Sialin (SLC17A5) functions as a nitrate transporter in the plasma membrane Lizheng Qina,1, Xibao Liub,1, Qifei Suna, Zhipeng Fana, Dengsheng Xiaa, Gang Dinga, Hwei Ling Ongb, David Adamsc, William A. Gahlc, Changyu Zhengb, Senrong Qia, Luyuan Jina, Chunmei Zhanga, Liankun Gud, Junqi Hee, Dajun Dengd,2, Indu S. Ambudkarb,2, and Songlin Wanga,e,2 aSalivary Gland Disease Center and Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction, Capital Medical University School of Stomatology, Beijing 100050, China; bMolecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, Bethesda, MD 20892; cMedical Genetics Branch, National Human Genome Research Institute, Bethesda, MD 20892; dKey Laboratory of Carcinogenesis and Translational Research, Peking University Cancer Hospital and Institute, Beijing 100142, China; and eDepartment of Biochemistry and Molecular Biology, Capital Medical University School of Basic Medicine, Beijing 100069, China Edited by Mark Gladwin, University of Pittsburg Medical Center, Pittsburgh, PA and accepted by the Editorial Board June 5, 2012 (received for review October 13, 2011) In vivo recycling of nitrate (NO −) and nitrite (NO −) is an important Nitrate uptake into salivary glands represents the key initial step 3 2 − alternative pathway for the generation of nitric oxide (NO) and in NO clearance from the serum; however, the mechanism me- 3 − maintenance of systemic nitrate–nitrite–NO balance. More than diating transport of NO3 in salivary gland epithelial cells has not − − fl 25% of the circulating NO3 is actively removed and secreted by yet been established. In this study, we examined NO3 in ux in − + salivary glands. Oral commensal bacteria convert salivary NO3 to salivary gland cells. Here we report that the sialic acid (SA)/H − SLC17A5 NO2 , which enters circulation and leads to NO generation. The cotransporter, sialin ( ), mutations in which result in Salla − transporters for NO3 in salivary glands have not yet been identi- disease and ISSD, is involved in nitrate uptake into salivary glands. fied. Here we report that sialin (SLC17A5), mutations in which cause Our data suggest a similar function for sialin in several other cell fi Salla disease and infantile sialic acid storage disorder (ISSD), func- types as well, including broblasts. We show that sialin is endoge- − + nously localized in the lysosomes as well as in the plasma membrane tions as an electrogenic 2NO3 /H cotransporter in the plasma − fi of salivary gland cells, where it functions as an electrogenic 2NO / membrane of salivary gland acinar cells. We have identi ed an ex- + 3 − H cotransporter mediating influx of nitrate into the cell. We also tracellular pH-dependent anion current that is carried by NO3 or − provide evidence that plasma membrane sialin is a multifunctional sialic acid (SA), but not by Br , and is accompanied by intracellular − + acidification. Both responses were reduced by knockdown of sialin anion transporter that can mediate electrogenic A /H cotransport expression and increased by the plasma membrane-targeted sialin of anions such as SA, glutamate, and aspartate. Importantly, we mutant (L22A-L23A). Fibroblasts from patients with ISSD displayed have assessed the function of sialin in vivo in pig salivary glands and − provide evidence for the physiological relevance of sialin in medi- reduced SA- and NO -induced currents compared with healthy − 3 ating nitrate uptake NO influx into pig salivary glands. In ag- controls. Furthermore, expression of disease-associated sialin mu- 3 + gregate, our findings suggest that sialin is a versatile anion tants in fibroblasts and salivary gland cells suppressed the H -de- − transporter, and that functional defects in the protein may have pendent NO conductance. Importantly, adenovirus-dependent 3 a deleterious impact on several critical physiological functions. expression of the sialinH183R mutant in vivo in pig salivary glands − − decreased NO3 secretion in saliva after intake of a NO3 -rich diet. Results Taken together, these data demonstrate that sialin mediates nitrate − Coupled NO Currents and Intracellular Acidification in Human influx into salivary gland and other cell types. We suggest that the 3 − + Submandibular Gland Cells. Human submandibular gland cell line 2NO /H transport function of sialin in salivary glands can contrib- 3 (HSG) cells did not display constitutive currents in standard ex- ute significantly to clearance of serum nitrate, as well as nitrate − − tracellular solution. Replacement of Cl with 150 mM NO3 recycling and physiological nitrite-NO homeostasis. produced a relatively slow, but significant, spontaneous increase in the outward current that was dramatically enhanced by decreasing pH | proton theexternalpHfrom7.4to4.0(Fig.1A); the current density in the − ± n = − − 150 mM NO3 solution was 42.4 5.4 pA/pF ( 21). Both the he anions NO3 and NO2 were once thought to be inert end constitutive and low pH-induced currents had similar outwardly Tproducts of NO metabolism. However, it is now evident that rectifying characteristics with a reversal potential of −15 ± 2mV nitrate and nitrite can be recycled in vivo to form NO, and thus (n = 19) (Fig. 1B). Low pH also increased the outward current in − these anions complement the nitric oxide synthase (NOS)-de- normal standard extracellular solution (Cl -containing), although − pendent activity (1). The nitrate-nitrite-NO pathway is emerging the amplitude of the current was smaller than that seen with NO3 as a potential therapeutic target in such diseases as myocardial (23.1 ± 4.3 pA/pF; n = 18) (Fig. 1C), and the current reversed at infarction, stroke, gastric ulcers, and pulmonary hypertension (1, 2). There are two major sources of nitrate and nitrite: the L-arginine–NO synthase pathway and diet. Dietary intake of ni- − Author contributions: L.Q., X.L., D.D., I.S.A., and S.W. designed research; L.Q., X.L., Q.S., Z. trate leads to a relatively rapid increase in NO3 concentration F., D.X., G.D., H.L.O., D.A., W.A.G., C. Zheng, S.Q., L.J., L.G., and S.W. performed research; in serum. Although a large part of the anion is excreted via the L.Q., X.L., C. Zheng, and L.J. contributed new reagents/analytic tools; L.Q., X.L., C. Zheng, kidneys, up to 25% of the circulating nitrate is actively taken up L.J., C. Zhang, J.H., D.D., I.S.A., and S.W. analyzed data; and L.Q., X.L., D.D., I.S.A., and S.W. by the salivary glands and concentrated ∼10-fold in the saliva wrote the paper. secreted from the glands (3–5). Conditions that compromise The authors declare no conflict of interest. − salivary gland function have been linked to decreased NO se- This article is a PNAS Direct Submission. M.G. is a guest editor invited by the Editorial − 3 Board. cretion from the salivary glands and increased NO3 levels in the serum and urine (5, 6). Although nitrate can be reduced to nitrite See Commentary on page 13144. by the commensal bacteria in the oral cavity, most of the salivary 1L.Q. and X.L. contributed equally to this work. nitrite escapes gastric conversion to NO and enters the systemic 2To whom correspondence may be addressed. E-mail: [email protected], iambudkar@dir. circulation, where it generates NO. Thus, salivary nitrate is nidcr.nih.gov, or [email protected]. − recycled back to NO and is critical for the maintenance of 2 − This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. physiological levels of NO and NO2 in the serum (1, 2, 7). 1073/pnas.1116633109/-/DCSupplemental. 13434–13439 | PNAS | August 14, 2012 | vol. 109 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1116633109 Downloaded by guest on September 28, 2021 SEE COMMENTARY − − Fig. 1. Coupled NO3 currents and intracellular acidification in HSG cells. NO3 currents in HSG cells (A–F) and primary huSMG cells (G) measured by the whole-cell patch-clamp technique. NaCl was replaced with NaNO3, NaBr, or Na-gluconate as indicated. Changes in extracellular pH are shown in the traces − (bar). I-V curves are shown in B and D.(H) External pH and NO3 (5 mM)-dependent acidification of HSG cells measured using BCECF fluorescence. (I)pH − dependence of NO3 currents in HSG cells. (J) Intracellular pH changes under the same experimental conditions as shown in I.(K) Data from I and J were used − to determined the relationship of NO3 currents and intracellular acidification (Hill coefficient: 2.0 ± 0.1). − − 0 mV (Fig. 1D). Decreasing the external pH in Br - or gluconate- conditions demonstrated pH- and NO3 -dependent intracellular containing medium did not generate any detectable current (Fig. 1 acidification (Fig. 1J).Thedatawerefitted (R2 = 0.999) using the E and F). Similar findings were observed in primary human sub- Hill equation, yielding a Hill coefficient of 2.0 ± 0.1 (Fig. 1K), mandibular gland (huSMG) cells (Fig. 1G), human parotid gland consistent with the electrophysiological data. These data strongly − + ductal cells, and freshly dispersed acini cells prepared from mouse suggest that an electrogenic 2NO /H cotransporter is involved in − − 3 salivary glands. Cl channels in salivary gland cells are quite per- mediating NO uptake in salivary gland epithelial cells. − − 3 meable to NO and Br , unlike the activity described here (8–11). 3 − Nonetheless, NO conductance was inhibited by several anion Involvement of Sialin in 2NO −/H+ Cotransport. To examine nitrate 3 − 3 − channel blockers that block various Cl channels (Fig. S1). Fur- transport via salivary gland, [NO ] was measured in the serum − − 3 thermore, unlike known Cl channels in salivary gland cells, NO and saliva of miniature pigs fed with regular or nitrate-rich 3 − conductance was not regulated by cAMP, intracellular or extra- fodder. In both cases, the [NO ] level in saliva exceeded that in PHYSIOLOGY + 3 cellular Ca2 , or muscarinic or purinergic receptor agonists (Fig.