John F. Heneghan

Species differences in Cl− affinity and in electrogenicity of SLC26A6-mediated oxalate/Cl− exchange correlate with the distinct human and mouse susceptibilities to nephrolithiasis

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− exchange for Cl−, sulphate or formate, but rates of oxalate/Cl− exchange roughly equivalent to those of mouse slc2a6. Both transporters exhibit highly cooperative dependence of oxalate efflux rate on extracellular [Cl−], but whereas the K1/2 for extracellular [Cl−] is only 8 mm for mouse slc26a6, that for human SLC26A6 is 62 mm. This latter value approximates the reported mean luminal [Cl−] of postprandial human jejunal chyme, and reflects contributions from both transmembrane and C‐terminal cytoplasmic domains of human SLC26A6. Human SLC26A6 variant V185M exhibits altered [Cl−] dependence and reduced rates of oxalate/Cl− exchange. Whereas mouse slc26a6 mediates bidirectional electrogenic oxalate/Cl− exchange, human SLC26A6‐mediated oxalate transport appears to be electroneutral. We hypothesize that the low extracellular Cl− affinity and apparent electroneutrality of oxalate efflux characterizing human SLC26A6 may partially explain the high human susceptibility to nephrolithiasis relative to that of mouse. SLC26A6 sequence variant(s) are candidate risk modifiers for nephrolithiasis.

APOL1 kidney disease risk variants cause cytotoxicity by depleting cellular potassium and inducing stress-activated protein kinases

Significance People of recent African ancestry develop chronic kidney disease and end stage kidney failure at rates five times that of European-Americans. Two coding variants in the apolipoprotein-L1 (APOL1) gene account for nearly all this excess risk. The mechanisms by which APOL1 variants cause kidney failure are not understood. Recent evidence suggests that APOL1 transports cations, including K+, across lipid bilayers. Here, we show that tetracycline-induced expression of APOL1 kidney risk variants in T-REx-293 cells causes significant net efflux of intracellular K+, which, in turn, activates the stress-activated protein kinases (SAPKs) p38 MAPK and JNK, ultimately resulting in cytotoxicity. We propose that APOL1 nephropathy may be mediated by APOL1 risk variant-induced loss of intracellular K+ and aberrant activation of SAPK signaling. Two specific genetic variants of the apolipoprotein L1 (APOL1) gene are responsible for the high rate of kidney disease in people of recent African ancestry. Expression in cultured cells of these APOL1 risk variants, commonly referred to as G1 and G2, results in significant cytotoxicity. The underlying mechanism of this cytotoxicity is poorly understood. We hypothesized that this cytotoxicity is mediated by APOL1 risk variant-induced dysregulation of intracellular signaling relevant for cell survival. To test this hypothesis, we conditionally expressed WT human APOL1 (G0), the APOL1 G1 variant, or the APOL1 G2 variant in human embryonic kidney cells (T-REx-293) using a tetracycline-mediated (Tet-On) system. We found that expression of either G1 or G2 APOL1 variants increased apparent cell swelling and cell death compared with G0-expressing cells. These manifestations of cytotoxicity were preceded by G1 or G2 APOL1-induced net efflux of intracellular potassium as measured by X-ray fluorescence, resulting in the activation of stress-activated protein kinases (SAPKs), p38 MAPK, and JNK. Prevention of net K+ efflux inhibited activation of these SAPKs by APOL1 G1 or G2. Furthermore, inhibition of SAPK signaling and inhibition of net K+ efflux abrogated cytotoxicity associated with expression of APOL1 risk variants. These findings in cell culture raise the possibility that nephrotoxicity of APOL1 risk variants may be mediated by APOL1 risk variant-induced net loss of intracellular K+ and subsequent induction of stress-activated protein kinase pathways.

The Ca2+ channel β subunit determines whether stimulation of Gq-coupled receptors enhances or inhibits N current

In superior cervical ganglion (SCG) neurons, stimulation of M1 receptors (M1Rs) produces a distinct pattern of modulation of N-type calcium (N-) channel activity, enhancing currents elicited with negative test potentials and inhibiting currents elicited with positive test potentials. Exogenously applied arachidonic acid (AA) reproduces this profile of modulation, suggesting AA functions as a downstream messenger of M1Rs. In addition, techniques that diminish AAs concentration during M1R stimulation minimize N-current modulation. However, other studies suggest depletion of phosphatidylinositol-4,5-bisphosphate during M1R stimulation suffices to elicit modulation. In this study, we used an expression system to examine the physiological mechanisms regulating modulation. We found the β subunit (CaVβ) acts as a molecular switch regulating whether modulation results in enhancement or inhibition. In human embryonic kidney 293 cells, stimulation of M1Rs or neurokinin-1 receptors (NK-1Rs) inhibited activity of N channels formed by CaV2.2 and coexpressed with CaVβ1b, CaVβ3, or CaVβ4 but enhanced activity of N channels containing CaVβ2a. Exogenously applied AA produced the same pattern of modulation. Coexpression of CaVβ2a, CaVβ3, and CaVβ4 recapitulated the modulatory response previously seen in SCG neurons, implying heterogeneous association of CaVβ with CaV2.2. Further experiments with mutated, chimeric CaVβ subunits and free palmitic acid revealed that palmitoylation of CaVβ2a is essential for loss of inhibition. The data presented here fit a model in which CaVβ2a blocks inhibition, thus unmasking enhancement. Our discovery that the presence or absence of palmitoylated CaVβ2a toggles M1R- or NK-1R–mediated modulation of N current between enhancement and inhibition identifies a novel role for palmitoylation. Moreover, these findings predict that at synapses, modulation of N-channel activity by M1Rs or NK-1Rs will fluctuate between enhancement and inhibition based on the presence of palmitoylated CaVβ2a.

SLC26 anion exchangers of guinea pig pancreatic duct: molecular cloning and functional characterization.

The secretin-stimulated human pancreatic duct secretes HCO(3)(-)-rich fluid essential for normal digestion. Optimal stimulation of pancreatic HCO(3)(-) secretion likely requires coupled activities of the cystic fibrosis transmembrane regulator (CFTR) anion channel and apical SLC26 Cl(-)/HCO(3)(-) exchangers. However, whereas stimulated human and guinea pig pancreatic ducts secrete ∼140 mM HCO(3)(-) or more, mouse and rat ducts secrete ∼40-70 mM HCO(3)(-). Moreover, the axial distribution and physiological roles of SLC26 anion exchangers in pancreatic duct secretory processes remain controversial and may vary among mammalian species. Thus the property of high HCO(3)(-) secretion shared by human and guinea pig pancreatic ducts prompted us to clone from guinea pig pancreatic duct cDNAs encoding Slc26a3, Slc26a6, and Slc26a11 polypeptides. We then functionally characterized these anion transporters in Xenopus oocytes and human embryonic kidney (HEK) 293 cells. In Xenopus oocytes, gpSlc26a3 mediated only Cl(-)/Cl(-) exchange and electroneutral Cl(-)/HCO(3)(-) exchange. gpSlc26a6 in Xenopus oocytes mediated Cl(-)/Cl(-) exchange and bidirectional exchange of Cl(-) for oxalate and sulfate, but Cl(-)/HCO(3)(-) exchange was detected only in HEK 293 cells. gpSlc26a11 in Xenopus oocytes exhibited pH-dependent Cl(-), oxalate, and sulfate transport but no detectable Cl(-)/HCO(3)(-) exchange. The three gpSlc26 anion transporters exhibited distinct pharmacological profiles of (36)Cl(-) influx, including partial sensitivity to CFTR inhibitors Inh-172 and GlyH101, but only Slc26a11 was inhibited by PPQ-102. This first molecular and functional assessment of recombinant SLC26 anion transporters from guinea pig pancreatic duct enhances our understanding of pancreatic HCO(3)(-) secretion in species that share a high HCO(3)(-) secretory output.

Regulated transport of sulfate and oxalate by SLC26A2/DTDST.

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.

The GPA-dependent, spherostomatocytosis mutant AE1 E758K induces GPA-independent, endogenous cation transport in amphibian oocytes.

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 (36)Cl(-) influx, trans-anion-dependent (36)Cl(-) efflux, and Cl(-)/HCO(3)(-) exchange activities at near wild-type levels. AE1 E758K expression was also associated with GPA-dependent increases of DIDS-sensitive pH-independent SO(4)(2-) uptake and oxalate uptake with altered pH dependence. In marked contrast, the bumetanide- and ouabain-insensitive (86)Rb(+) 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. (86)Rb(+) 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 (36)Cl(-) influx differed from that of AE1 E758K-associated (86)Rb(+) influx, as well as from that of wild-type AE1-mediated Cl(-) transport. Thus AE1 E758K-expressing oocytes displayed GPA-dependent surface polypeptide expression and anion transport, accompanied by substantially GPA-independent, pharmacologically distinct Rb(+) 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.

Pendrin function and regulation in Xenopus oocytes.

SLC26A4/PDS mutations cause Pendred Syndrome and non-syndromic deafness. but some aspects of function and regulation of the SLC26A4 polypeptide gene product, pendrin, remain controversial or incompletely understood. We have therefore extended the functional analysis of wildtype and mutant pendrin in Xenopus oocytes, with studies of isotopic flux, electrophysiology, and protein localization. Pendrin mediated electroneutral, pH-insensitive, DIDS-insensitive anion exchange, with extracellular K(1/2) (in mM) of 1.9 (Cl-), 1.8 (I-), and 0.9 (Br-). The unusual phenotype of Pendred Syndrome mutation E303Q (loss-of-function with normal surface expression) prompted systematic mutagenesis at position 303. Only mutant E303K exhibited loss-of-function unrescued by forced overexpression. Mutant E303C was insensitive to charge modification by methanethiosulfonates. The corresponding mutants SLC26A2 E336Q, SLC26A3 E293Q, and SLC26A6 E298Q exhibited similar loss-of-function phenotypes, with wildtype surface expression also documented for SLC26A2 E336Q. The strong inhibition of wildtype SLC26A2, SLC26A3, and SLC26A6 by phorbol ester contrasts with its modest inhibition of pendrin. Phorbol ester inhibition of SLC26A2, SLC26A3, and SLC26A6 was blocked by coexpressed kinase-dead PKCδ but was without effect on pendrin. Mutation of SLC26A2 serine residues conserved in PKCδ -sensitive SLC26 proteins but absent from pendrin failed to reduce PKCδ sensitivity of SLC26A2 (190).

BH3 domain-independent apolipoprotein L1 toxicity rescued by BCL2 prosurvival proteins.

The potent trypanolytic properties of human apolipoprotein L1 (APOL1) can be neutralized by the trypanosome variant surface antigen gene product known as serum resistance-associated protein. However, two common APOL1 haplotypes present uniquely in individuals of West African ancestry each encode APOL1 variants resistant to serum resistance-associated protein, and each confers substantial resistance to human African sleeping sickness. In contrast to the dominantly inherited anti-trypanosomal activity of APOL1, recessive inheritance of these two trypanoprotective APOL1 alleles predisposes to kidney disease. Proposed mechanisms of APOL1 toxicity have included BH3 domain-dependent autophagy and/or ion channel activity. We probed these potential mechanisms by expressing APOL1 in Xenopus laevis oocytes. APOL1 expression in oocytes increased ion permeability and caused profound morphological deterioration (toxicity). Coexpression of BCL2 family members rescued APOL1-associated oocyte toxicity in the order MCL1 ∼ BCLW > BCLXL ∼ BCL2A1 ≫ BCL2. Deletion of nine nominal core BH3 domain residues abolished APOL1-associated toxicity, but missense substitution of the same residues abolished neither oocyte toxicity nor its rescue by coexpressed MCL1. The APOL1 BH3 domain was similarly dispensable for the ability of APOL1 to rescue intact mice from lethal trypanosome challenge. Replacement of most extracellular Na(+) by K(+) also reduced APOL1-associated oocyte toxicity, allowing demonstration of APOL1-associated increases in Ca(2+) and Cl(-) fluxes and oocyte ion currents, which were similarly reduced by MCL1 coexpression. Thus APOL1 toxicity in Xenopus oocytes is BH3-independent, but can nonetheless be rescued by some BCL2 family proteins.

Loss-of-function and gain-of-function phenotypes of stomatocytosis mutant RhAG F65S

Four patients with overhydrated cation leak stomatocytosis (OHSt) exhibited the heterozygous RhAG missense mutation F65S. OHSt erythrocytes were osmotically fragile, with elevated Na and decreased K contents and increased cation channel-like activity. Xenopus oocytes expressing wild-type RhAG and RhAG F65S exhibited increased ouabain and bumetanide-resistant uptake of Li(+) and (86)Rb(+), with secondarily increased (86)Rb(+) influx sensitive to ouabain and to bumetanide. Increased RhAG-associated (14)C-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li(+), (86)Rb(+), and (14)C-MA were pharmacologically distinct, and Li(+) uptakes associated with RhAG and RhAG F65S were differentially inhibited by NH(4)(+) and Gd(3+). RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH(3)/NH(4)(+), but alkalinized and depolarized by subsequent bath exposure to 5 mM methylammonium chloride (MA/MA(+)). RhAG F65S-expressing oocytes exhibited near-wild-type responses to NH(4)Cl, but MA/MA(+) elicited attenuated alkalinization and strong hyperpolarization. Expression of RhAG or RhAG F65S increased steady-state cation currents unaltered by bath Li(+) substitution or bath addition of 5 mM NH(4)Cl or MA/MA(+). These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH(3)/NH(4)(+) and MA/MA(+); 2) RhAG F65S exhibits gain-of-function phenotypes of increased cation conductance/permeability, and loss-of-function phenotypes of decreased and modified MA/MA(+) transport, and decreased NH(3)/NH(4)(+)-associated depolarization; and 3) RhAG transports NH(3)/NH(4)(+) and MA/MA(+) by distinct mechanisms, and/or the substrates elicit distinct cellular responses. Thus, RhAG F65S is a loss-of-function mutation for amine transport. The altered oocyte intracellular pH, membrane potential, and currents associated with RhAG or RhAG F65S expression may reflect distinct transport mechanisms.

L- and N-current but not M-current inhibition by M1 muscarinic receptors requires DAG lipase activity†

Stimulation of postsynaptic M1 muscarinic receptors (M1Rs) increases firing rates of both sympathetic and central neurons that underlie increases in vasomotor tone, heart rate, and cognitive memory functioning. At the cellular level, M1R stimulation modulates currents through various voltage‐gated ion channels, including KCNQ K+ channels (M‐current) and both L‐ and N‐type Ca2+ channels (L‐ and N‐current) by a pertussis toxin‐insensitive, slow signaling pathway. Depletion of phosphatidylinositol‐4,5‐bisphosphate (PIP2) during M1R stimulation suffices to inhibit M‐current. We found previously that following PIP2 hydrolysis by phospholipase C, activation of phospholipase A2 and liberation of a lipid metabolite, most likely arachidonic acid (AA) are necessary for L‐ and N‐current modulation. Here we examined the involvement of a third lipase, diacylglycerol lipase (DAGL), in the slow pathway. We documented the presence of DAGL in superior cervical ganglion neurons, and then tested the highly selective DAGL inhibitor, RHC‐80267, for its capacity to antagonize M1R‐mediated modulation of whole‐cell Ca2+ currents. RHC‐80267 significantly reduced L‐ and N‐current inhibition by the muscarinic agonist oxotremorine‐M (Oxo‐M) but did not affect their inhibition by exogenous AA. Moreover, voltage‐dependent inhibition of N‐current by Oxo‐M remained in the presence of RHC‐80267, indicating selective action on the slow pathway. RHC also blocked inhibition of recombinant N‐current. In contrast, RHC‐80267 had no effect on native M‐current inhibition. These data are consistent with a role for DAGL in mediating L‐ and N‐current inhibition. These results extend our previous findings that the signaling pathway mediating L‐ and N‐current inhibition diverges from the pathway initiating M‐current inhibition. J. Cell. Physiol. 216: 91–100, 2008.