Identification and characterization of a novel mammalian Mg2+ transporter with channel-like properties

Abstract

BackgroundIntracellular magnesium is abundant, highly regulated and plays an important role in biochemical functions. Despite the extensive evidence for unique mammalian Mg2+ transporters, few proteins have been biochemically identified to date that fulfill this role. We have shown that epithelial magnesium conservation is controlled, in part, by differential gene expression leading to regulation of Mg2+ transport. We used this knowledge to identify a novel gene that is regulated by magnesium.ResultsOligonucleotide microarray analysis was used to identify a novel human gene that encodes a protein involved with Mg2+-evoked transport. We have designated this magnesium transporter (MagT1) protein. MagT1 is a novel protein with no amino acid sequence identity to other known transporters. The corresponding cDNA comprises an open reading frame of 1005 base pairs encoding a protein of 335 amino acids. It possesses five putative transmembrane (TM) regions with a cleavage site, a N-glycosylation site, and a number of phosphorylation sites. Based on Northern analysis of mouse tissues, a 2.4 kilobase transcript is present in many tissues. When expressed in Xenopus laevis oocytes, MagT1 mediates saturable Mg2+ uptake with a Michaelis constant of 0.23 mM. Transport of Mg2+ by MagT1 is rheogenic, voltage-dependent, does not display any time-dependent inactivation. Transport is very specific to Mg2+ as other divalent cations did not evoke currents. Large external concentrations of some cations inhibited Mg2+ transport (Ni2+, Zn2+, Mn2+) in MagT1-expressing oocytes. Ca2+and Fe2+ were without effect. Real-time reverse transcription polymerase chain reaction and Western blot analysis using a specific antibody demonstrated that MagT1 mRNA and protein is increased by about 2.1-fold and 32%, respectively, in kidney epithelial cells cultured in low magnesium media relative to normal media and in kidney cortex of mice maintained on low magnesium diets compared to those animals consuming normal diets. Accordingly, it is apparent that an increase in mRNA levels is translated into higher protein expression.ConclusionThese studies suggest that MagT1 may provide a selective and regulated pathway for Mg2+ transport in epithelial cells.

Huntingtin-interacting Proteins, HIP14 and HIP14L, Mediate Dual Functions, Palmitoyl Acyltransferase and Mg2+ Transport

“…HIP14 and HIP14L Are Magnesium-responsive Genes-With the knowledge that differential gene expression is involved with selective control of epithelial cell magnesium conservation, our strategy was to use microarray analysis to identify cDNAs that were up-regulated with low magnesium (12). We used RNA from immortalized mouse distal convoluted tubule epithelial cells cultured in media containing normal magnesium concentration or nominally magnesium-free media for 5 h prior to RNA harvest.…”

“…Oligonucleotide Microarray Analysis-Microarray analysis was performed according to the protocol recommended by Affymetrix (www.affymetric.com) using MG U74 Bv2 and MG U74 Cv2 arrays (Affymetrix, Santa Clara, CA) as described previously (12). DNA fragments representing transcripts that were up-regulated with low magnesium were selected and prioritized according to properties characteristic of membrane transport proteins.…”

“…Oocytes were studied 3-5 days following injec-tion. Permeability ratios were calculated using the Nernst relation and apparent K m and V max values with Eadie-Hofstee analysis using non-linear regression analysis (12).…”

“…In an attempt to identify genes underlying cellular changes resulting from adaptation to low extracellular magnesium, we used oligonucleotide microarray analysis to screen for magnesium-regulated transcripts in epithelial cells (12). One transcript, HIP14L, was significantly up-regulated by low extracellular magnesium suggesting that the synthesis was regulated by changes in cell magnesium.…”

Transport of cations and anions across forestomach epithelia: conclusions from in vitro studies

“…Ammonia also interferes with ruminal Mg 21 absorption (Martens and Rayssiguier, 1980), with effects on both the electrogenic and the electroneutral component (S. LeonhardMarek and H. Martens, unpublished data (Goytain and Quamme, 2005;Kozak et al, 2005). It thus seems difficult to hypothesize mechanisms of adaptation that would explain why the effect of ammonia on ruminal Mg absorption disappears within some days as observed by Gä bel and Martens (1986).…”