'Active' sugar transport in eukaryotes.
Sugar transporters in prokaryotes and eukaryotes belong to a large family of membrane proteins containing 12 transmembrane alpha-helices. They are divided into two classes: one facilitative (uniporters) and the other concentrative (cotransporters or symporters). The concentrative transporters are energised by either H+ or Na+ gradients, which are generated and maintained by ion pumps. The facilitative and H(+)-driven sugar transporters belong to a gene family with a distinctive secondary structure profile. The Na(+)-driven transporters belong to a separate, small gene family with no homology at either the primary or secondary structural levels. It is likely that the Na(+)- and H(+)-driven sugar cotransporters share common transport mechanisms. To explore these mechanisms, we have expressed cloned eukaryote Na+/sugar cotransporters (SGLT) in Xenopus laevis oocytes and measured the kinetics of sugar transport using two-electrode voltage-clamp techniques. For SGLT1, we have developed a six-state ordered model that accounts for the experimental data. To test the model we have carried out the following experiments. (i) We measured pre-steady-state kinetics of SGLT1 using voltage-jump techniques. In the absence of sugar, SGLT1 exhibits transient carrier currents that reflect voltage-dependent conformational changes of the protein. Time constants for the carrier currents give estimates of rate constants for the conformational changes, and the charge movements, integrals of the transient currents, give estimates of the number and valence of SGLT1 proteins in the plasma membrane. Ultrastructural studies have confirmed these estimates of SGLT1 density. (ii) We have perturbed the kinetics of the cotransporter by site-directed mutagenesis of selected residues.(ABSTRACT TRUNCATED AT 250 WORDS)
Wright
E M
EM
Department of Physiology, UCLA School of Medicine 90024-1751.
Loo
D D
DD
Panayotova-Heiermann
M
M
Lostao
M P
MP
Hirayama
B H
BH
Mackenzie
B
B
Boorer
K
K
Zampighi
G
G
D000818:Animals; D001693:Biological Transport, Active; D050260:Carbohydrate Metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008297:Male; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017433:Protein Structure, Secondary; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D051269:Sodium-Glucose Transporter 1; D014982:Xenopus laevis
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; C495674:SLC5A1 protein, human; D051269:Sodium-Glucose Transporter 1; D012964:Sodium; D005947:Glucose