Characterization of organic cation/carnitine transporter family in human sperm

doi:10.1016/S0006-291X(03)00930-6

Biochemical and Biophysical Research Communications

Volume 306, Issue 1, 20 June 2003, Pages 121-128

https://doi.org/10.1016/S0006-291X(03)00930-6 Get rights and content

Abstract

Spermatozoan maturation, motility, and fertility are, in part, dependent upon the progressive increase in epididymal and spermatozoal carnitine, critical for mitochondrial fatty acid oxidation, as sperm pass from the caput to the cauda of the epididymis. We demonstrate that the organic cation/carnitine transporters, OCTN1, OCTN2, and OCTN3, are expressed in sperm as three distinct proteins with an expected molecular mass of 63 kDa, using Western blot analysis and our transporter-specific antibodies. Carnitine uptake studies in normal control human sperm samples further support the presence of high-affinity (OCTN2) carnitine uptake (K_m of 3.39 ± 1.16 μM; V_max of 0.23 ± 0.14 pmol/min/mg sperm protein; and mean ± SD; n=12), intermediate-affinity (OCTN3) carnitine uptake (K_m of 25.9 ± 14.7 μM; V_max of 1.49 ± 1.03 pmol/min/mg protein; n=26), and low-affinity (OCTN1) carnitine uptake (K_m of 412.6 ± 191 μM; V_max of 32.7 ± 20.5 pmol/min/mg protein; n=18). Identification of individuals with defective sperm carnitine transport may provide potentially treatable etiologies of male infertility, responsive to l-carnitine supplementation.

Section snippets

Materials and methods

Sperm preparation and analysis. All studies were performed with the approval of the Institutional Review Board of the Hospital for Sick Children, Toronto. Residual unused portions of semen samples were obtained, with informed consent, from men, with normal semen parameters, who were donating samples for artificial insemination at the Sunnybrook and Women’s College Hospital Fertility Centre (Toronto, Canada). Samples were examined and selected according to World Health Organization [41]

Western blot of OCTN1, OCTN2, and OCTN3

To date, three proteins have been shown to transport l-carnitine, namely OCTN1, OCTN2, and OCTN3. Importantly, we have demonstrated the expression of all three, each with an expected molecular weight of ∼63 kDa, in human sperm by Western blot analysis using our anti-murine transporter-specific antibodies (Fig. 1).

Carnitine uptake in sperm

Carnitine uptake studies in human sperm from normal control samples support the presence of a high-affinity transport process with an apparent K_m of 3.39 ± 1.16 μM and a maximal velocity

Acknowledgements

This work was supported in part by a grant from the Physicians’ Services Incorporated Foundation of Ontario and the Heart and Stroke Foundation of Ontario NA # 4964. We thank Nelson Palomaris, Domingo Molingbayan, Jennifer Willis, and Dr. Wei Qiu for their helpful assistance.

References (70)

C.J Rebouche et al.
Tissue distribution of carnitine biosynthetic enzymes in man
Biochim. Biophys. Acta
(1980)
C.A Stanley
New genetic defects in mitochondrial fatty acid oxidation and carnitine deficiency
Adv. Pediatr.
(1987)
C.J Rebouche et al.
Sodium gradient-stimulated transport of l-carnitine into renal brush border membrane vesicles: kinetics, specificity, and regulation by dietary carnitine
Arch. Biochem. Biophys.
(1984)
X Wu et al.
cDNA sequence, transport function, and genomic organization of human OCTN2, a new member of the organic cation transporter family
Biochem. Biophys. Res. Commun.
(1998)
I Tamai et al.
Molecular and functional identification of sodium ion-dependent, high affinity human carnitine transporter OCTN2
J. Biol. Chem.
(1998)
M.J James et al.
Kinetics of carnitine uptake by rat epididymal cells. Androgen-dependence and lack of stereospecificity
FEBS Lett.
(1981)
I Tamai et al.
Molecular and functional characterization of organic cation/carnitine transporter family in mice
J. Biol. Chem.
(2000)
P.J Huth et al.
Properties of carnitine transport in rat kidney cortex slices
Biochim. Biophys. Acta
(1980)
K.J Ullrich
Specificity of transporters for ‘organic anions’ and ‘organic cations’ in the kidney
Biochim. Biophys. Acta
(1994)
I Tamai et al.
Cloning and characterization of a novel human pH-dependent organic cation transporter, OCTN1
FEBS Lett.
(1997)

A.M Lamhonwah et al.

A third human carnitine/organic cation transporter (OCTN3) as a candidate for the 5q31 Crohn’s disease locus (IBD5)

Biochem. Biophys. Res. Commun.

(2003)

X Wu et al.

Structural and functional characteristics and tissue distribution pattern of rat OCTN1, an organic cation transporter, cloned from placenta

Biochim. Biophys. Acta

(2000)

D.E Brooks et al.

The uptake of l-(methyl-³H) carnitine by the rat epididymis

Biochem. Biophys. Res. Commun.

(1973)

N.R Marquis et al.

The distribution of carnitine, acetylcarnitine and carnitine acetylcarnitine transferase in rat tissues

J. Biol. Chem.

(1965)

E.R Casillas

Accumulation of carnitine by bovine spermatozoa during maturation in the epididymis

J. Biol. Chem.

(1973)

M Kuwajima et al.

Animal model of systemic carnitine deficiency: analysis in C3H-H-2 degrees strain of mouse associated with juvenile visceral steatosis

Biochem. Biophys. Res. Commun.

(1991)

K Toshimori et al.

Dysfunctions of the epididymis as a result of primary carnitine deficiency in juvenile visceral steatosis mice

FEBS Lett.

(1999)

I Tein et al.

The human plasmalemmal carnitine transporter defect is expressed in cultured lymphoblasts: a new non-invasive method for diagnosis

Clin. Chim. Acta

(1996)

M.M Bradford

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding

Anal. Biochem.

(1976)

D.E Brooks

Carnitine in the male reproductive tract and its relation to the metabolism of the epididymis and spermatozoa

E.R Casillas

The distribution of carnitine in male reproductive tissues and its effect on palmitate oxidation by spermatozoal particles

Biochim. Biophys. Acta

(1972)

E.R Casillas

Accumulation of carnitine by bovine spermatozoa during maturation in the epididymis

J. Biol. Chem.

(1973)

P.M Robitaille et al.

Phosphorus-31 nuclear magnetic resonance studies of spermatozoa from the boar, ram, goat and bull

Comp. Biochem. Physiol. B

(1987)

A.M Lamhonwah et al.

Carnitine uptake defect: frameshift mutations in the human plasmalemmal carnitine transporter gene

Biochem. Biophys. Res. Commun.

(1998)

S.D Cederbaum et al.

Carnitine membrane transporter deficiency: a long-term follow up and OCTN2 mutation in the first documented case of primary carnitine deficiency

Mol. Genet. Metab.

(2002)

A.M Lamhonwah et al.

GFP-human high-affinity carnitine transporter OCTN2 protein: subcellular localization and functional restoration of carnitine uptake in mutant cell lines with the carnitine transporter defect

Biochem. Biophys. Res. Commun.

(1999)

K Jarvi et al.

Heterogeneity of reproductive tract abnormalities in men with absence of the vas deferens: role of cystic fibrosis transmembrane conductance regulator gene mutations

Fertil. Steril.

(1998)

J Bremer

Carnitine—metabolism and functions

Physiol. Rev.

(1983)

R.R Ramsay

The carnitine acyltransferases: modulators of acyl-CoA-dependent reactions

Biochem. Soc. Trans.

(2000)

D.C De Vivo et al.

Primary and secondary disorders of carnitine metabolism

Int. Pediatr.

(1990)

B.T Hinton et al.

The concentration of carnitine in the luminal fluid of the testis and epididymis of the rat and other mammals

J. Reprod. Fertil.

(1979)

I Tein et al.

Impaired skin fibroblast carnitine uptake in primary systemic carnitine deficiency manifested by childhood carnitine-responsive cardiomyopathy

Pediatr. Res.

(1990)

J Bahl et al.

Carnitine transport in isolated adult rat heart myocytes and the effect of 7,8-diOH chlorpromazine

Circ. Res.

(1981)

C.J Rebouche et al.

Carnitine transport in cultured muscle cells and skin fibroblasts from patients with primary systemic carnitine deficiency

In Vitro

(1982)

W.R Treem et al.

Primary carnitine deficiency due to a failure of carnitine transport in kidney, muscle, and fibroblasts

N. Engl. J. Med.

(1988)

Cited by (45)

The potential therapeutic effects of ergothioneine in pre-eclampsia
2018, Free Radical Biology and Medicine
Citation Excerpt :
The latter was an early paper and used radioisotopes to study the subcellular distribution of labelled ERG; 10 min after injection approximately 15% of the label was associated with a crude mitochondrial fraction, but although the tables refer to mitochondrial localisation, the authors referred in the text (with no evidence either way) to “transfer of radioactivity to the mitochondria which probably represents the distribution of 3H or 3H2O formed by degradation”. The papers of Lamhomwah and Xuan based their evidence mainly on the use of antibodies, which are notoriously unreliable, and the uptake studies used carnitine, a very poor substrate for OCTN1 [75–77,79,80]. While ERG is uncharged at neutral pH, it is probably fair to say that the question of whether ERG is in fact concentrated in mitochondria is actually rather open, and if it does which transporters were used to get and/or accumulate it there.
Ergothioneine (ERG), is a water-soluble amino acid that is derived entirely from dietary sources. It has received much attention as a therapeutic agent due to its anti-oxidant properties, and there are claims of preferential accumulation within high oxidative stress organs. Pre-eclampsia, a condition accompanied by increased oxidative stress, is one of the leading causes of maternal morbidity and mortality. Despite intense research efforts, its aetiologies remain somewhat unclear and there are still no effective treatment options. Clinical trials of the anti-oxidants vitamin C and vitamin E have proven largely ineffective with little improvement in clinical outcome or even a negative response. This could be explained in part by their inability to permeate the plasma and mitochondrial membranes and scavenge mitochondria-derived superoxide species, and for the former by the fact that it is actually a pro-oxidant in the presence of unliganded iron. ERG accumulates within tissues through the action of a specific organic cation transporter, SLC22A4 (previously referred to as OCTN1), which is possibly also expressed in mammalian mitochondria. Mitochondrial dysfunction has been implicated in a variety of vascular diseases including pre-eclampsia. This review discusses the use of ERG as a possibly mitochondrial-targeted anti-oxidant, focusing on its physical properties, potential mechanisms of action, safety profile and administration in relation to pregnancies complicated by pre-eclampsia.
Uptake Transporters
2018, Comprehensive Toxicology: Third Edition
Membrane transporters are recognized to be an important class of proteins for regulating cellular and physiologic solute and fluid balance. Transporter proteins can be generally separated into two major classes—uptake and efflux transporters. Not only are transporter proteins critical to human physiology in the maintenance of normal homeostasis via transport of endogenous substrates, but they are also, in many cases, important to the disposition of numerous xenobiotic compounds such as environmental toxins, dietary constituents, drugs, and their metabolites. In particular, over the past two decades, members of the solute carrier (SLC) families, including the organic anion transporting polypeptide (SLCO) family, the organic cation/anion/zwitterion transporter (SLC22) family, and the sodium bile salt cotransport (SLC10) family, have been determined to have important physiological, pathological, and therapeutic implications. In this article, we focus on uptake transporter proteins and provide a comprehensive overview of the significant roles these proteins play in normal physiology and the drug disposition process.
Adaptor protein is a new therapeutic target in chronic kidney disease
2017, Kidney International
Ergothinoneine, derived from food, has powerful antioxidant effects. In this issue, Shinozaki et al. showed a novel kidney-intestine interaction, mediated by the postsynaptic density 95/disk-large/ZO-1 domain–containing protein (PDZK1)–organic cation transporter 1 (OCTN1)-ergothinoneine axis. In chronic kidney disease, decreased PDZK1 disturbed the localization of OCTN1 on the intestine, resulting in decreased serum ergothinoneine, which canceled the compensatory expression of OCTN1 in the kidney. Because PDZK1 regulates the function of several transporters, targeting PDZK1 may be a new therapeutic target in chronic kidney disease.
Wide tolerance to amino acids substitutions in the OCTN1 ergothioneine transporter
2016, Biochimica et Biophysica Acta - General Subjects
Citation Excerpt :
Expression of OCTN1 in Xenopus oocytes increased the transport of compounds such as [3H]pyrilamine, [3H]quinidine, and [3H]verapamil and zwitterionic L-[3H]carnitine [12]. When expressed in human and rodent cells, OCTN1 mediates a Na+-independent and pH-dependent transport of the prototypical organic cation tetraethylammonium, while interacts with carnitine with very low affinity [13–15]. OCTN1 affinity for carnitine has been linked to the presence of functional variants of the amino acid in position 503 of the SLC22A4 gene: individuals homozygous for the more prevalent 503 L showed a Km of 507 μM toward carnitine, while in individuals homozygous for the 503F variant the Km was 1360 μM [16,17].
Organic cation transporters transfer solutes with a positive charge across the plasma membrane. The novel organic cation transporter 1 (OCTN1) and 2 (OCTN2) transport ergothioneine and carnitine, respectively. Mutations in the SLC22A5 gene encoding OCTN2 cause primary carnitine deficiency, a recessive disorders resulting in low carnitine levels and defective fatty acid oxidation. Variations in the SLC22A4 gene encoding OCTN1 are associated with rheumatoid arthritis and Crohn disease.
Here we evaluate the functional properties of the OCTN1 transporter using chimeric transporters constructed by fusing different portion of the OCTN1 and OCTN2 cDNAs. Their relative abundance and subcellular distribution was evaluated through western blot analysis and confocal microscopy.
Substitutions of the C-terminal portion of OCTN1 with the correspondent residues of OCTN2 generated chimeric OCTN transporters more active than wild-type OCTN1 in transporting ergothioneine. Additional single amino acid substitutions introduced in chimeric OCTN transporters further increased ergothioneine transport activity. Kinetic analysis indicated that increased transport activity was due to an increased V_max, with modest changes in K_m toward ergothioneine.
Our results indicate that the OCTN1 transporter is tolerant to extensive amino acid substitutions. This is in sharp contrast to the OCTN2 carnitine transporter that has been selected for high functional activity through evolution, with almost all substitutions reducing carnitine transport activity.
The widespread tolerance of OCTN1 to amino acid substitutions suggests that the corresponding SLC22A4 gene may have derived from a recent duplication of the SLC22A5 gene and might not yet have a defined physiological role.
Over-expression in Escherichia coli, purification and reconstitution in liposomes of the third member of the OCTN sub-family: The mouse carnitine transporter OCTN3
2012, Biochemical and Biophysical Research Communications
Citation Excerpt :
The coding genes for OCTN’s have been identified only in higher mammals. Differently from OCTN1 and 2, the gene of OCTN3 has not yet been annotated in Homo sapiens genome [13,14], even though, the OCTN3 protein has been detected in human skin and sperm using antibody against mouse (m)OCTN3 [15–18]. The functional information concerning the OCTN3 transporter comes from experiments performed with murine cells [16,19–21] or in cell lines in which the transporter was artificially expressed [13].
pET-21a(+)-mOCTN3-6His was constructed and used for over-expression in Escherichia coli Rosetta(DE3)pLysS. After IPTG induction a protein with apparent molecular mass of 53 kDa was collected in the insoluble fraction of the cell lysate and purified by Ni²⁺-chelating chromatography with a yield of 2 mg/l of cell culture. The over-expressed protein was identified with mOCTN3 by anti-His antibody and reconstitution in liposomes. mOCTN3 required peculiar conditions for optimal expression and reconstitution in liposomes. The protein catalyzed a time dependent [³H]carnitine uptake which was stimulated by intraliposomal ATP and nearly independent of the pH. The K_m for carnitine was 36 μM. [³H]carnitine transport was inhibited by carnitine analogues and some Cys and NH₂ reagents. This paper represents the first outcome in over-expressing, in active form, the third member of the OCTN sub-family, mOCTN3, in E. coli.
Colon OCTN2 gene expression is up-regulated by peroxisome proliferator-activated receptor γ in humans and mice and contributes to local and systemic carnitine homeostasis
2010, Journal of Biological Chemistry
Citation Excerpt :
Three OCTNs have been identified so far: OCTN1 (SLC22A4), OCTN2 (SLC22A5), and OCTN3 (SLC22A21) (8–10). OCTN1 and OCTN2 are highly expressed in several tissues, such as kidney, intestine, skeletal muscle, heart, liver, and brain (8, 11–14), whereas OCTN3 is almost exclusively expressed in testes (10, 15). Because of its high binding affinity for carnitine and its wide expression, OCTN2 is the most important carnitine transporter.
In the large intestine organic cation transporter type-2 (OCTN2) is recognized as a transporter of compounds such as carnitine and colony sporulation factor, promoting health of the colon intestinal epithelium. Recent reports suggest that OCTN2 expression in small intestine is under control of peroxisome proliferator-activated receptor-α (PPARα). However, PPARα contribution to colonic OCTN2 expression remains controversial. Here we examined the transcriptional regulation of colon OCTN2 gene by PPARγ. To exclude any additional modulation of other PPAR to OCTN2 expression, we used both in vivo and in vitro PPAR-null models and specific PPAR inhibitors. The PPARγ agonists thiazolidinediones increased both OCTN2 mRNA and protein expression in colonic epithelial cell lines independently by PPARα expression. The induction was blocked only by PPARγ antagonists or by γORF4, a PPARγ isoform with dominant negative activity, suggesting a PPARγ-dependent mechanism. A conserved noncanonical PPAR-responsive element was found by computational analysis in the first intron of human OCTN2 gene and validated by EMSA assay. Promoter-reporter assays further confirmed transcriptional functionality of the putative PPAR response element, whereas selective mutation caused complete loss of responsiveness to PPARγ activation. Finally, adenovirus-mediated overexpression of constitutively active PPARγ mutant increased colon OCTN2 expression in PPARα^−/− mice. Interestingly, animals overexpressing colon PPARγ showed a significant increase in plasma carnitine, thus demonstrating the functional contribution of large intestine to systemic carnitine homeostasis. This study reveals a PPARγ-dependent absorption machinery in colon that is likely involved in the health of colon epithelium, in the microbiota-host interactions and in the absorption of nutraceuticals and drugs.

View all citing articles on Scopus

View full text

Characterization of organic cation/carnitine transporter family in human sperm

Abstract

Section snippets

Materials and methods

Western blot of OCTN1, OCTN2, and OCTN3

Carnitine uptake in sperm

Acknowledgements

Biochim. Biophys. Acta

Adv. Pediatr.

Arch. Biochem. Biophys.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

Biochim. Biophys. Acta

Biochim. Biophys. Acta

FEBS Lett.

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

FEBS Lett.

Clin. Chim. Acta

Anal. Biochem.

Biochim. Biophys. Acta

J. Biol. Chem.

Comp. Biochem. Physiol. B

Biochem. Biophys. Res. Commun.

Mol. Genet. Metab.

Biochem. Biophys. Res. Commun.

Fertil. Steril.

Carnitine—metabolism and functions

Physiol. Rev.

The carnitine acyltransferases: modulators of acyl-CoA-dependent reactions

Biochem. Soc. Trans.

Primary and secondary disorders of carnitine metabolism

Int. Pediatr.

The concentration of carnitine in the luminal fluid of the testis and epididymis of the rat and other mammals

J. Reprod. Fertil.

Impaired skin fibroblast carnitine uptake in primary systemic carnitine deficiency manifested by childhood carnitine-responsive cardiomyopathy

Pediatr. Res.

Carnitine transport in isolated adult rat heart myocytes and the effect of 7,8-diOH chlorpromazine

Circ. Res.

Carnitine transport in cultured muscle cells and skin fibroblasts from patients with primary systemic carnitine deficiency

In Vitro

Primary carnitine deficiency due to a failure of carnitine transport in kidney, muscle, and fibroblasts

N. Engl. J. Med.