitive, and cAMP sensitive but [K.sup.+] independent. Sodium uptake increases with decreasing osmolarity between 120 and 70 mosmol/kg[H.sub.2]O (r = 0.95, P [is less than] 0.01). Immunohistochemical analysis shows that in hypotonic conditions mBSC1-A4 protein is expressed in the plasma membrane. These studies indicate that the mBSC1-A4 isoform of the SLC12A1 gene encodes a hypotonically activated, cAMP- and furosemide-sensitive [Na.sup.+]-[Cl.sup.-] cotransporter. Thus it is possible that alternative splicing of the BSC1 gene could provide the molecular mechanism enabling the [Na.sup.+]-[Cl.sup.-]-to-[Na.sup.+]-[K.sup.+]-[2Cl.sup.-] switching in thick ascending limb cells. bumetanide; protein kinase A; adenosine 3',5'-cyclic monophosphate; thick ascending limb of Henle

Leipziger, Jens, Gordon G. MacGregor, Gordon J. Cooper, Jason Xu, Steven C. Hebert, and Gerhard Giebisch. PKA site mutations of ROMK2 channels shift the pH dependence to more alkaline values. Am J Physiol Renal Physiol 279: F919-F926, 2000.--Close similarity between the rat native low-conductance [K.sup.+] channel in the apical membrane of renal cortical collecting duct principal cells and the cloned rat ROMK channel strongly suggest that the two are identical. Prominent features of ROMK regulation are a steep pH dependence and activation by protein kinase A (PKA)-dependent phosphorylation. In this study, we investigated the pH dependence of cloned renal [K.sup.+] channel (ROMK2), wild-type (R2-WT), and PKA site mutant channels (R2-S25A, R2-S200A, and R2-S294A). [Ba.sup.2+]-sensitive outward whole cell currents (holding voltage -50 mV) were measured in two-electrode voltage-clamp experiments in Xenopus laevis oocytes expressing either R2-WT or mutant channels. Intracellular pH (p[H.sub.i]) was measured with pH-sensitive microelectrodes in a different group of oocytes from the same batch on the same day. Resting p[H.sub.i] of R2-WT and PKA site mutants was the same: 7.32 [+ or -] 0.02 (n = 22). The oocytes were acidified by adding 3 mM Na butyrate with external pH ([pH.sub.o]) adjusted to 7.4, 6.9, 6.4, or 5.4. At [pH.sub.o] 7.4, butyrate led to a rapid ([Tau]: 163 [+ or -] 14 s, where [Tau] means time constant, n = 4) and stable acidification of the oocytes ([Delta] p[H.sub.i] 0.13 [+ or -] 0.02 pH units, where [Delta] means change, n = 12). Intracellular acidification reversibly inhibited ROMK2-dependent whole cell current. The effective acidic dissociation constant ([pK.sub.a]) value of R2-WT was 6.92 [+ or -] 0.03 (n = 8). Similarly, the effective [pK.sub.a] value of the N-terminal PKA site mutant R2-S25A was 6.99 [+ or -] 0.02 (n = 6). The effective [pK.sub.a] values of the two COOH-terminal PKA site mutant channels, however, were significantly shifted to alkaline values; i.e., 7.15 [+ or -] 0.06 (n = 5) for R2-S200A and 7.16 [+ or -] 0.03 (n = 8) for R2-S294A. The apparent [Delta] pH shift between the R2-WT and the R2-S294A mutant was 0.24 pH units. In excised inside-out patches, alkaline pH 8.5 activated R2-S294A channel current by 32 [+ or -] 6.7%, whereas in R2-WT channel patches alkalinzation only marginally increased current by 6.5 [+ or -] 1% (n = 5). These results suggest that channel phosphorylation may substantially influence the pH sensitivity of ROMK2 channel. Our data are consistent with the hypothesis that in the native channel PKA activation involves a shift of the [pK.sub.a] value of ROMK channels to more acidic values, thus relieving a [H.sup.+]-mediated inhibition of ROMK channels. phosphorylation; potassium ion channel; inward-rectifier; potassium ion secretion; intracellular pH

Characterization of the thiazidesensitive [Na.sup.+]-[Cl.sup.-] cotransporter: a new model for ions and diuretics interaction. Am J Physiol Renal Physiol 279: F161-F169, 2000.--The thiazide-sensitive [Na.sup.+]-[Cl.sup.-] cotransporter (TSC) is the major pathway for salt reabsorption in the apical membrane of the mammalian distal convoluted tubule. When expressed in Xenopus laevis oocytes, rat TSC exhibits high affinity for both cotransported ions, with the Michaelis-Menten constant ([K.sub.m]) for [Na.sup.+] of 7.6 [+ or -] 1.6 mM and for [Cl.sup.-] of 6.3 [+ or -] 1.1 mM, and Hill coefficients for [Na.sup.+] and [Cl.sup.-] consistent with electroneutrality. The affinities of both [Na.sup.+] and [Cl.sup.-] were increased by increasing concentration of the counterion. The [IC.sub.50] values for thiazides were affected by both extracellular [Na.sup.+] and [Cl.sup.-]. The higher the [Na.sup.+] or [Cl.sup.-] concentration, the lower the inhibitory effect of thiazides. Finally, rTSC function is affected by extracellular osmolarity. We propose a transport model featuring a random order of binding in which the binding of each ion facilitates the binding of the counterion. Both ion binding sites alter thiazide-mediated inhibition of transport, indicating that the thiazide-binding site is either shared or modified by both [Na.sup.+] and [C1.sup.-]. metolazone; distal tubule; osmolarity; salt reabsorption

Tanemoto, Masayuki, Carlos G. Vanoye, Ke Dong, Richard Welch, Takaaki Abe, Steven C. Hebert, and Jason Z. Xu. --Rat homolog of sulfonylurea receptor 2B determines glibenclamide sensitivity of ROMK2 in Xenopus laevis oocyte. Am J Physiol Renal Physiol 278: F659-F666, 2000.--Recent studies showed that coexpression of Kir6.1 or Kir6.2 with the sulfonylurea receptor (SUR1, SUR2A, or SUR2B) reconstituted an inwardly rectifying, ATP-sensitive [K.sup.+] channel that was inhibited by glibenclamide (2, 15-17). Here we report the isolation of a rat homolog of mouse SUR2B (denoted rSUR2B) from a rat kidney cDNA library. The rSUR2B sequence contains a 4,635-bp open reading frame that encodes a 1,545-amino acid polypeptide, showing 67% shared identity with SUR1 (a pancreatic [Beta]-cell isoform) and 98% with both SUR2A (a brain isoform) and SUR2B (a vascular smooth muscle isoform). Consistent with the predicted structures of other members of the ATP-binding cassette (ABC) superfamily, the sequence of rSUR2B contains 17 putative membrane-spanning segments. Also, predicted Walker A and B consensus binding motifs, present in other ABC members, are conserved in the rSUR2B sequence. RT-PCR revealed that rSUR2B is widely expressed in various rat tissues including brain, colon, heart, kidney, liver, skeletal muscle, and spleen. The intrarenal distribution of the rSUR2B transcript was investigated using RT-PCR and Southern blot of microdissected tubules. The rSUR2B transcript was detected in proximal tubule, cortical thick ascending limb, distal collecting tubule, cortical collecting duct, and outer medullary collecting duct, but not medullary thick ascending limb. This distal distribution overlaps with that of ROMK. Coexpression of rSUR2B with ROMK2 cRNA (in 1:10 ratio) in Xenopus laevis oocytes resulted in whole cell [Ba.sup.2+]-sensitive [K.sup.+] currents that were inhibited by glibenclamide (50% inhibition with 0.2 mM glibenclamide). In contrast, rSUR2B did not confer significant glibenclamide sensitivity to oocytes coinjected with ROMK1 or ROMK3. The interaction between ROMK2 and rSUR2B was further studied by coimmunoprecipitation of in vitro translated rSUR2B and ROMK2. In agreement with the functional data, the rSUR2B protein was coimmunoprecipitated with ROMK2 in the ROMK2-rSUR2B cotranslated samples. Our data demonstrate that ROMK2, but not ROMK1 and ROMK3, can interact with rSUR2B to confer a sulfonylurea-sensitive [K.sup.+] channel, implicating SUR proteins in forming and regulating renal ATP-sensitive [K.sup.+] channels. The ROMK isoform specificity of glibenclamide effects suggests that the [NH.sub.2] terminus of the ROMK protein mediates rSUR2B-ROMK2 interactions. ROMK potassium channel; sulfonylurea receptor; glibenclamide; kidney

Loffing, Johannes, Dominique Loffing-Cueni, Andreas Macher, Steven C. Hebert, Beatriz Olson, Mark A. Knepper, Bernard C. Rossier, and Brigitte Kaissling. Localization of epithelial sodium channel and aquaporin-2 in rabbit kidney cortex. Am J Physiol Renal Physiol 272: F530-F539, 2000.--The amiloride-sensitive epithelial sodium channel (ENaC) and the vasopressin-dependent water channel aquaporin-2 (AQP2) mediate mineralocorticoid-regulated sodium- and vasopressin-regulated water reabsorption, respectively. Distributions of ENaC and AQP2 have been shown by immunohistochemistry in rats. Functional data from rabbits suggest a different distribution pattern of these channels than in rats. We studied, by immunohistochemistry in the rabbit kidney cortex, the distributions of ENaC and AQP2, in conjunction with marker proteins for distal segments. In rabbit cortex ENaC is restricted to the connecting tubule (CNT) cells and cortical collecting duct (CCD) cells. The intracellular distribution of ENaC shifts from the apical membrane in the most upstream CNT cells to a cytoplasmic location further downstream in the CNT and in the CCD cells. AQP2 is detected in the CCD cells exclusively. The anatomic subdivisions in the rabbit distal nephron coincide exactly with distributions of apical transport systems. The differences between rabbits and rats in the distribution patterns of ENaC and AQP2 may explain functional differences in renal salt and water handling between these species. immunohistochemistry; morphology; intercalated cells; bumetamide-sensitive sodium-potassium chloride cotransporter; thiazide-sensitive sodium chloride cotransporter; diuretics

Researchers have linked Dent's disease, a type of hypercalciuric nephrolithiasis, to a defect in a gene called CLCN5. CLCN5 encodes CLC-5, a putative chloride channel. Dent's disease was originally viewed as a dysfunction in renal calcium transport and not of chloride or fluid volume. Two other types of hypercalciuric nephrolithiasis were found to be associated with mutations in the CLCN5 gene.