Mechanistic insights and functional determinants of the transport cycle of the ascorbic acid transporter SVCT2. Activation by sodium and absolute dependence on bivalent cations.
We characterized the human Na(+)-ascorbic acid transporter SVCT2 and developed a basic model for the transport cycle that challenges the current view that it functions as a Na(+)-dependent transporter. The properties of SVCT2 are modulated by Ca(2+)/Mg(2+) and a reciprocal functional interaction between Na(+) and ascorbic acid that defines the substrate binding order and the transport stoichiometry. Na(+) increased the ascorbic acid transport rate in a cooperative manner, decreasing the transport K(m) without affecting the V(max), thus converting a low affinity form of the transporter into a high affinity transporter. Inversely, ascorbic acid affected in a bimodal and concentration-dependent manner the Na(+) cooperativity, with absence of cooperativity at low and high ascorbic acid concentrations. Our data are consistent with a transport cycle characterized by a Na(+):ascorbic acid stoichiometry of 2:1 and a substrate binding order of the type Na(+):ascorbic acid:Na(+). However, SVCT2 is not electrogenic. SVCT2 showed an absolute requirement for Ca(2+)/Mg(2+) for function, with both cations switching the transporter from an inactive into an active conformation by increasing the transport V(max) without affecting the transport K(m) or the Na(+) cooperativity. Our data indicate that SVCT2 may switch between a number of states with characteristic properties, including an inactive conformation in the absence of Ca(2+)/Mg(2+). At least three active states can be envisioned, including a low affinity conformation at Na(+) concentrations below 20 mM and two high affinity conformations at elevated Na(+) concentrations whose Na(+) cooperativity is modulated by ascorbic acid. Thus, SVCT2 is a Ca(2+)/Mg(2+)-dependent transporter.
Godoy
Alejandro
A
Departamento de Fisiopatología, Universidad de Concepción, Concepción, Casilla 160C, Chile.
Ormazabal
Valeska
V
Moraga-Cid
Gustavo
G
Zúñiga
Felipe A
FA
Sotomayor
Paula
P
Barra
Valeria
V
Vasquez
Osmán
O
Montecinos
Viviana
V
Mardones
Lorena
L
Guzmán
Catherine
C
Villagrán
Marcelo
M
Aguayo
Luis G
LG
Oñate
Sergio A
SA
Reyes
Alejandro M
AM
Cárcamo
Juan G
JG
Rivas
Coralia I
CI
Vera
Juan Carlos
JC
D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D002412:Cations; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008545:Melanoma / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D029363:Organic Anion Transporters, Sodium-Dependent / Q000737:chemistry / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000737:chemistry*; D060347:Sodium-Coupled Vitamin C Transporters; D027981:Symporters / Q000737:chemistry / Q000502:physiology*
D002412:Cations; D029363:Organic Anion Transporters, Sodium-Dependent; C555163:SLC23A2 protein, human; D060347:Sodium-Coupled Vitamin C Transporters; D027981:Symporters; D012964:Sodium; D008274:Magnesium; D001205:Ascorbic Acid; D002118:Calcium