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1.  Investigation of Kir4.1 potassium channel as putative antigen of multiple sclerosis: a cohort study 
The Lancet. Neurology  2014;13(8):795-806.
Antibody-dependent pathogenicity is suggested in multiple sclerosis (MS) by intrathecal immunoglobulin production, IgG and complement deposition in the most common immunopathological lesion subtype (pattern II), and by a recent report that 47% of MS patients’ sera contain a glial potassium-channel-specific-IgG(inwardly-rectifying, Kir4.1). Our study’s aims were to determine, in MS serum and CSF, the frequency and specificity of Kir4.1-binding-IgG and, in demyelinating MS lesions, whether Kir4.1-immunoreactivity is retained or lost.
We tested by ELISA(Kir4.1-peptide 83–120) sera from 286 clinically and geographically diverse MS patients (229 population-based and 57 clinic-based),99 healthy controls and 109 disease controls, and cerebrospinal fluid [CSF] from 25 MS and 22 controls. CSFs and clinic-based MS-subset serum (50)were tested on functional Kir4.1-expressing cells, using methodologies validated for detecting clinically-pertinent neural plasma membrane-reactive autoantibodies: immunofluorescence and immunoprecipitation (solubilized recombinant human Kir4.1). We evaluated Kir4.1-immunoreactivity in brain from 15 archival histopathologically-confirmed MS cases(22 plaques: 8 early active, 8 inactive, 6 remyelinated; 13 periplaque regions)and compared 3 non-neurological cases (8 normal-appearing white/gray matter regions).
Kir4.1-peptide-ELISA reactivity was rare and did not differ significantly for 286 MS or 208 control sera (both 1%); no CSF was positive. IgGin 0/50 clinic-based MS sera immunoprecipitated Kir4.1, but control Kir4.1-specific-IgG did. By immunofluorescence,1/50 MS sera yielded faint plasmalemmal staining on both Kir4.1-expressing and non-expressing cells; 16/50 bound faintly to intracellular components. In all cases, IgG binding was quenched by absorption with liver powder or non-transfected cell lysates. Control Kir4.1-specific-IgG binding was quenched only by Kir4.1 protein-containing lysates. IgG in 0/25 MS CSFs bound to Kir4.1-transfected cells, live or fixed. Glial Kir4.1-immunoreactivity was increased relative to baseline normal brain expression (3 controls) in early active and remyelinated MS lesions, and in periplaque white matter (15 patients).
We did not find Kir4.1-specific-IgG in MS sera or CSF, nor Kir4.1 loss from glial cells in active demyelinating MS lesions. Serological testing for Kir4.1-IgG is unlikely to aid MS diagnosis. The target antigen of MS remains elusive.
The National Institutes of Health, the National Multiple Sclerosis Society and the Mayo Clinic Robert and Arlene Kogod Center on Aging.
PMCID: PMC4144430  PMID: 25008548
2.  Functional analysis of non-synonymous single nucleotide polymorphisms in human SLC26A9 
Human Mutation  2012;33(8):1275-1284.
Slc26 anion transporters play crucial roles in transepithelial Cl− absorption and HCO3− secretion; Slc26 protein mutations lead to several diseases. Slc26a9 functions as a Cl− channel and electrogenic Cl−-HCO3− exchanger, and can interact with CFTR. Slc26a9(−/−) mice have reduced gastric acid secretion, yet no human disease is currently associated with SLC26A9 coding mutations. Therefore, we tested the function of non-synonymous, coding, single nucleotide polymorphisms (cSNPs) of SLC26A9. Presently, eight cSNPs are NCBI-documented: Y70N, T127N, I384T, R575W, P606L, V622L, V744M and H748R. Using two-electrode voltage-clamp and anion selective electrodes, we measured the biophysical consequences of these cSNPs. Y70N (cytoplasmic N-terminus) displays higher channel activity and enhanced Cl−-HCO3− exchange. T127N (transmembrane) results in smaller halide currents but not for SCN−. V622L (STAS domain) and V744M (STAS adjacent) decreased plasma membrane expression which partially accounts for decreased whole cell currents. Nevertheless, V622L transport is reduced to ~50%. SLC26A9 polymorphisms lead to several function modifications (increased activity, decreased activity, altered protein expression) which could lead to a spectrum of pathophysiologies. Thus, knowing an individual’s SLC26A9 genetics becomes important for understanding disease potentially caused by SLC26A9 mutations or modifying diseases, e.g., cystic fibrosis. Our results also provide a framework to understand SLC26A9 transport modalities and structure-function relationships.
PMCID: PMC3399991  PMID: 22544634
SLC26A9; single nucleotide polymorphisms; voltage clamp; Cl− channel; intracellular pH; Xenopus oocytes
3.  Synthesis, biological and antitumor activity of a highly potent 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate inhibitor with proton-coupled folate transporter and folate receptor selectivity over the reduced folate carrier that inhibits β-glycinamide ribonucleotide formyltransferase 
Journal of medicinal chemistry  2011;54(20):7150-7164.
2-Amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine antifolates with a thienoyl side chain (compounds 1–3, respectively) were synthesized for comparison with compound 4, the previous lead compound of this series. Conversion of hydroxyl acetylen-thiophene carboxylic esters to thiophenyl-α-bromomethylketones and condensation with 2,4-diamino-6-hydroxypyrimidine afforded the 6-substituted pyrrolo[2,3-d]pyrimidine compounds of type 18 and 19. Coupling with L-glutamate diethyl ester, followed by saponification, afforded 1–3. Compound 3 selectively inhibited proliferation of cells expressing folate receptors (FRs) α or β, or the proton-coupled folate transporter (PCFT), including human tumor cells KB and IGROV1 much more potently than 4. Compound 3 was more inhibitory than 4 toward β-glycinamide ribonucleotide formyltransferase (GARFTase). Both 3 and 4 depleted cellular ATP pools. In SCID mice with IGROV1 tumors, 3 was more efficacious than 4. Collectively, our results show potent antitumor activity for 3 in vitro and in vivo, associated with its selective membrane transport by FRs and PCFT over RFC and inhibition of GARFTase, clearly establishing the 3-atom bridge as superior to the 1, 2 and 4-atom bridge lengths for the activity of this series.
PMCID: PMC3209708  PMID: 21879757
4.  Slc26A9 - anion exchanger, channel and Na+ transporter 
The Journal of membrane biology  2009;228(3):125-140.
The SLC26 gene family encodes anion transporters with diverse functional attributes: (a) anion exchanger, (b) anion sensor and (c) anion conductance (likely channel). We have cloned and studied Slc26a9, a paralog expressed mostly in lung and stomach. Immunohistochemistry shows that Slc26a9 is present at apical and intracellular membranes of lung and stomach epithelia. Using expression in Xenopus laevis oocytes and ion-sensitive microelectrodes, we discovered that Slc26a9 has a novel function not found in any other Slc26 proteins – cation coupling. Intracellular pH and voltage measurements show that Slc26a9 is a nCl--HCO3- exchanger, suggesting roles in gastric HCl secretion or pulmonary HCO3- secretion; Na+ electrodes and uptakes reveal that Slc26a9 has a cation-dependence. Single channel measurements indicate that Slc26a9 displays discrete open and close states. These experiments show that Slc26a9 has three discrete physiological modes: nCl--HCO3- exchanger, Cl- channel, and Na+-anion cotransporter. Thus, the Slc26a9 transporter-channel is uniquely suited for dynamic and tissue-specific physiology or regulation in epithelial tissues.
PMCID: PMC2733867  PMID: 19365592
intracellular pH; Cl-; Na+; HCO3-; Xenopus oocyte expression; epithelial localization

Results 1-4 (4)