Marc J. Bergeron

Chloride extrusion enhancers as novel therapeutics for neurological diseases

The K+-Cl− cotransporter KCC2 is responsible for maintaining low Cl− concentration in neurons of the central nervous system (CNS), which is essential for postsynaptic inhibition through GABAA and glycine receptors. Although no CNS disorders have been associated with KCC2 mutations, loss of activity of this transporter has emerged as a key mechanism underlying several neurological and psychiatric disorders, including epilepsy, motor spasticity, stress, anxiety, schizophrenia, morphine-induced hyperalgesia and chronic pain. Recent reports indicate that enhancing KCC2 activity may be the favored therapeutic strategy to restore inhibition and normal function in pathological conditions involving impaired Cl− transport. We designed an assay for high-throughput screening that led to the identification of KCC2 activators that reduce intracellular chloride concentration ([Cl−]i). Optimization of a first-in-class arylmethylidine family of compounds resulted in a KCC2-selective analog (CLP257) that lowers [Cl−]i. CLP257 restored impaired Cl− transport in neurons with diminished KCC2 activity. The compound rescued KCC2 plasma membrane expression, renormalized stimulus-evoked responses in spinal nociceptive pathways sensitized after nerve injury and alleviated hypersensitivity in a rat model of neuropathic pain. Oral efficacy for analgesia equivalent to that of pregabalin but without motor impairment was achievable with a CLP257 prodrug. These results validate KCC2 as a druggable target for CNS diseases.

Homooligomeric and heterooligomeric associations between K+-Cl- cotransporter isoforms and between K+-Cl- and Na+-K+-Cl- cotransporters.

Little is known regarding the quaternary structure of cation-Cl– cotransporters (CCCs) except that the Na+-dependent CCCs can exist as homooligomeric units. Given that each of the CCCs exhibits unique functional properties and that several of these carriers coexist in various cell types, it would be of interest to determine whether the four K+-Cl– cotransporter (KCC) isoforms and their splice variants can also assemble into such units and, more importantly, whether they can form heterooligomers by interacting with each other or with the secretory Na+-K+-Cl– cotransporter (NKCC1). In the present work, we have addressed these questions by conducting two groups of analyses: 1) yeast two-hybrid and pull-down assays in which CCC-derived protein segments were used as both bait and prey and 2) coimmunoprecipitation and functional studies of intact CCCs coexpressed in Xenopus laevis oocytes. Through a combination of such analyses, we have found that KCC2 and KCC4 could adopt various oligomeric states (in the form of KCC2-KCC2, KCC4-KCC4, KCC2-KCC4, and even KCC4-NKCC1 complexes), that their carboxyl termini were probably involved in carrier assembly, and that the KCC4-NKCC1 oligomers, more specifically, could deploy unique functional features. Through additional coimmunoprecipitation studies, we have also found that KCC1 and KCC3 had the potential of assembling into various types of CCC-CCC oligomers as well, although the interactions uncovered were not characterized as extensively, and the protein segments involved were not identified in yeast two-hybrid assays. Taken together, these findings could change our views on how CCCs operate or are regulated in animal cells by suggesting, in particular, that cation-Cl– cotransport achieves higher levels of functional diversity than foreseen.

Trypanosoma cruzi-Mediated IFN-γ-Inducible Nitric Oxide Output in Macrophages Is Regulated by iNOS mRNA Stability

Although the effects of activated macrophages (Μφ) on the intracellular parasite Trypanosoma cruzi are well documented, little is known about how host-Μφ functions are affected by this pathogen before activation. This study is aimed at assessing the capacity of T. cruzi infection to modulate J77.4 murine Μφ NO generation following IFN-γ stimulation, and identifying mechanisms regulating this modulation. Results show that parasite infection potentiates Μφ to produce inducible NO synthase (iNOS) mRNA and protein as well as NO following IFN-γ stimulation above IFN-γ alone controls. This potentiation occurs through the concomitant activation of NF-κB, ERK1/ERK2 MAPK, and stress-activated protein kinase signaling pathways. Activation of the JAK/STAT pathway by IFN-γ then leads to STAT1α translocation and the transcription of a stable iNOS mRNA species. A decreased rate of iNOS mRNA degradation results in elevated levels of iNOS protein and NO production. Maximal iNOS expression is likely achieved through NF-κB activation by T. cruzi, whereas iNOS mRNA stability results from ERK1/ERK2 MAPK and stress-activated protein kinase activation by the infection. Taken together, our data show that T. cruzi-infected Μφ NO generation is controlled at both pre- and posttranscriptional levels and relies on signaling pathway cross-talk. This is the first report of a parasite pathogen capable of heightening host mRNA stability.

Identification of Key Functional Domains in the C Terminus of the K+-Cl– Cotransporters

The K+-Cl– cotransporter (KCC) isoforms constitute a functionally heterogeneous group of ion carriers. Emerging evidence suggests that the C terminus (Ct) of these proteins is important in conveying isoform-specific traits and that it may harbor interacting sites for 4β-phorbol 12-myristate 13-acetate (PMA)-induced effectors. In this study, we have generated KCC2-KCC4 chimeras to identify key functional domains in the Ct of these carriers and single point mutations to determine whether canonical protein kinase C sites underlie KCC2-specific behaviors. Functional characterization of wild-type (wt) and mutant carriers in Xenopus laevis oocytes showed for the first time that the KCCs do not exhibit similar sensitivities to changes in osmolality and that this distinguishing feature as well as differences in transport activity under both hypotonic and isotonic conditions are in part determined by the residue composition of the distal Ct. At the same time, several mutations in this domain and in the proximal Ct of the KCCs were found to generate allosteric-like effects, suggesting that the regions analyzed are important in defining conformational ensembles and that isoform-specific structural configurations could thus account for variant functional traits as well. Characterization of the other mutants in this work showed that KCC2 is not inhibited by PMA through phosphorylation of its canonical protein kinase C sites. Intriguingly, however, the substitutions N728S and S940A were seen to alter the PMA effect paradoxically, suggesting again that allosteric changes in the Ct are important determinants of transport activity and, furthermore, that the structural configuration of this domain can convey specific functional traits by defining the accessibility of cotransporter sites to regulatory intermediates such as PMA-induced effectors.

Molecular Mechanisms of Cl- Transport by the Renal Na+-K+-Cl- Cotransporter IDENTIFICATION OF AN INTRACELLULAR LOCUS THAT MAY FORM PART OF A HIGH AFFINITY Cl--BINDING SITE

The 2nd transmembrane domain (tm) of the secretory Na+-K+-Cl- cotransporter (NKCC1) and of the kidney-specific isoform (NKCC2) has been shown to play an important role in cation transport. For NKCC2, by way of illustration, alternative splicing of exon 4, a 96-bp sequence from which tm2 is derived, leads to the formation of the NKCC2A and F variants that both exhibit unique affinities for cations. Of interest, the NKCC2 variants also exhibit substantial differences in Cl- affinity as well as in the residue composition of the first intracellular connecting segment (cs1a), which immediately follows tm2 and which too is derived from exon 4. In this study, we have prepared chimeras of the shark NKCC2A and F (saA and saF) to determine whether cs1a could play a role in Cl- transport; here, tm2 or cs1a in saF was replaced by the corresponding domain from saA (generating saA/F or saF/A, respectively). Functional analyses of these chimeras have shown that cs1a-specific residues account for most of the A-F difference in Cl- affinity. For example, Km(Cl-)s were ∼8 mm for saF/A and saA, and ∼70 mm for saA/F and saF. Intriguingly, variant residues in cs1a also affected cation transport; here, Km(Na+)s for the chimeras and for saA were all ∼20 mm, and Km(Rb+) all ∼2 mm. Regarding tm2, our studies have confirmed its importance in cation transport and have also identified novel properties for this domain. Taken together, our results demonstrate for the first time that an intracellular loop in NKCC contributes to the transport process perhaps by forming a flexible structure that positions itself between membrane spanning domains.

Novel Insights Regarding the Operational Characteristics and Teleological Purpose of the Renal Na+-K+-Cl2 Cotransporter (NKCC2s) Splice Variants

The absorptive Na+-K+-Cl− cotransporter (NKCC2) is a polytopic protein that forms homooligomeric complexes in the apical membrane of the thick ascending loop of Henle (TAL). It occurs in at least four splice variants (called B, A, F, and AF) that are identical to one another except for a short region in the membrane-associated domain. Although each of these variants exhibits unique functional properties and distributions along the TAL, their teleological purpose and structural organization remain poorly defined. In the current work, we provide additional insight in these regards by showing in mouse that the administration of either furosemide or an H2O-rich diet, which are predicted to alter NKCC2 expression in the TAL, exerts differential effects on mRNA levels for the variants, increasing those of A (furosemide) but decreasing those of F and AF (furosemide or H2O). Based on a yeast two-hybrid mapping analysis, we also show that the formation of homooligomeric complexes is mediated by two self-interacting domains in the COOH terminus (residues 671 to 816 and 910 to 1098), and that these complexes could probably include more than one type of variant. Taken together, the data reported here suggest that A, F, and AF each play unique roles that are adapted to specific physiological needs, and that the accomplishment of such roles is coordinated through the splicing machinery as well as complex NKCC2–NKCC2 interactions.

Phosphoregulation of K+‐Cl− cotransporter 4 during changes in intracellular Cl− and cell volume

It has long been stated that the K+‐Cl− cotransporters (KCCs) are activated during cell swelling through dephosphorylation of their cytoplasmic domains by a protein phosphatase (PP) but that other enzymes are involved by targeting this PP or the KCCs directly. To date, however, the role of signaling intermediates in KCC regulation has been deduced from indirect evidence rather than in vitro phosphorylation studies, and examined after simulation of ion transport through cell swelling or N‐ethylmaleimide treatment. In this study, the oocyte expression system was used to examine the effects of changes in cell volume (CVOL) and intracellular [Cl−] ([Cl−]i) on the activity and phosphorylation levels (PLEV) of KCC4, and determine whether these effects are mediated by PP1 or phorbol myristate acetate (PMA)‐sensitive effectors. We found that (1) low [Cl−]i or low CVOL leads to decreased activity but increased PLEV, (2) high CVOL leads to increased activity but no decrease in PLEV and (3) calyculin A (Cal A) or PMA treatment leads to decreased activity but no increase in PLEV. Thus, we have shown for the first time that one of the KCCs can be regulated through direct phosphorylation, that changes in [Cl−]i or CVOL modify the activity of signaling enzymes at carrier sites, and that the effectors directly involved do not include a Cal A‐sensitive PP in contrast to the widely held view. J. Cell. Physiol. 219: 787–796, 2009.

Frog Oocytes to Unveil the Structure and Supramolecular Organization of Human Transport Proteins

Structural analyses of heterologously expressed mammalian membrane proteins remain a great challenge given that microgram to milligram amounts of correctly folded and highly purified proteins are required. Here, we present a novel method for the expression and affinity purification of recombinant mammalian and in particular human transport proteins in Xenopus laevis frog oocytes. The method was validated for four human and one murine transporter. Negative stain transmission electron microscopy (TEM) and single particle analysis (SPA) of two of these transporters, i.e., the potassium-chloride cotransporter 4 (KCC4) and the aquaporin-1 (AQP1) water channel, revealed the expected quaternary structures within homogeneous preparations, and thus correct protein folding and assembly. This is the first time a cation-chloride cotransporter (SLC12) family member is isolated, and its shape, dimensions, low-resolution structure and oligomeric state determined by TEM, i.e., by a direct method. Finally, we were able to grow 2D crystals of human AQP1. The ability of AQP1 to crystallize was a strong indicator for the structural integrity of the purified recombinant protein. This approach will open the way for the structure determination of many human membrane transporters taking full advantage of the Xenopus laevis oocyte expression system that generally yields robust functional expression.

Synthesis, maturation and trafficking of human Na+-dicarboxylate cotransporter NaDC1 requires the chaperone activity of cyclophilin B

Renal excretion of citrate, an inhibitor of calcium stone formation, is controlled mainly by reabsorption via the apical Na+-dicarboxylate cotransporter NaDC1 (SLC13A2) in the proximal tubule. Recently, it has been shown that the protein phosphatase calcineurin inhibitors cyclosporin A (CsA) and FK-506 induce hypocitraturia, a risk factor for nephrolithiasis in kidney transplant patients, but apparently through urine acidification. This suggests that these agents up-regulate NaDC1 activity. Using the Xenopus lævis oocyte and HEK293 cell expression systems, we examined first the effect of both anti-calcineurins on NaDC1 activity and expression. While FK-506 had no effect, CsA reduced NaDC1-mediated citrate transport by lowering heterologous carrier expression (as well as endogenous carrier expression in HEK293 cells), indicating that calcineurin is not involved. Given that CsA also binds specifically to cyclophilins, we determined next whether such proteins could account for the observed changes by examining the effect of selected cyclophilin wild types and mutants on NaDC1 activity and cyclophilin-specific siRNA. Interestingly, our data show that the cyclophilin isoform B is likely responsible for down-regulation of carrier expression by CsA and that it does so via its chaperone activity on NaDC1 (by direct interaction) rather than its rotamase activity. We have thus identified for the first time a regulatory partner for NaDC1, and have gained novel mechanistic insight into the effect of CsA on renal citrate transport and kidney stone disease, as well as into the regulation of membrane transporters in general.

Reply to The small molecule CLP257 does not modify activity of the K + –Cl − co-transporter KCC2 but does potentiate GABA A receptor activity

Reply to The small molecule CLP257 does not modify activity of the K + –Cl − co-transporter KCC2 but does potentiate GABA A receptor activity