Key Points Dehydrated hereditary stomatocytosis is characterized by abnormal RBC morphology but may involve pseudohyperkalemia and perinatal edema. This syndrome is associated with germline mutations in PIEZO1, encoding a transmembrane protein that induces mechanosensitive currents.

The unusually low 78% amino acid identity between the orthologous human SLC26A6 and mouse slc26a6 polypeptides prompted systematic comparison of their anion transport functions in Xenopus oocytes. Multiple human SLC26A6 variant polypeptides were also functionally compared.

We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca 2؉ -gated K ؉ (K Ca ) channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci.(U. S. A. 94, 11651-11656). mIK1 mRNA was detected at varied levels in many tissue types. mIK1 K Ca channel activity expressed in Xenopus oocytes closely resembled the K ca of red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation, inward rectification, and Ca 2؉ concentration dependence. mIK1 also resembled the erythroid channel in its pharmacological properties, mediating whole cell and unitary currents sensitive to low nM concentrations of both clotrimazole (CLT) and its des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to low nM concentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression conferred on oocytes a novel, Ca 2؉ -dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes increased outward currents that were Ca 2؉ -dependent, CLT-sensitive, and reversed near the K ؉ equilibrium potential. mIK1 mRNA levels in ES cells increased steadily during erythroid differentiation in culture, in contrast to other K Ca mRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood stem cells in liquid culture.Terminal differentiation of erythroid precursor cells is marked by enucleation and reduction in cell volume. A major component of cell volume reduction is achieved by reduction of cell K ϩ content. Mature, circulating erythrocytes retain two major ion transport pathways mediating K ϩ efflux (1). These are: 1) electroneutral K-Cl cotransport and 2) a voltage-insensitive, Ca 2ϩ -activated potassium (K ϩ ) channel of intermediate conductance (2-4), also known as the Gardos channel (5). The Gardos channel is thought to play a major role in volume regulation in normal (6) and sickle (SS) 1 human erythrocytes (7,8).Especially in the chronically hypoxic environment of adherent or trapped sickle cells, the Gardos channel appears to mediate the major component of K ϩ loss from the erythrocyte (9), leading to an increased concentration of intracellular hemoglobin S, and exponentially decreasing the lag time for accelerated hemoglobin S polymerization (10). The Gardos channel's biophysical and pharmacological properties have been characterized in excised inside-out human red cell membrane patches, in which Ca 2ϩ -activated K (K Ca ) currents show inwardly rectifying properties with a unitary slope conductance ranging from 15 to 40 picosiemens, depending on the ionic conditions used (11-13). The channel is sensitive to block by charybdotoxin (14 -16), but insensitive to the SK channel blocker, apamin, and to the K ATP channel blockers, the antihypoglycemic drugs (17). 2Sickle...

The cytoplasmic C-terminal portion of the polycystin-1 polypeptide (PKD1(1-226)) regulates several important cell signaling pathways, and its deletion suffices to cause autosomal dominant polycystic kidney disease. However, a functional link between PKD1 and the ion transport processes required to drive renal cyst enlargement has remained elusive. We report here that expression at the Xenopus oocyte surface of a transmembrane fusion protein encoding the C-terminal portion of the PKD1 cytoplasmic tail, PKD1 (115-226

Functional evaluation of chemically modified human erythrocytes has led to the proposal that amino acid residue E681 of the band 3 anion exchanger AE1 lies on the anion translocation pathway and is a proton carrier required for H+/SO4 2− cotransport. We have tested in Xenopus oocytes the functional consequences of mutations in the corresponding residue E699 of mouse AE1. Most mutations tested abolished AE1-mediated Cl− influx and efflux. Only the E699Q mutation increased stilbene disulfonate-sensitive efflux and influx of SO4 2−. E699Q-mediated Cl− influx was activated by elevation of intracellular SO4 2−, but E699Q-mediated Cl− efflux was undetectable. The DNDS (4,4′-dinitrostilbene-2,2′-disulfonic acid) sensitivity of E699Q-mediated SO4 2− efflux was indistinguishable from that of wt AE1-mediated Cl− efflux. The extracellular anion selectivity of E699Q-mediated SO4 2− efflux was similar to that of wt AE1-mediated Cl− efflux. The stoichiometry of E699Q-mediated exchange of extracellular Cl− with intracellular SO4 2− was 1:1. Whereas SO4 2− injection into oocytes expressing wt AE1 produced little change in membrane potential or resistance, injection of SO4 2−, but not of Cl− or gluconate, into oocytes expressing E699Q depolarized the membrane by 17 mV and decreased membrane resistance by 66%. Replacement of bath Cl− with isethionate caused a 28-mV hyperpolarization in SO4 2−-loaded oocytes expressing E699Q, but had no effect on oocytes expressing wt AE1. Extracellular Cl−-dependent depolarization of SO4 2−-preloaded oocytes was blocked by DNDS. AE1 E699Q-mediated inward current measured in the presence of extracellular Cl− was of magnitude sufficient to account for measured 35SO4 2− efflux. Thus, AE1 E699Q-mediated SO4 2−/ Cl− exchange operated largely, if not exclusively, as an electrogenic, asymmetric, 1:1 anion exchange. The data confirm the proposal that E699 resides on or contributes to the integrity of the anion translocation pathway of AE1. A single amino acid change in the sequence of AE1 converted electroneutral to electrogenic anion exchange without alteration of SO4 2−/Cl− exchange stoichiometry.

The general anesthetic ketamine has been repurposed by physicians as an anti-depressant and by the public as a recreational drug. However, ketamine use can cause extensive pathological changes, including ketamine cystitis. The mechanisms of ketamine's antidepressant and adverse effects remain poorly understood. Here we present evidence that ketamine is an effective L-type Ca 2+ channel (Cav1.2) antagonist that directly inhibits calcium influx and smooth muscle contractility, leading to voiding dysfunction. Ketamine prevents Cav1.2-mediated induction of immediate early genes and transcription factors, and inactivation of Cav1.2 in smooth muscle mimics the ketamine cystitis phenotype. Our results demonstrate that ketamine inhibition of Cav1.2 signaling is an important pathway mediating ketamine cystitis. In contrast, Cav1.2 agonist Bay k8644 abrogates ketamine-induced smooth muscle dysfunction. Indeed, Cav1.2 activation by Bay k8644 decreases voiding frequency while increasing void volume, indicating Cav1.2 agonists might be effective drugs for treatment of bladder dysfunction.

The mouse is refractory to lithogenic agents active in rats and humans, and so has been traditionally considered a poor experimental model for nephrolithiasis. However, recent studies have identified slc26a6 as an oxalate nephrolithiasis gene in the mouse. Here we extend our earlier demonstration of different anion selectivities of the orthologous mouse and human SLC26A6 polypeptides to investigate the correlation between species-specific differences in SLC26A6 oxalate/anion exchange properties as expressed in Xenopus oocytes and in reported nephrolithiasis susceptibility. We find that human SLC26A6 mediates minimal rates of Cl

Nephrolithiasis in the Slc26a6(-/-) 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 SO(4)(2-) transporter SLC26A2 prompted us to characterize transport of oxalate and other anions by human SLC26A2 and mouse Slc26a2 expressed in Xenopus oocytes. We found that hSLC26A2-mediated [(14)C]oxalate uptake (K(1/2) of 0.65 +/- 0.08 mM) was cis-inhibited by external SO(4)(2-) (K(1/2) of 3.1 mM). hSLC26A2-mediated bidirectional oxalate/SO(4)(2-) exchange exhibited extracellular SO(4)(2-) K(1/2) of 1.58 +/- 0.44 mM for exchange with intracellular [(14)C]oxalate, and extracellular oxalate K(1/2) of 0.14 +/- 0.11 mM for exchange with intracellular (35)SO(4)(2-). Influx rates and K(1/2) values for mSlc26a2 were similar. hSLC26A2-mediated oxalate/Cl(-) exchange and bidirectional SO(4)(2-)/Cl(-) exchange were not detectably electrogenic. Both SLC26A2 orthologs exhibited nonsaturable extracellular Cl(-) dependence for efflux of intracellular [(14)C]oxalate, (35)SO(4)(2-), or (36)Cl(-). Rate constants for (36)Cl(-) efflux into extracellular Cl(-), SO(4)(2-), and oxalate were uniformly 10-fold lower than for oppositely directed exchange. Acidic extracellular pH (pH(o)) inhibited all modes of hSLC26A2-mediated anion exchange. In contrast, acidic intracellular pH (pH(i)) selectively activated exchange of extracellular Cl(-) for intracellular (35)SO(4)(2-) but not for intracellular (36)Cl(-) or [(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 (35)SO(4)(2-) and [(14)C]oxalate. A386V surface abundance was reduced, but R279W surface abundance was at wild-type levels.