NEUTRALIZATION OF CONSERVATIVE CHARGED TRANSMEMBRANE RESIDUES IN THE NA+ GLUCOSE COTRANSPORTER SGLT1/

Our goal was to identify pairs of charged residues in the membrane dom ains of the Na+/glucose cotransporter (SGLT1) that form salt bridges, to obtain information about packing of the transmembrane helices. The strategy was to neutralize Glu225, Asp273, Asp294, and Lys321 in helic es 6-8, express the mutants in oocytes, measure [C-14]-alpha MDG uptak e, and then attempt to find second-site mutations of opposite charge t hat restored function, alpha MDG uptake by E225A was identical to that by SGLT1, whereas transport was reduced by over 90% for D273A, D294A, and K321A and was not restored in the double mutants D273A/K321A or D 294A/K321A. This suggested that K321 did not form salt bridges with D2 73 or D294 and that E225 was not involved in salt-bridging. Neutraliza tion of K321 dramatically changed the Na+ uniport and Na+/glucose cotr ansport kinetics. The maximum rate of uniport and no in K321A increase d 3-5-fold with a decrease in the apparent affinity for Na+ (kappa(0.5 )(Na+) 70 vs 3 mM) change in apparent H+ affinity (kappa(0.5)(H+) 0.5 mu M). The change in Na+ affinity caused a +50 mV shift in the charge/ voltage (Q/V) and relaxation time constant (tau)/voltage curves in the presteady-state kinetics. The presteady-state kinetics in H+ remained unchanged, The lower Nat affinity resulted also in a 200-fold increas e in the apparent K-0.5 for alpha MDG and phlorizin. Replacements of K 321 with alanine, valine, glutamine, arginine, or glutamic acid residu es changed the steady-state kinetics in a similar way. Therefore, we s uggest that K321 determines, directly or indirectly, (i) the rate and selectivity of SGLT1 uniport activity and (ii) the apparent affinities of SGLT1 for Na+, and indirectly sugar in the cotransport mode.