A zinc-sensing receptor triggers the release of intracellular Ca ²⁺ and regulates ion transport

Abstract

Changes in extracellular zinc concentration participate in modulating fundamental cellular processes such as proliferation, secretion, and ion transport in a mechanism that is not well understood. Here, we show that a micromolar concentration of extracellular zinc triggers a massive release of calcium from thapsigarginsensitive intracellular pools in the colonocytic cell line HT29. Calcium release was blocked by a phospholipase-C inhibitor, indicating that formation of inositol 1,4,5-triphosphate is required for zinc-dependent calcium release. Zinc influx was not observed, indicating that extracellular zinc triggered the release. The Ca i 2؉ release was zinc specific and could not be triggered by other heavy metals. Furthermore, zinc failed to activate the Ca 2؉ -sensing receptor heterologously expressed in HEK293 cells. The zinc-induced Ca i 2؉ rise stimulated the activity of the Na ؉ ͞H ؉ exchanger in HT29 cells. Our results indicate that a previously uncharacterized extracellular, G protein-coupled, Zn 2؉ -sensing receptor is functional in colonocytes. Because Ca i 2؉ rise is known to regulate key cellular and signal-transduction processes, the zinc-sensing receptor may provide the missing link between extracellular zinc concentration changes and the regulation of cellular processes.Z inc is an essential micronutrient involved in structural and regulatory cellular functions. Zinc interacts with zinc-finger domains and acts as a cofactor of numerous enzymes (1, 2). Zinc ions also specifically bind to many membrane receptors, transporters, and channels, thereby modulating their activity (3). Therefore, it is not surprising that zinc deficiency affects multiple organs, including the digestive (4), immune (5), and neuronal (2) systems. A severe lack of zinc also is linked to the attenuation of growth and sexual development (2). Conversely, an excess of extracellular zinc is considered toxic. Indeed, brain ischemia is accompanied by a massive release of synaptic zinc permeating into neurons, leading to neuronal cell death (6-8). Furthermore, striking changes in plasma-zinc concentration occur during diverse pathophysiological syndromes including myocardial infarction, hepatic renal failure, and neoplastic processes (9). Despite the large fluctuations in extracellular zinc concentrations and their subsequent clinical importance, little is known about cellular signaling mechanisms that sense changes in extracellular-zinc concentration.The calcium-sensing receptor serves as an example for the importance of an ion-sensing mechanism. It is a G proteincoupled receptor that senses changes in extracellular calcium and regulates diverse cellular functions (10, 11). Although the existence of other ion-sensing mechanisms have been suggested (12), none have been characterized fully yet.Gastrointestinal pathology, manifested in severe diarrhea, is an important hallmark of zinc deficiency. Indeed, zinc has a major role in the duration and severity of diarrheal diseases (4). The morbidity and mortality caused by diarrheal diseas...

The zinc sensing receptor, ZnR/GPR39, triggers metabotropic calcium signalling in colonocytes and regulates occludin recovery in experimental colitis

“…Consistently, zinc supplementation to Crohn's disease patients during clinical remission or in animal models of experimental colitis reduced intestinal permeability and mucosal damage [12][13][14][15]. A specific Zn 2þ sensing receptor (ZnR) was functionally described in this tissue [16,17] and was later associated with GPR39 as the molecular moiety mediating its activity [18,19]. The ZnR/GPR39 triggers cellular Ca 2þ signalling pathways and enhances cell proliferation, differentiation and survival [20].…”

Identification of the Zn2+ Binding Site and Mode of Operation of a Mammalian Zn2+ Transporter

“…Fluorescent Imaging of Ion Transport-Zn 2ϩ transport was determined in cells loaded with either 3.5 M Fura-2 AM (TEFLabs), 0.5 M FluoZin-3 AM, or 5 M Newport Green AM (Invitrogen), using an imaging system as described previously (21). To verify that the fluorescence changes were related to intracellular Zn 2ϩ , the cell-permeable heavy metal chelator N,N,NЈ,NЈ-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN; 20 M) was used.…”

Lithium-Calcium Exchange Is Mediated by a Distinct Potassium-independent Sodium-Calcium Exchanger

“…Cell Cultures and Plasmid Transfection-HEK293-T cells (human embryonic kidney cell line) were cultured in Dulbecco's modified Eagle's medium as described previously (15). Briefly, cells grown on glass coverslips were transfected with 8 g of NCLX, control, or rat NCX1 (kindly provided by Dr. Hanna Rahamimoff, The Hebrew University, Jerusalem, Israel) using standard calcium phosphate (CaPO 4 ) precipitation as described previously (12).…”

“…The first consisted of a Zeiss Axiovert 100 microscope (Zeiss) and the second consisted of an Olympus IX (Olympus) inverted microscope, Polychrome II monochromator (T.I.L.L. Photonics) equipped with a cooled CCD camera (PCO Imaging) as described previously (15). Fluorescent imaging measurements were acquired using Axon Imaging Workbench 2 software (Axon Instruments, Foster City, CA).…”