Biomedical subjects

Kunihiko Tanaka

Publications and source records attributed to Kunihiko Tanaka.

At least 19 recordsLinked to original sources

PubMed2026

Selective saccular plasticity under microgravity links peripheral transcriptomic remodeling to postflight vestibular dysfunction.

Long-duration exposure to microgravity disrupts human balance and spatial orientation, yet the molecular mechanisms underlying vestibular adaptation to spaceflight remain poorly understood. Here, we tested the hypothesis that the saccule, the primary gravity-sensing otolith organ, undergoes selective remodeling during spaceflight and contributes to transient postflight postural instability. Using a cross-species approach, we combined transcriptomic analysis of mouse otolith organs with physiological assessments in astronauts. Laser microdissection-based RNA sequencing of mouse otolith sensory epithelia after a 35-d spaceflight revealed pronounced, organ-specific transcriptomic remodeling in the saccule, whereas the utricle remained stable. Principal component and clustering analyses demonstrated that the saccular transcriptome shifted toward an utricle-like profile under microgravity, accompanied by changes in genes related to synaptic and neuronal function. Promoter motif analysis identified NFAT-associated transcriptional networks, suggesting Ca2+-dependent regulation of synaptic plasticity as a potential molecular substrate of gravity-dependent adaptation. In parallel, vestibular testing in astronauts following long-duration missions (157 to 328 d) revealed selective attenuation of saccule-mediated cervical vestibular-evoked myogenic potentials and increased postural sway immediately after return to Earth, while utricle-mediated responses and semicircular canal function were preserved. Both saccular function and postural stability recovered within approximately 10 d. Notably, early postflight postural instability was partially mitigated by noisy galvanic vestibular stimulation, consistent with stochastic resonance-mediated sensory enhancement. Together, these findings identify the saccule as a plastic gravity sensor and establish a mechanistic link between peripheral molecular remodeling and functional balance deficits after spaceflight, providing a framework for developing countermeasures to facilitate vestibular readaptation during human space exploration.

Animals↗

PubMed2007

Determination of the external loops and the cellular orientation of the N- and the C-termini of the human organic anion transporter hOAT1.

The OAT (organic anion transporter) family mediates the absorption, distribution and excretion of a diverse array of environmental toxins and clinically important drugs. OAT dysfunction significantly contributes to renal, hepatic, neurological and fetal toxicity and disease. As a first step to establish the topological model of hOAT1 (human OAT1), we investigated the external loops and the cellular orientation of the N- and the C-termini of this transporter. Combined approaches of immunofluorescence studies and site-directed chemical labelling were used for such purpose. Immunofluorescence microscopy of Myc-tagged hOAT1 expressed in cultured cells identified that both the N- and the C-termini of the transporter were located in the cytoplasm. Replacement of Lys59 in the predicted extracellular loop I with arginine resulted in a mutant (K59R), which was largely inaccessible for labelling by membrane-impermeable NHS (N-hydroxysuccinimido)-SS (dithio)-biotin present in the extracellular medium. This result suggests that loop I faces outside of the cell membrane. A single lysine residue introduced into putative extracellular loops III, V and VI of mutant K59R, which is devoid of extracellular lysine, reacted readily with membrane-impermeable NHS-SS-biotin, suggesting that these putative extracellular loops are in the extracellular domains of the protein. These studies provided the first experimental evidence on the extracellular loops and the cellular orientation of the N- and the C-termini of hOAT1.

Amino Acid Sequence↗

PubMed2006

Long-term hypergravity induces plastic alterations in vestibulo-cardiovascular reflex in conscious rats.

To test the hypothesis that an altered gravitational environment induces plastic changes in the vestibulo-cardiovascular reflex, arterial pressure (AP) and hypothalamic glutamate concentration were examined in 2 groups of conscious rats, i.e., a 3-G group and a 1-G group, in which rats were maintained under a 3-G and 1-G environment for 2 weeks, respectively. The vestibulo-cardiovascular reflex was stimulated by a gravitational change induced by a parabolic flight that consisted of 3 phases: "pull-up", during which the G load gradually increased to 2G; a 20s "push-over" into microgravity; and "pull-out", during which the G load increased to 1.8. In the 1-G group, the AP increased by 11.9+/-1.2 mmHg during the pull-up hypergravity period. The AP response was significantly attenuated in the 3-G group (4.0+/-0.8 mmHg). During the push-over microgravity period, the AP decreased from the peak level in the pull-up period and recovered to the pre-parabolic control level (-1.8+/-2.4 mmHg). In rats of the 3-G group, the AP was not altered by push-over microgravity. These AP responses were associated with a significant increase in the glutamate concentration in the hypothalamus (4.4+/-0.7%). The glutamate response was also significantly attenuated in the 3-G group compared with that in the 1-G group. These results indicate that an altered gravitational environment induces plastic alterations in the vestibulo-cardiovascular reflex.

Animals↗

PubMed2006

Functional role of the C terminus of human organic anion transporter hOAT1.

Human organic anion transporter hOAT1 plays critical roles in the body disposition of environmental toxins and clinically important drugs. In the present study, we examined the role of the C terminus of hOAT1 in its function. Combined approaches of cell surface biotinylation and transport analysis were employed for such purposes. It was found that deletion of the last 15 amino acids (residues 536-550) or the last 30 amino acids (residues 521-550) had no significant effect on transport activity. However, deletion of the entire C terminus (residues 506-550) completely abolished transport activity. Alanine scanning mutagenesis within the region of amino acids 506-520 led to the discovery of two critical amino acids: Glu-506 and Leu-512. Substitution of negatively charged Glu-506 with neutral amino acids alanine or glutamine resulted in complete loss of transport activity. However, such loss of transport activity could be rescued by substitution of Glu-506 with another negatively charged amino acid aspartic acid, suggesting the importance of negative charge at this position for maintaining the correct tertiary structure of the transporter, possibly by forming a salt bridge with a positively charged amino acid. Substitution of Leu-512 with amino acids carrying progressively smaller side chains including isoleucine, valine, and alanine resulted in mutants (L512I, L512V, and L512A) with increasingly impaired transport activity. However, the cell surface expression of these mutants was not affected. Kinetic analysis of mutant L512V revealed that the reduced transport activity of this mutant resulted mainly from a reduced maximum transport velocity Vmax without affecting the binding affinity (1/Km) of the transporter for its substrates, suggesting that the size of the side chain at position 512 critically affects transporter turnover number. Together, our results are the first to highlight the central role of the C terminus of hOAT1 in the function of this transporter.

Amino Acid Sequence↗

PubMed2006

Neuromuscular electrical stimulation and an ottoman-type seat effectively improve popliteal venous flow in a sitting position.

The aim of this study was to examine the effects of the sitting posture on the lower limb venous flow and to explore the beneficial effects of neuromuscular electrical stimulation (NMES) and an ottoman-type seat on the venous flow. Healthy adult volunteers without a history of lower limb surgery or thromboembolism were recruited, and the flow velocity, cross-sectional area, and flow volume of the popliteal vein were measured using Doppler ultrasound. A posture change from the prone position to the sitting position on the ottoman-type seat decreased the flow velocity and increased the cross-sectional area of the popliteal vein, and the flow volume was not altered over 120 min. The flow velocity was further decreased, and the cross-sectional area was further increased by subjects sitting on a regular driving seat when compared with the values obtained with an ottoman-type seat. The popliteal flow velocity in the NMES leg was significantly higher than in the non-NMES leg throughout the 120-min testing in the sitting position, but no difference in the cross-sectional area was found between the NMES and the non-NMES legs. Thus lower limb venous stasis elicited by the sitting posture was improved by the ottoman-type seat and NMES.

Adult↗

PubMed2005

Roles of the vestibular system in controlling arterial pressure in conscious rats during a short period of microgravity.

In order to evaluate the roles of the vestibular system in controlling arterial pressure (AP) during exposure to a short period of microgravity (microG), the AP was measured in conscious free-moving rats having intact vestibular systems and those having vestibular lesions (FM-Intact and FM-VL groups, respectively). During free drop-induced microG, the AP increased in the FM-Intact group; it was 38+/-4 mmHg more than the AP observed during 1G. However, the increase in AP was significantly lower in the FM-VL group (20+/-2 mmHg). Further, to examine the sudden effect of a body floating in the midair in response to the AP during exposure to muG a body stabilizer was placed on the back of rats having intact vestibular systems and those having vestibular lesions (STAB-Intact and STAB-VL groups, respectively). The increase in the AP was significantly depressed in the STAB-Intact group; when compared with that in the FM-Intact group, but the increase was still significant (27+/-2 mmHg). On the other hand, the increase in the AP was completely eliminated in the STAB-VL group (7+/-5 mmHg). These results indicate that the AP increases during exposure to muG in conscious rats, and the vestibular system and body stability are significantly involved in this response.

Animals↗

PubMed2005

Effects of high NaCl diet on arterial pressure in Sprague-Dawley rats with hepatic and sinoaortic denervation.

The Na(+) receptor that exists in the hepatoportal region plays an important role in postprandial natriuresis and the regulation of Na(+) balance during NaCl load. Thus it would be considered that a dysfunction of the hepatic Na(+) receptor might result in the elevation of arterial pressure under a condition of high NaCl diet. To elucidate this hypothesis, arterial pressure was continuously measured during three weeks of high NaCl diet (8% NaCl) in four groups of rats: (i) intact rats, (ii) rats with hepatic denervation (HD), (iii) rats with sinoaortic denervation (SAD), and (iv) rats with SAD+HD. During a 1-week normal NaCl diet period, there was no difference in arterial pressure among the four groups. A high NaCl diet had no influence on arterial pressure in intact or HD rats; however, it significantly increased by 11 +/- 3 mmHg in SAD rats. The addition of HD to SAD had no synergistic effect on arterial pressure; i.e., in SAD+HD rats, mean arterial pressure increased by 13 +/- 1 mmHg. In conclusion, sinoaortic baroreceptor, but not hepatic Na(+) receptor, has a significant role in the long-term regulation of arterial pressure on a high NaCl diet.

Animals↗

PubMed2005

Regional difference of blood flow in anesthetized rats during reduced gravity induced by parabolic flight.

To examine a hypothesis that change in regional blood flow due to decreased hydrostatic pressure gradient and redistribution of blood during reduced gravity (rG) is different between organs, changes in cerebrocortical blood flow (CBF) and blood flow in the temporal muscle (MBF) with exposure to rG were measured in anesthetized rats in head-up tilt and flat positions during parabolic flight. Carotid arterial pressure (CAP), jugular venous pressure (JVP), and abdominal aortic pressure were also measured simultaneously. In the head-up tilt group, CBF increased by 15 +/- 3% within 3 s of entry into rG and rapidly recovered during rG. MBF also increased, but the change was significantly greater than that of CBF. JVP increased by 1.8 +/- 0.5 mmHg, probably due to loss of hydrostatic pressure gradient, since the measuring point of JVP was 2-3 cm above the hydrostatic indifference point. CAP and abdominal aortic pressure increased by 16.7 +/- 2 and 7.7 +/- 2 mmHg, respectively, compared with the 1-G condition. Muscle vascular resistance [(CAP-JVP)/MBF] decreased on entry into rG, but no significant change was observed in cerebrocortical vascular resistance [(CAP-JVP)/CBF]. In the flat group, no significant change was observed in all the variables. The results indicate that arteriolar vasodilatation occurs in the temporal muscle but not in the cerebral cortex. Thus the blood flow control mechanism at the onset of rG is different between intra- and extracranial organs.

Adaptation, Physiological↗

PubMed2005

Human organic anion transporter hOAT1 forms homooligomers.

Human organic anion transporter hOAT1 belongs to a superfamily of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. To gain insight into the regulation of hOAT1, detailed information on its structural assembly is essential. In the present study, we investigate the quaternary structure of hOAT1 using combined approaches of chemical cross-linking, gel filtration chromatography, co-immunoprecipitation, cell surface biotinylation, and metabolic labeling. Chemical cross-linking of intact membrane proteins from LLC-PK1 cells stably expressing hOAT1 converted quantitatively hOAT1 monomer to putative trimer and higher order of oligomer, indicating that hOAT1 is present in the membrane as multimeric complexes. When co-expressed in LLC-PK1 cells, FLAG-tagged hOAT1 co-immunoprecipitated with myc-tagged hOAT1. The hOAT1 oligomer was also detected in gel filtration chromatography of total membranes from hOAT1-expressing LLC-PK1 cells. Cell surface biotinylation with membrane-impermeable reagents and metabolic labeling with [(35)S]methionine followed by immunoprecipitation showed that the oligomeric hOAT1 did not contain any other proteins. Taken together, this is the first study demonstrating that hOAT1 exists in the plasma membrane as a homooligomer, possibly trimer, and higher order of oligomer.

Animals↗

PubMed2005

Controlling arterial blood pressure using a computer-brain interface.

There has been recent interest in the concept of connecting a computer to the brain to control brain functions. However, there are challenges that must be overcome in developing such a computer-brain interface, including a selection of nucleus that is stimulated, and an implantable electrode and electrical stimulator. Another important issue is the designing of the controller, that is, determining how to encode as an electrical signal the information to be sent to the brain. We have applied system identification theory, a method for evaluating dynamic characteristics of a system, to the arterial blood pressure control system of the brain. Our results show that (1) the stimulation-arterial blood pressure response relationship can be described as a mathematical model, which gives a good prediction of the arterial blood pressure response, facilitating the designing of a computer-brain interface, and (2) the arterial blood pressure can be actually controlled using a computer-brain interface.

Animals↗

PubMed2005

Change in intrathoracic pressure in rats with spontaneous and controlled ventilation during microgravity by parabolic flight.

We previously reported that the intrathoracic pressure (ITP) decreases and the transmural pressure of the aortic wall (TMP) increases during 4.5 s of microgravity (muG) induced by free drop. To examine the ITP response to a longer period of muG in the absence of the respiratory rate (RR) decrease, i.e., bradypnea, which occurs at the onset of muG, we measured the aortic blood pressure at the diaphragma level (AP) and ITP. We then calculated the TMP at the aortic arch level during 20 s of muG induced by parabolic flight in anesthetized rats (n = 7) with either spontaneous ventilation (SPN-V) or controlled ventilation (CONT-V). In the SPN-V group, the bradypnea was observed in all rats after the onset of the muG (RR change -13.9 +/- 2.9/min). The ITP during muG (-9.3 +/- 0.9 mmHg) was significantly lower than that during 1 G (-7.7 +/- 0.9 mmHg), and the TMP was significantly increased during muG (112 +/- 6 mmHg) compared to 1 G (103 +/- 5 mmHg). Similar changes in ITP and TMP were observed in the CONT-V group: During muG and 1G, respectively, the ITP was -8.4 +/- 0.6 mmHg and -5.9 +/- 0.7 mmHg, and the TMP was 112 +/- 6 mmHg and 101 +/- 6 mmHg, whereas no change in RR was observed because of the controlled ventilation. These results show that the ITP decreases and the TMP increases during muG, and they are not affected by a disturbance of respiratory rhythm.

Anesthesia↗

PubMed2005

Intracranial pressure dynamics assessed by noninvasive ultrasound during 30 days of bed rest.

INTRODUCTION: Intracranial pressure (ICP) may be an important contributor to symptoms of space adaptation syndrome during the initial days of microgravity exposure. The temporary nature of these symptoms suggests that some physiologic adaptation or compensation occurs. Fluid shifts similar to those in microgravity can be simulated on Earth using head-down tilt (HDT) bed rest. This study was performed to calibrate a new noninvasive ICP instrument and to investigate ICP adaptation during 30 d of HDT bed rest. METHODS: A noninvasive ultrasound technique that measures small skull expansions with fluctuations in ICP was used to measure cranial oscillations before and near the end of 30-d HDT bed rest in eight healthy, male volunteers. Pulse phase-locked loop (PPLL) output voltage and arterial BP were continuously monitored and correlated. RESULTS: The amplitude of intracranial distance pulsation decreased during 30-d bed rest. Prior to bed rest, the PPLL amplitude was 25 +/- 9 mV and this amplitude was reduced by 60% to 9 +/- 4 mV (a value consistent with that of upright posture) at the end of HDT bed rest (p = 0.01). DISCUSSION: PPLL measurements of skull pulsations are acutely posture dependent, being significantly higher in supine and HDT as compared with upright posture. A cephalad fluid shift is probably the responsible mechanism. Our results indicate that there are adaptations to intracranial pooling of blood and tissue fluid during bed rest that reduce skull pulsation amplitudes to values similar to those obtained in normal upright posture. Detailed studies of the time course of cranial vessel and bone adaptations may provide insights into the potential adaptative mechanisms.

Aerospace Medicine↗

PubMed2005

Mechanical counter-pressure vs. gas-pressurized spacesuit gloves: grip and sensitivity.

INTRODUCTION: An elastic mechanical counter pressure (MCP) glove for spacesuits is under development. In this study we compared handgrip and pinch grip strength levels for the MCP glove and the current extravehicular mobility unit (EMU) gas-pressurized glove. METHODS: We employed handgrip and pinch grip dynamometers to assess strength levels and von Frey monofilaments to evaluate hand sensitivity. Tests were conducted with the gloved hand inserted in an evacuation chamber at 200 mmHg below atmospheric pressure to simulate conditions in space. RESULTS: Average bare hand strength was 463 N and decreased to 240 N for EMU and 250 N for MCP. Pinch grip and key grip testing showed no difference among conditions. However, there was a significant decrease in palmar grip strength from 111 N barehanded to 67 N in both gloves. Barehanded endurance time was 160 s and dropped to 63 and 69 s for EMU and MCP, respectively. Sensitivity was significantly better for MCP compared with the EMU. DISCUSSION: The MCP glove improved hand sensitivity when compared with the EMU glove and performed as well as the EMU glove in terms of overall handgrip strength, endurance at 25% of maximum handgrip strength, pinch grip, palmar grip, and key grip tests. Improvements in fabric composition and glove design may further improve ergonomic and other functional parameters of the MCP glove.

Adult↗

PubMed2004

Vestibulosympathetic reflex mediates the pressor response to hypergravity in conscious rats: contribution of the diencephalon.

To investigate the mechanism of arterial pressure (AP) regulation during hypergravity, the AP response to gravitational force was examined in conscious rats and the AP was found to increase, depending on the degree of gravity load induced by centrifugation. At 20 s after application of 2, 3, or 5 G, the AP increased by 9+/-2, 20+/-3, or 24+/-3 mm Hg, respectively. The AP increase during first 60 s was suppressed by vestibular lesion or pretreatment with hexamethonium, suggesting that the vestibular system and sympathetic nerve system be involved, respectively, in the afferent and efferent pathways. To further examine the central pathway of this response, Fos expression in the brain was examined after exposure to 5 G for 90 min. Intense Fos expression was seen in the medial vestibular nucleus, paraventricular hypothalamic nucleus, autonomic nuclei in the brain stem in intact rats, but not in rats with vestibular lesion. To examine the involvement of the diencephalic nuclei in this pressor response, AP was measured under hypergravity in rats with midcollicular transection. In these rats, the AP change was minimal at 2, 3, and 5 G, indicating that nuclei rostral to the transection level were involved in the pressor response. These results indicate that output from the vestibular system project to the diencephalon, and activation of diencephalic nuclei is indispensable to the pressor response via the sympathetic nerve system.

Analysis of Variance↗

PubMed2004

Sequence of forebrain activation induced by intraventricular injection of hypertonic NaCl detected by Mn2+ contrasted T1-weighted MRI.

In order to define the sequence of forebrain activation involved in osmoregulation, central activation in response to intracerebroventricular injection of NaCl solution (10 microl of 0.15, 0.5, or 1.5 M) was detected using manganese-contrasted magnetic resonance imaging (MRI) in anesthetized rats. Changes in renal sympathetic nerve activity (RNA) were also measured, and the time courses of forebrain activation and RNA changes compared. NaCl injection resulted in rapid activation of the subfornical organ (SFO), organum vasculosum lamina terminalis (OVLT), and periventricular regions and the lateral hypothalamic area (LHA), then of the paraventricular hypothalamic nucleus (PVN) and supraoptic nucleus (SON). The delay in activation in the PVN and SON showed a wide variation from 0 to 5.78 min, and the average delay in the PVN (2.88+/-0.34 min) and SON (2.90+/-0.39 min) was significantly greater than that in the SFO (0.40+/-0.10 min) and OVLT (0.74+/-0.13 min). NaCl (1.5 M) injection elicited a rapid, large increase in RNA, which consisted of two components, an early rapid increase at 99 s after injection (160+/-27%) and a slower increase at 9 min after injection (209+/-34%). These results suggest that the PVN and SON are activated not only by the afferent input from the SFO and OVLT but also by diffusion of the hypertonic stimulus to these regions and probably by their intrinsic osmosensitivity. The PVN might be responsible for the second slower component of the RNA response, but cannot be responsible for the first component.

Animals↗

PubMed2004

Cysteine residues in the organic anion transporter mOAT1.

Mouse organic anion transporter 1 (mOAT1) belongs to a family of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, anti-tumour drugs, antibiotics, anti-hypertensives and anti-inflammatories. mOAT1-mediated transport of organic anion PAH ( p -aminohippurate) in HeLa cells was inhibited by the cysteine-modifying reagent PCMBS (p-chloromercuribenzenesulphonate). Therefore the role of cysteine residues in the function of mOAT1 was examined by site-directed mutagenesis. All 13 cysteine residues in mOAT1 were replaced by alanine, singly or in combination. Single replacement of these residues had no significant effect on mOAT1-mediated PAH transport, indicating that no individual cysteine residue is necessary for function. Multiple replacements at a C-terminal region (C335/379/427/434A; Cys(335/379/427/434)-->Ala) resulted in a substantial decrease in transport activity. A simultaneous replacement of all 13 cysteine residues (C-less) led to a complete loss of transport function. The decreased or lack of transport activity of the mutants C335/379/427/434A and C-less was due to the impaired trafficking of the mutant transporters to the cell surface. These results suggest that although cysteine residues are not required for function in mOAT1, their presence appears to be important for the targeting of the transporter to the plasma membrane. We also showed that, although all cysteine mutants of mOAT1 were sensitive to the inhibition by PCMBS, C49A was less sensitive than the wild-type mOAT1, suggesting that the modification of Cys49 may play a role in the inhibition of mOAT1 by PCMBS.

4-Chloromercuribenzenesulfonate↗

PubMed2004

Acute hemodynamic responses in the head during microgravity induced by free drop in anesthetized rats.

To examine acute hemodynamic responses to microgravity (microG) in the head, we measured carotid artery pressure (CAP) and jugular vein pressure (JVP) to calculate cephalic perfusion pressure (CPP = CAP - JVP) and recorded images of microvessels in the iris to evaluate capillary blood flow velocity (CBFV) and capillary diameter (CD) in anesthetized rats during 4.5 s of microG induced by free drop. Rats were placed in 30 degrees head-up whole body-tilted (HU, n = 7) or horizontal (flat, n = 6) position. In the flat group, none of the measured variables was significantly affected by microG, whereas in the HU group, CAP, JVP, and CPP increased, respectively, by 23.4 +/- 2.6, 1.3 +/- 0.2, and 22.9 +/- 3.1 mmHg, and CBFV and CD increased, respectively, by 33 +/- 8 and 9 +/- 3%, showing an increase in capillary blood flow. To further examine the mechanisms underlying these CAP and JVP increases, another experiment was performed in which CAP and JVP were measured in anesthetized rats (n = 6) during a postural change from HU to flat. In these animals, the change in JVP was similar to that observed during actual microG, but no change in CAP was seen, indicating that the JVP increase during actual microG is caused by disappearance of the gravitational pressure gradient in the head-to-foot axis, whereas the CAP increase is not. In conclusion, actual microG elicits an increase in CPP due to a greater increase in CAP than JVP, resulting in increased capillary blood flow. Although the increase in JVP is explained by the disappearance of gravitational pressure gradient in the head-to-foot axis as a result of microG, the larger increase in CAP is not.

Anesthesia↗

PubMed2004

Role of glycosylation in the organic anion transporter OAT1.

Organic anion transporters (OAT) play essential roles in the body disposition of clinically important anionic drugs, including antiviral drugs, antitumor drugs, antibiotics, antihypertensives, and anti-inflammatories. We reported previously (Kuze, K., Graves, P., Leahy, A., Wilson, P., Stuhlmann, H., and You, G. (1999) J. Biol. Chem. 274, 1519-1524) that tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited organic anion transport in COS-7 cells expressing a mouse organic anion transporter (mOAT1), suggesting an important role of glycosylation in mOAT1 function. In the present study, we investigated the effect of disrupting putative glycosylation sites in mOAT1 as well as its human counterpart, hOAT1, by mutating asparagine to glutamine and assessing mutant transporters in HeLa cells. We showed that the putative glycosylation site Asp-39 in mOAT1 was not glycosylated but the corresponding site (Asp-39) in hOAT1 was glycosylated. Disrupting Asp-39 resulted in a complete loss of transport activity in both mOAT1 and hOAT1 without affecting their cell surface expression, suggesting that the loss of function is not because of deglycosylation of Asp-39 per se but rather is likely because of the change of this important amino acid critically involved in the substrate binding. Single replacement of asparagines at other sites had no effect on transport activity indicating that glycosylation at individual sites is not essential for OAT function. In contrast, a simultaneous replacement of all asparagines in both mOAT1 and hOAT1 impaired the trafficking of the transporters to the plasma membrane. In summary, we provided the evidence that 1) Asp-39 is crucially involved in substrate recognition of OAT1, 2) glycosylation at individual sites is not required for OAT1 function, and 3) glycosylation plays an important role in the targeting of OAT1 onto the plasma membrane. This study is the first molecular identification and characterization of glycosylation of OAT1 and may provide important insights into the structure-function relationships of the organic anion transporter family.

Animals↗