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Nephrolithiasis in the [Slc26a6.sup.-/-] mouse is accompanied by 50-75% reduction in intestinal oxalate secretion with unchanged intestinal oxalate absorption. The molecular identities of enterocyte pathways for oxalate absorption and for Slc26a6-independent oxalate secretion remain undefined. The reported intestinal expression of S[O.sup.2-.sub.4] transporter SLC26A2 prompted us to characterize transport of oxalate and other anions by human SLC26A2 and mouse S1c26a2 expressed in Xenopus oocytes. We found that hSLC26A2-mediated [[sup.14]C]oxalate uptake ([K.sub.1/2] of 0.65 [+ or -] 0.08 mM) was cis-inhibited by external S[O.sup.2-.sub.4] ([K.sub.1/2] of 3.1 mM). hSLC26A2-mediated bidirectional oxalate/S[O.sup.2-.sub.4] exchange exhibited extracellular S[O.sup.2-.sub.4] [K.sub.1/2] of 1.58 [+ or -] 0.44 mM for exchange with intracellular [[sup.14]C]oxalate, and extracellular oxalate [K.sub.1/2] of 0.14 [+ or -] 0.11 mM for exchange with intracellular [sup.35]S[O.sup.2-.sub.4]. Influx rates and [K.sub.1/2] values for mSlc26a2 were similar, hSLC26A2-mediated oxalate/[Cl.sup.-] exchange and bidirectional S[O.sup.2-.sub.4]/[Cl.sup.-] exchange were not detectably electrogenic. Both SLC26A2 orthologs exhibited nonsaturable extracellular [Cl.sup.-] dependence for efflux of intracellular [[sup.14]C]oxalate, [sup.35]S[O.sup.2-.sub.4], or [sup.36][Cl.sup.-]. Rate constants for [sup.36][Cl.sup.-] efflux into extracellular [Cl.sup.-], S[O.sup.2-.sub.4], and oxalate were uniformly 10-fold lower than for oppositely directed exchange. Acidic extracellular pH ([pH.sub.o]) inhibited all modes of hSLC26A2-mediated anion exchange. In contrast, acidic intracellular pH ([pH.sub.i]) selectively activated exchange of extracellular [Cl.sup.-] for intracellular [sup.35]S[O.sup.2-.sub.4] but not for intracellular [sup.36][Cl.sup.-] or [[sup.14]C]oxalate. Protein kinase C inhibited hSLC26A2 by reducing its surface abundance. Diastrophic dysplasia mutants R279W and A386V of hSLC26A2 exhibited similar reductions in uptake of both [sup.35]S[O.sup.2-.sub.4] and [[sup.14]C]oxalate. A386V surface abundance was reduced, but R279W surface abundance was at wild-type levels. chondrodysplasia; nephrolithiasis; oxalosis; anion exchange; Xenopus oocyte doi: 10.1152/ajpcell.00004.2010.

The previously undescribed heterozygous missense mutation E758K was discovered in the human AE1/SLC4A1/band 3 gene in two unrelated patients with well-compensated hereditary spherostomatocytic anemia (HSt). Oocyte surface expression of AE1 E758K, in contrast to that of wild-type AE1, required coexpressed glycophorin A (GPA). The mutant polypeptide exhibited, in parallel, strong GPA dependence of DIDS-sensitive [sup.36][Cl.sup.-] influx, trans-anion-dependent [sup.36][Cl.sup.-] efflux, and [Cl.sup.-]/HC[O.sub.3.sup.-] exchange activities at near wild-type levels. AE1 E758K expression was also associated with GPA-dependent increases of DIDS-sensitive pH-independent S[O.sub.4.sup.2-] uptake and oxalate uptake with altered pH dependence. In marked contrast, the bumetanide- and ouabain-insensitive [sup.86][Rb.sup.+] influx associated with AE1 E758K expression was largely GPA-independent in Xenopus oocytes and completely GPA-independent in Ambystoma oocytes. AE1 E758K-associated currents in Xenopus oocytes also exhibited little or no GPA dependence. [sup.86][Rb.sup.+] influx was higher but inward cation current was lower in oocytes expressing AE1 E758K than previously reported in oocytes expressing the AE1 HSt mutants S731P and H734R. The pharmacological inhibition profile of AE1 E758K-associated [sup.36][Cl.sup.-] influx differed from that of AE1 E758K-associated [sup.86][Rb.sup.+] influx, as well as from that of wild-type AE1mediated [Cl.sup.-] transport. Thus AE1 E758K-expressing oocytes displayed GPA-dependent surface polypeptide expression and anion transport, accompanied by substantially GPA-independent, pharmacologically distinct [Rb.sup.+] flux and by small, GPA-independent currents. The data strongly suggest that most of the increased cation transport associated with the novel HSt mutant AE1 E758K reflects activation of endogenous oocyte cation permeability pathways, rather than cation translocation through the mutant polypeptide. chloride-bicarbonate exchange; Xenopus oocytes; Ambystoma oocytes; erythrocyte band 3, glycophorin A doi: 10.1152/ajpcell.00444.2009

The genome of zebrafish (Danio rerio) encodes two unlinked genes equally closely related to the SLC4A2/AE2 anion exchanger genes of mammals. One of these is the recently reported zebrafish ae2 gene (Shmukler BE, Kurschat CE, Ackermann GE, Jiang L, Zhou Y, Barut B, Stuart-Tilley AK, Zhao J, Zon LI, Drummond IA, Vandorpe DH, Paw BH, Alper SL. Am J Physiol Renal Physiol Renal Physiol 289: F835-F849, 2005), now called ae2.1. We now report the structural and functional characterization of Ae2.2, the product of the second zebrafish Ae2 gene, ae2.2. The ae2.2 gene of zebrafish linkage group 24 encodes a polypeptide of 1,232 aa in length, sharing 70% amino acid identity with zebrafish Ae2.1 and 67% identity with mouse AE2a. Zebrafish Ae2.2 expressed in Xenopus oocytes encodes a 135-kDa polypeptide that mediates bidirectional, DIDS-sensitive [Cl.sup.-]/[Cl.sup.-] exchange and [Cl.sup.-]/HC[O.sup.-.sub.3] exchange. Ae2.2-mediated [Cl.sup.-]/[Cl.sup.-] exchange is cation independent, voltage insensitive, and electroneutral. Acute regulation of anion exchange mediated by Ae2.2 includes activation by N[H.sup.+.sub.4] and independent inhibition by acidic intracellular pH and by acidic extracellular pH. In situ hybridization reveals low-level expression of Ae2.2 mRNA in zebrafish embryo, most notably in posterior tectum, eye, pharynx, epidermal cells, and axial vascular structures, without notable expression in the Ae2.1-expressing pronephric duct. Knockdown of Ae2.2 mRNA, of Ae2.1 mRNA, or of both with nontoxic or minimally toxic levels of N-morpholino oligomers produced no grossly detectable morphological phenotype, and preserved normal structure of the head and the pronephric duct at 24 h postfertilization. chloride/bicarbonate exchanger; Xenopus oocyte; isotopic flux; in situ hybridization; two-electrode voltage clamp; N-morpholino oligomer

The shark liver antimicrobial polyaminosterol squalamine is an angiogenesis inhibitor under clinical investigation as an anti-cancer agent and as a treatment for the choroidal neovascularization associated with macular degeneration of the retina. The related polyaminosterol MSI-1436 is an appetite suppressant that decreases systemic insulin resistance. However, the mechanisms of action of these polyaminosterols are unknown. We report effects of MSI-1436 on Xenopus oocytes consistent with the existence of a receptor for polyaminosterols. MSI-1436 activates bidirectional, trans-chloride-independent Cl- flux in Xenopus oocytes. At least part of this DIDS-sensitive [Cl.sup.-] flux is conductive, as measured using two-electrode voltage-clamp and on-cell patch-clamp techniques. MSI-1436 also elevates cytosolic [Ca.sup.2+] concentration ([[Ca.sup.2+]]) and increases bidirectional [sup.45][Ca.sup.2+] flux. Activation of [Cl.sup.-] flux and elevation of cytosolic [[Ca.sup.2+]] by MSI-1436 both are accelerated by lowering bath [Ca.sup.2+] and are not acutely inhibited by extracellular EGTA. Elevation of cytosolic [[Ca.sup.2+]] by MSI-1436 requires heparin-sensitive intracellular [Ca.sup.2+] stores. Although injected EGTA abolishes the increased conductive [Cl.sup.-] flux, that [Cl.sup.-] flux is not dependent on heparin-sensitive stores. In low-bath [Ca.sup.2+] conditions, several structurally related polyaminosterols act as strong agonists or weak agonists of conductive [Cl.sup.-] flux in oocytes. Weak agonist polyaminosterols antagonize the strong agonist, MSI1436, but upon addition of the conductive [Cl.sup.-] transport inhibitor DIDS, they are converted into strong agonists. Together, these properties operationally define a polyaminosterol receptor at or near the surface of the Xenopus oocyte, provide an initial description of receptor signaling, and suggest routes toward further understanding of a novel class of appetite suppressants and angiogenesis inhibitors. MSI-1436; calcium-dependent chloride conductance; Xenopus oocyte; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid

Although the zebrafish has been used increasingly for the study of pronephric kidney development, studies of renal ion transporters and channels of the zebrafish remain few. We report the cDNA cloning and characterization of the AE2 anion exchanger ortholog from zebrafish kidney, slc4a2/ae2. The ae2 gene in linkage group 2 encodes a polypeptide of 1,228 aa exhibiting 64% aa identity with mouse AE2a. The exonintron boundaries of the zebrafish ae2 gene are nearly identical to those of the rodent and human genes. Whole-mount in situ hybridization detects ae2 mRNA in prospective midbrain as early as the five-somite stage, then later in the pronephric primordia and the forming pronephric duct, where it persists through 72 h post-fertilization (hpf). Zebrafish Ae2 expressed in Xenopus laevis oocytes mediates [Na.sup.+]-independent, electroneutral [sup.36][Cl.sup.-]/[Cl.sup-] exchange moderately sensitive to inhibition by DIDS, is inhibited by acidic intracellular pH and by acidic extracellular pH, but activated by (acidifying) ammonium and by hypertonicity. Zebrafish Ae2 also mediates [Cl.sup.-]/HC[O.sup.-.sub.3] exchange in X. laevis oocytes and accumulates in or near the plasma membrane in transfected HEK-293 cells. In 24-48 hpf zebrafish embryos, the predominant but not exclusive localization of Ae2 polypeptide is the apical membrane of pronephric duct epithelial cells. Thus Ae2 resembles its mammalian orthologs in function, mechanism, and acute regulation but differs in its preferentially apical expression in kidney. These results will inform tests of the role of Ae2 in zebrafish kidney development and function. AE2; chloride/bicarbonate exchanger

Goss, Greg G., Lianwei Jiang, David H. Vandorpe, Dawn Kieller, Marina N. Chernova, Marilyn Robertson, and Seth L. Alper. Role of JNK in hypertonic activation of [Cl.sup.-] -dependent [Na.sup.+]/[H.sup.+] exchange in Xenopus oocytes. Am J Physiol Cell Physiol 281: C1978-C1990, 2001.--In the course of studying the hypertonicity-activated ion transporters in Xenopus oocytes, we found that activation of endogenous oocyte [Na.sup.+]/[H.sup.+] exchange activity (xoNHE) by hypertonic shrinkage required [CI.sup.-], with an E[C.sub.5o] for bath [Cl.sup.-] of ~3 mM. This requirement for chloride was not supported by several nonhalide anions and was not shared by xoNHE activated by acid loading. Hypertonicity-activated xoNHE exhibited an unusual rank order of inhibitory potency among amiloride derivatives and was blocked by [Cl.sup.-] transport inhibitors. Chelation of intracellular [Ca.sup.2+] by injection of EGTA blocked hypertonic activation of xoNHE, although many inhibitors of [Ca.sup.2+]-related signaling pathways were without inhibitory effect. Hypertonicity activated oocyte extracellular signal-regulated kinase 1/2 (ERK1/2), but inhibitors of neither ERK1/2 nor p38 prevented hypertonic activation of xoNHE. However, hypertonicity also stimulated a Cl--dependent increase in c-Jun N[H.sub.2] -terminal kinase (JNK) activity. Inhibition of JNK activity prevented hypertonic activation of xoNHE but not activation by acid loading. We conclude that hypertonic activation of [Na.sup.+]/[H.sup.+] exchange in Xenopus oocytes requires [Cl.sup.-] and is mediated by activation of JNK. [Na.sup.+]/[H.sup.+] exchange; c-Jun N[H.sub.2]-terminal kinase; extracellular signal-regulated kinase; p38; transport; SP600125; U-0126 Received 11 June 2001; accepted in final form 16 August 2001

The presence of epithelial Na+ channel (ENaC) poly(A)+RNA in permanent lines of inner medullary collecting duct (mIMCD)-K2 cells were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Analysis of Na+ absorption across mIMCD-K2 cells indicated the role of a cyclic guanylic acid-gated, nonselective cation (CNG) channels in mediating Na+ trasport across mIMCD-K2 cells. Furthermore, IMCD cells also expressed ENaC channels which mediate Na+ absorption together with CNG channels.