CYSTEINE SCANNING MUTAGENESIS OF THE SEGMENT BETWEEN PUTATIVE TRANSMEMBRANE HELIX-IV AND HELIX-V OF THE HIGH-AFFINITY NA+ GLUCOSE COTRANSPORTER SGLT1 - EVIDENCE THAT THIS REGION PARTICIPATES IN THE NA+ AND VOLTAGE-DEPENDENCE OF THE TRANSPORTER/
B. Lo et M. Silverman, CYSTEINE SCANNING MUTAGENESIS OF THE SEGMENT BETWEEN PUTATIVE TRANSMEMBRANE HELIX-IV AND HELIX-V OF THE HIGH-AFFINITY NA+ GLUCOSE COTRANSPORTER SGLT1 - EVIDENCE THAT THIS REGION PARTICIPATES IN THE NA+ AND VOLTAGE-DEPENDENCE OF THE TRANSPORTER/, The Journal of biological chemistry, 273(45), 1998, pp. 29341-29351
Site-directed mutagenesis and chemical modification of specific cystei
ne amino acid side chains by methanethiosulfonate (MTS) derivatives we
re combined to elucidate structure/function relationships of the clone
d rabbit Na+/glucose cotransporter, SGLT1, Each amino acid in the regi
on (residues 162-173) between putative transmembrane helices IV and V
of SGLT1 was replaced individually with Cys, Mutant proteins were expr
essed in Xenopus laevis oocytes and studied using the two-electrode vo
ltage clamp method. At certain key positions, Cys substitution resulte
d in 1) a change in the apparent affinity for sugar, 2) an alteration
in the voltage dependence of the transient currents, and 3) a sensitiv
ity to inhibition by either the ethylamine (MTSEA) or the ethylsulfona
te MTS derivatives. For the three Cys mutants inhibited by MTSEA (F163
C, A166C, and L173C), inhibition of steady state transport is related
to changes in membrane potential-dependent transitions within the Na+/
glucose transport cycle. MTSEA shifted the transient currents of these
Cys mutants toward more negative membrane potentials (Delta V-0.5 = -
18 mV for F163C and A166C, -12 mV for L173C), When the mutations were
combined to produce double and triple Cys mutants, the degree to which
the transient currents were shifted along the membrane potential axis
by MTSEA correlated with the number of cysteines, In this way it was
possible to manipulate the voltage dependence of the transient current
s over a range spanning 91 mV. Examination of the Na+ dependence of th
e transient currents indicates that a 91-mV shift is equivalent to tha
t caused by a 10-fold reduction in the external Na+ concentration. We
conclude that this region has a role in determining the Na+ binding an
d voltage-sensing properties of SGLT1 and that it forms an alpha-helix
with one surface possibly lining a Na+ pore within SGLT1.