Vibrio alginolyticus mutants resistant to phenamil, a specific inhibitor of the sodium-driven flagellar motor

The polar flagella of Vibrio alginolyticus are driven by sodium motive force and those motors are specifically and strongly inhibited by phenamil, an amiloride analog that is thought to interact with a sodium channel of the flagellar motor. To study the sodium ion coupling site, we isolated motility mutants resistant to phenamil and named the phenotype Mpa(r) for motility resistant to phenamil. The motility of the wild-type (Mpa(s)) was inhibited by 50 μM phenamil, whereas Mpa(r) strains were still motile in the presence of 200 μM phenamil. The K(i) value for phenamil in the Mpa(r) strain was estimated to be five times larger than that in the Mpa(s) strain. However, the sensitivities to amiloride or benzamil, another amiloride analog, were not distinctly changed in the Mpa(r) strain. The rotation rate of the wild-type Na⁺-driven motor fluctuates greatly in the presence of phenamil, which can be explained in terms of a relatively slow dissociation rate of phenamil from the motor. We therefore studied the stability of the rotation of the Mpa(r) and Mpa(s) motors by phenamil. The speed fluctuations of the Mpa(r) motors were distinctly reduced relative to the Mpa(s) motors. The steadier rotation of the Mpa(r) motors can be explained by an increase in the phenamil dissociation rate from a sodium channel of the motor, which suggests that a phenamil-specific binding site of the motor is mutated in the Mpa(r) strain.