The rotation of the Na+-driven flagellar motor is specifically and strongly inhibited by phenamil, an amiloride analog. Here, we provide the first evidence that phenamil interacts directly with the Na+-channel components (PomA and PomB) of the motor. The alterations in Mpar (motility resistant to phenamil) strains were mapped to the pomA and/or pomB genes. We cloned and sequenced pomA and pomB from two Mpar strains, NMB205 and NMB201, and found a substitution in pomA (Asp148 to Tyr; NMB205) and in pomB (Pro16 to Ser; NMB201). Both residues are predicted to be near the cytoplasmic ends of the putative transmembrane segments. Mutational analyses at PomA-Asp148 and PomB-Pro16 suggest that a certain structural change around these residues affects the sensitivity of the motor to phenamil. Co-expression of the PomA D148Y and PomB P16S proteins resulted in an Mpar phenotype which seemed to be less sensitive to phenamil than either of the single mutants, although motility was more severely impaired in the absence of inhibitors. These results support the idea that PomA and PomB interact with each other and suggest that multiple residues, including Asp148 of PomA and Pro16 of PomB, constitute a high-affinity phenamil-binding site at the inner face of the PomA/PomB channel complex.
Copyright 1999 Academic Press.