Neutralization of conservative charged transmembrane residues in the Na+/glucose cotransporter SGLT1.

摘要: Our goal was to identify pairs of charged residues in the membrane domains Na+/glucose cotransporter (SGLT1) that form salt bridges, obtain information about packing transmembrane helices. The strategy neutralize Glu225, Asp273, Asp294, and Lys321 helices 6-8, express mutants oocytes, measure [14C]-alphaMDG uptake, then attempt find second-site mutations opposite charge restored function. alphaMDG uptake by E225A identical SGLT1, whereas transport reduced over 90% for D273A, D294A, K321A not double D273A/K321A or D294A/K321A. This suggested K321 did bridges with D273 D294 E225 involved salt-bridging. Neutralization dramatically changed Na+ uniport cotransport kinetics. maximum rate increased 3-5-fold a decrease apparent affinity (70 vs 3 mM) no change H+ (0.5 microM). caused +50 mV shift charge/voltage (Q/V) relaxation time constant (tau)/voltage curves presteady-state kinetics remained unchanged. lower resulted also 200-fold increase K0.5 phlorizin. Replacements alanine, valine, glutamine, arginine, glutamic acid steady-state similar way. Therefore, we suggest determines, directly indirectly, (i) selectivity SGLT1 activity (ii) affinities Na+, indirectly sugar mode.