New creatine transporter assay and identification of distinct creatine transporter isoforms in muscle

Abstract

. New creatine transporter assay and identification of distinct creatine transporter isoforms in muscle. Am J Physiol Endocrinol Metab 283: E390-E401, 2002. First published February 26, 2002 10.1152/ajpendo.00428.2001.-Despite the pivotal role of creatine (Cr) and phosphocreatine (PCr) in muscle metabolism, relatively little is known about sarcolemmal creatine transport, creatine transporter (CRT) isoforms, and subcellular localization of the CRT proteins. To be able to quantify creatine transport across the sarcolemma, we have developed a new in vitro assay using rat sarcolemmal giant vesicles. The rat giant sarcolemmal vesicle assay reveals the presence of a specific high-affinity and saturable transport system for Cr in the sarcolemma (Michaelis-Menten constant 52.4 Ϯ 9.4 M and maximal velocity value 17.3 Ϯ 3.1 pmol ⅐ min Ϫ1 ⅐ mg vesicle protein Ϫ1 ), which cotransports Cr into skeletal muscle together with Na ϩ and Cl Ϫ ions. The regulation of Cr transport in giant vesicles by substrates, analogs, and inhibitors, as well as by phorbol 12-myristate 13-acetate and insulin, was studied. Two antibodies raised against COOH-and NH 2-terminal synthetic peptides of CRT sequences both recognize two major polypeptides on Western blots with apparent molecular masses of 70 and 55 kDa, respectively. The highest CRT expression occurs in heart, brain, and kidney, and although creatine kinase is absent in liver cells, CRT is also found in this tissue. Surprisingly, immunofluorescence staining of cultured adult rat heart cardiomyocytes with specific anti-CRT antibodies, as well as cell fractionation and cell surface biotinylation studies, revealed that only a minor CRT species with an intermediate molecular mass of ϳ58 kDa is present in the sarcolemma, whereas the previously identified major CRT-related protein species of 70 and 55 kDa are specifically located in mitochondria. Our studies indicate that mitochondria may represent a major compartment of CRT localization, thus providing a new aspect to the current debate about the existence and whereabouts of intracellular Cr and PCr compartments that have been inferred from [ 14 C]PCr/Cr measurements in vivo as well as from recent in vivo NMR studies.

Novel Mitochondrial Creatine Transport Activity

“…Furthermore, the existence of CRT splice variants has recently been suggested, based on genetics studies using rapid amplification of cDNA ends methods (17). Recent immunofluorescence studies have indicated a high degree of intracellular CRT localization (16), which is consistent with a mainly mitochondrial localization (15). This finding is in contrast to the general view that CRT, represented here by two major ϳ55-and ϳ70-kDa CRT-related proteins, is localized exclusively in the plasma membrane.…”

“…Instead, it was also recently reported that only a minor CRT protein species with an intermediate apparent molecular mass of ϳ58 kDa is located in the plasma membrane (15). This ϳ58-kDa polypeptide has been identified by surface biotinylation of intact cardiomyocytes, followed by Western blotting with anti-CRT antibodies, or alternatively by Western blotting of highly enriched plasma membrane fractions (15). Also in contrast to the prevailing view that mitochondria do not contain Cr, PCr uptake into isolated rat heart mitochondria had been reported earlier (18) and was attributed, however, to the activity of adenine nucleotide translocase.…”

“…Computational analysis revealed that these CRTs, like other neurotransmitter transporters, are integral membrane proteins containing 12 putative transmembrane domains (8). CRT expression has been studied by a few research groups (11)(12)(13)(14)(15)(16). The presence of two different gene products expressed in various tissues corresponding to two major polypeptides of ϳ55 and ϳ70 kDa has been described.…”

“…The two polypeptides are most likely generated by alternative splicing (12). This assumption is supported by the fact that antipeptide antibodies generated against the Nand C-terminal region of the cDNA-derived CRT polypeptide sequence, all recognize the same two proteins with molecular masses of ϳ55 and ϳ70 kDa on Western blots (11)(12)(13)(14)(15)(16). Furthermore, the existence of CRT splice variants has recently been suggested, based on genetics studies using rapid amplification of cDNA ends methods (17).…”

“…This finding is in contrast to the general view that CRT, represented here by two major ϳ55-and ϳ70-kDa CRT-related proteins, is localized exclusively in the plasma membrane. Instead, it was also recently reported that only a minor CRT protein species with an intermediate apparent molecular mass of ϳ58 kDa is located in the plasma membrane (15). This ϳ58-kDa polypeptide has been identified by surface biotinylation of intact cardiomyocytes, followed by Western blotting with anti-CRT antibodies, or alternatively by Western blotting of highly enriched plasma membrane fractions (15).…”