TABLE 1.

Single Cys replacements of conserved residues in TMS II of NhaA

For characterization of the mutations, EP432 cells transformed with the plasmids carrying the indicated mutations were used. The expression level was expressed as percentage of the control cells (EP432/pCL-AXH3). Na⁺/H⁺ and Li⁺/H⁺ antiporter activity at pH 8.5 was determined with 10 mm NaCl or LiCl. The activity is expressed as percentage of dequenching. EP432/pBR322 served as a negative control. The apparent K_m for the ions was determined at pH 8.5, as described under “Experimental Procedures.”

Mutation in TMS II	Expression	Growth			Activity		Apparent Km
		Na+ (7)	Na+ (8.3)	Li+ (7)	Na+	Li+	Na+	Li+
					%		mm
L60C	165	+++	++	+++	83	91	0.15	0.02
W62C	100	+++	++	+++	73	90	1.0	0.02
N64C	190	+++	++	+++	93	85	0.12	0.02
D65C	52	+++	++	+++	56	65	2.26	0.7
L67C	100	+++	++	+++	62	84	1.8	0.67
M68C	150	+++	++	+++	58	52	0.41	0.02
F71C	100	+	–	+++	83	55	0.83	0.14
F72C	76	+++	++	+++	67	43	0.54	0.02
G76C	82	+++	++	+++	80	73	0.18	0.02
E78C	22	+++	++	+++	40	24	1.12	0.68
E82C	17	+++	++	+++	33	12	2.61	NDa
Controls
pCL-AXH3	100	+++	++	+++	88	75	0.19	0.07
pBR322		–	–	–	–	–	–	–

^a ND, not determined.