Table 2. Na⁺-mediated [³H]leucine binding and the effect of K⁺ addition, and K⁺-dependent Na⁺/[³H]leucine displacement in Na1 site mutants.

For LeuT Na1 site mutants (and K398C), Na⁺-mediated [³H]leucine binding was assayed using 10× K_d of [³H]leucine in the presence of 0, 200, and 800 mM K⁺. For K⁺-dependent displacement of Na⁺-mediated [³H]leucine binding, 10× K_d of [³H]leucine and Na⁺ equivalent to the EC₅₀ value for each of the mutants were used. Na⁺ and K⁺ were substituted for Ch⁺ to preserve the ionic strength. Data were fitted to a Hill equation, yielding EC₅₀ and IC₅₀ values reported here as mean [s.e.m. interval], n=3–6 determined in triplicates. Comparing the Na⁺ EC₅₀ obtained for the mutants with that of WT showed significant difference for all (p<0.0001) except for E290Q (p>0.05), and for the K⁺ IC₅₀ all showed significant differences from WT (p<0.0001) expect for T254S (p>0.05) (Dunnett’s multiple comparison-corrected one-way analysis of variance [ANOVA]). N.d. indicates that the value is not determined. WT data are also reported in Figure 1.

	EC50 Na+ (mM)	IC50 K+ (mM)	EC50 Na++200 mM K+ (mM)	EC50 Na++800 mM K+ (mM)	EC50800/EC50
WT	7.7 [7.3; 8.1]	235 [228; 241]	19.4 [19.1; 19.8]	48.5 [47.5; 49.5]	~6
A22V	2.0 [2.0; 2.1]	49.0 [47.7; 50.4]	23.0 [21.7; 24.5]	70.7 [69.2; 72.2]	~35
A22S	53.3 [45.5; 62.5]	>1900	72.4 [62.3; 84.2]	117 [100; 137]	~2
N27Q	2.4 [2.3; 2.5]	>2,300	n.d.	4.4 [4.0; 4.8]	~2
T254S	15.1 [14.2; 16.0]	219 [209; 229]	43.5 [37.4; 50.5]	90.2 [73.8; 110]	~6
N286Q	54.6 [47.5; 62.6]	>2000	n.d.	121 [111; 133]	~2
E290Q	9.6 [8.8; 10.4]	n.d.	n.d.	144 [132; 157]	~15
K398C	7.7 [6.8; 8.8]	n.d	n.d.	51.5 [48.1; 55.1]	~7