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Amino acid transporter of mussel
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AMINO
Abstracts' / Comparative Biochemistry and Physiology Part A 126 (2000) SI-S163
ACID TRANSPORTER
Hosoi M), Goto C), T o y o h a r a H. ~
OF MUSSEL
T a k e u c h i K. ~, K u b o t a S. ~, S a k a g u c h i
M),
H a y a s h i I. ~, Y o k o y a m a
y b and
aDivision of Applied Biosciences, Graduate School of Agriculture, Kyoto University; bDepartment of Domestic Science, Kobe Shoin Women's College To understand the regulation of intracellular osmolyte concentration in marine mussel, Mytilus galloprovincialis (Lam.) for adapting the environmental salinity, we investigated changes in free amino acids pool of the tissues (mantle, gill and adductor muscle) and hemolyph exposed to abrupt changes in salinity. We found that free amino acids pool in tissues decreased significantly with the decrease in salinity, but slightly increase or decrease with the increase in salinity. Most prevalent free amino acid was taurine which was corresponded to 50-80% of total free amino acids pool and the content of taurine was subject to the change in salinity. For the better understanding of the mechanism to regulate the intracellular amino acid concentration, we tried to isolate the cDNA coding sodium and chloride-dependent taurine transporter which uptakes taurine from hemolyph into cell. In order to isolate the transporter eDNA, we designed a set of oligonucleotide primers based on the amino acids sequence which is highly conserved among taurine transporters in vertebrates. As a result of RT-PCR and 5"-, 3'-RACE, we isolated eDNA which possibly encodes a sodium and chloride-dependent amino acid ransporter. Comparison with amino acid sequences in the SWISS-PROT indicated that the deduced amino acids sequence is highly homologous to taurine transporter in vertebrates with about 50% amino acid identity. The cloned transporter has predictive 12 transmenbrane domains and particularly highly conserved amino acid sequences between the first and the second transmenbrane domains. Interestingly, the mussel transporter has additional amino acid sequences in places, of which function is unknown. Northern blot analysis showed that mRNA of the transporter in mantle, gill and adductor muscle was induced in accordance with the increase in the environmetnal osmolarity, while increase in mRNA was also detected for the hypo-osmotic adaptation. The transporter gene cloned from mussel in the present study has higher amino acid identity with mammalian and carp taurine transporter. We are now trying to determine substrates of the transporter by the transient expression experiment using COS-7 cells. Of interest, the expression of the gene was induced in response to hypo-osmotic stress as well as hyperosmotic stress. Further studies are required to understand the induction of the gene responding to the environmental salinity.
A R O L E O F E X T E R N A L A N D I N T E R N A L P H IN T H E R E G U L A T I O N O F T H E V O L U M E SENSITIVE K + CURRENT IN EHRLICH ASCITES TUMOUR CELLS i H o u g a a r d C., 2 J O r g e n s e n F. a n d 1 H o f f m a n n E . K . IA u g u s t K r o g h I n s t i t u t e , 13 U n i v e r s i t e t s p a r k e n , D K - 2 1 0 0 C o p e n h a g e n , D e n m a r k . 2 I M B , D e p a r t m e n t o f P h y s i o l o g y & P h a r m a c o l o g y , 21 W i n s l ~ w p a r k e n , D K - 5 0 0 0 O d e n s e , D e n m a r k . Previous studies have demonstrated that hypoosmotic swelling of Ehrlich ascites tumour cells results in cytosolic alkalinization followed by acidification and that volume recovery after cell swelling is strongly affected by the pH of the external solution [1,2]. These observations indicate that pH could be involved in the regulation of channel activity upon swelling of Ehrlich cells. In the present study we have investigated the possible role of pH in the modulation of the volumesensitive K + (IK,,o0 and CI- (Ict.,o0 currents in Ehrlich cells using the whole-cell patch-clamp technique. In order to investigate the effect of external pH (pHo) on IK.,.o~and Icl.,.ol the currents were activated by reducing the osmolarity of the external medium at various pHo values. The pH of the pipette solution (pH0 was in these experiments buffered at 7.4. Both the magnitude and the activation time of Ic~.,o~ appeared unaffected by pHo. In contrast, the current density of I~,~oLwas significantly reduced at pHo 6.4 and pHo 6.9 when compared to the control (pHo 7.4). Increasing pHo to 8.4 did not further increase the current density of IK,vo~but, however, significantly reduced the activation time of IK.~ot after hypoosmotic stimulation. A similar dependence of pHo on the K + current magnitude is found in e.g. TASK background two-pore channels [31. Since the swelling-activated K + channel, like the two-pore channels, is a small (4 pS) K + channel which lack intrinsic voltage dependence this might indicate that the K + channels underlying IK,,.,,~is of the two-pore type. To investigate the possible effect of pH i on the currents experiments were performed at pHo 7.4 but with pH~ buffered in the range 6.4-8.4. lc+.,.o~ appeared unaffected by the pHi, however the current density of I~.,.o~ was significantly reduced at pHi 6.4 and 7.0 when compared to control (pHi 7.4). At pHi 7.9 the magnitude of IK.,.o~was not further increased but the activation time of the current was significantly reduced. Increasing pHi to 8.4 under isotonic conditions resulted in the spontanous activation of a IK with similar current-voltage relation and pharmacological profile as IK,vo~indicating that the K ÷ channels underlying IK.,,,~ can be activated by increasing pH~ without any cell swelling. Noise analysis of IK.,o~demonstrated that both pHo and phi affected the number of single gating channels. Decreasing pH reduced the number of gating channels whereas increasing pH from 7.4 to 7.9 or 8.4 increased the number of channel openings. Whether the effect of pH is a direct effect on the K + channels or due to modulation of the signalling pathway is at present unclear. [1] Livne, A. and Hoffmann, E.K. (1990) J. Membrane Biol. 114:153-157 121 Kramhefft, B., Lambert, I.H., Hoffmann, E.K. and J~argensen, F. (1986) Am. J. Physiol. 251:C369-C379 131 Duprat, F., Lesage, F., Fink, M., Reyes, R., Heurteaux, C and Lazdunski, M. (1997) EMBO J. L6:5464-5471.
AMINO
Abstracts' / Comparative Biochemistry and Physiology Part A 126 (2000) SI-S163
ACID TRANSPORTER
Hosoi M), Goto C), T o y o h a r a H. ~
OF MUSSEL
T a k e u c h i K. ~, K u b o t a S. ~, S a k a g u c h i
M),
H a y a s h i I. ~, Y o k o y a m a
y b and
aDivision of Applied Biosciences, Graduate School of Agriculture, Kyoto University; bDepartment of Domestic Science, Kobe Shoin Women's College To understand the regulation of intracellular osmolyte concentration in marine mussel, Mytilus galloprovincialis (Lam.) for adapting the environmental salinity, we investigated changes in free amino acids pool of the tissues (mantle, gill and adductor muscle) and hemolyph exposed to abrupt changes in salinity. We found that free amino acids pool in tissues decreased significantly with the decrease in salinity, but slightly increase or decrease with the increase in salinity. Most prevalent free amino acid was taurine which was corresponded to 50-80% of total free amino acids pool and the content of taurine was subject to the change in salinity. For the better understanding of the mechanism to regulate the intracellular amino acid concentration, we tried to isolate the cDNA coding sodium and chloride-dependent taurine transporter which uptakes taurine from hemolyph into cell. In order to isolate the transporter eDNA, we designed a set of oligonucleotide primers based on the amino acids sequence which is highly conserved among taurine transporters in vertebrates. As a result of RT-PCR and 5"-, 3'-RACE, we isolated eDNA which possibly encodes a sodium and chloride-dependent amino acid ransporter. Comparison with amino acid sequences in the SWISS-PROT indicated that the deduced amino acids sequence is highly homologous to taurine transporter in vertebrates with about 50% amino acid identity. The cloned transporter has predictive 12 transmenbrane domains and particularly highly conserved amino acid sequences between the first and the second transmenbrane domains. Interestingly, the mussel transporter has additional amino acid sequences in places, of which function is unknown. Northern blot analysis showed that mRNA of the transporter in mantle, gill and adductor muscle was induced in accordance with the increase in the environmetnal osmolarity, while increase in mRNA was also detected for the hypo-osmotic adaptation. The transporter gene cloned from mussel in the present study has higher amino acid identity with mammalian and carp taurine transporter. We are now trying to determine substrates of the transporter by the transient expression experiment using COS-7 cells. Of interest, the expression of the gene was induced in response to hypo-osmotic stress as well as hyperosmotic stress. Further studies are required to understand the induction of the gene responding to the environmental salinity.
A R O L E O F E X T E R N A L A N D I N T E R N A L P H IN T H E R E G U L A T I O N O F T H E V O L U M E SENSITIVE K + CURRENT IN EHRLICH ASCITES TUMOUR CELLS i H o u g a a r d C., 2 J O r g e n s e n F. a n d 1 H o f f m a n n E . K . IA u g u s t K r o g h I n s t i t u t e , 13 U n i v e r s i t e t s p a r k e n , D K - 2 1 0 0 C o p e n h a g e n , D e n m a r k . 2 I M B , D e p a r t m e n t o f P h y s i o l o g y & P h a r m a c o l o g y , 21 W i n s l ~ w p a r k e n , D K - 5 0 0 0 O d e n s e , D e n m a r k . Previous studies have demonstrated that hypoosmotic swelling of Ehrlich ascites tumour cells results in cytosolic alkalinization followed by acidification and that volume recovery after cell swelling is strongly affected by the pH of the external solution [1,2]. These observations indicate that pH could be involved in the regulation of channel activity upon swelling of Ehrlich cells. In the present study we have investigated the possible role of pH in the modulation of the volumesensitive K + (IK,,o0 and CI- (Ict.,o0 currents in Ehrlich cells using the whole-cell patch-clamp technique. In order to investigate the effect of external pH (pHo) on IK.,.o~and Icl.,.ol the currents were activated by reducing the osmolarity of the external medium at various pHo values. The pH of the pipette solution (pH0 was in these experiments buffered at 7.4. Both the magnitude and the activation time of Ic~.,o~ appeared unaffected by pHo. In contrast, the current density of I~,~oLwas significantly reduced at pHo 6.4 and pHo 6.9 when compared to the control (pHo 7.4). Increasing pHo to 8.4 did not further increase the current density of IK,vo~but, however, significantly reduced the activation time of IK.~ot after hypoosmotic stimulation. A similar dependence of pHo on the K + current magnitude is found in e.g. TASK background two-pore channels [31. Since the swelling-activated K + channel, like the two-pore channels, is a small (4 pS) K + channel which lack intrinsic voltage dependence this might indicate that the K + channels underlying IK,,.,,~is of the two-pore type. To investigate the possible effect of pH i on the currents experiments were performed at pHo 7.4 but with pH~ buffered in the range 6.4-8.4. lc+.,.o~ appeared unaffected by the pHi, however the current density of I~.,.o~ was significantly reduced at pHi 6.4 and 7.0 when compared to control (pHi 7.4). At pHi 7.9 the magnitude of IK.,.o~was not further increased but the activation time of the current was significantly reduced. Increasing pHi to 8.4 under isotonic conditions resulted in the spontanous activation of a IK with similar current-voltage relation and pharmacological profile as IK,vo~indicating that the K ÷ channels underlying IK.,,,~ can be activated by increasing pH~ without any cell swelling. Noise analysis of IK.,o~demonstrated that both pHo and phi affected the number of single gating channels. Decreasing pH reduced the number of gating channels whereas increasing pH from 7.4 to 7.9 or 8.4 increased the number of channel openings. Whether the effect of pH is a direct effect on the K + channels or due to modulation of the signalling pathway is at present unclear. [1] Livne, A. and Hoffmann, E.K. (1990) J. Membrane Biol. 114:153-157 121 Kramhefft, B., Lambert, I.H., Hoffmann, E.K. and J~argensen, F. (1986) Am. J. Physiol. 251:C369-C379 131 Duprat, F., Lesage, F., Fink, M., Reyes, R., Heurteaux, C and Lazdunski, M. (1997) EMBO J. L6:5464-5471.