Hi, I wrote a discussion based on my graduation research. I hope to get feedback and of course suggestion on improving this article to make it a better one. Thanks in advance.Here it goes:Amiloride, a well known inhibitor for Na+-coupled systems in various organisms, was reported in a previous report to be a potent inhibitor for the flagellar motors of alkalophilic Bacillus by competing with Na+ in the medium. Like amiloride, results show that naphthamil also caused the growth inhibition of alkalophilic Bacillus spp. However, it does not affect the interpretation of the mode of action on the motors, since the motility inhibition by naphthamil begins almost simultaneously, but growth inhibition by 50[font='Ｍ]μ[/font]M naphthamil was observed only about 2hrs after the addition of naphthamil. Inhibition caused by addition of 20[font='Ｍ]μ[/font]M naphthamil was less significant, and thus it implies that naphthamil causes less inhibition on the cell growth at lower concentrations. It was reported that cells grown in the presence of amiloride showed some morphological abnormalities, suggesting that one of the site for this secondary effect of amiloride occurs in the sequence of cell wall synthesis. Studies showed some morphological abnormalities such as elongation occurred on the cells after adding naphthamil, inferring that the secondary effect affects the sequence of cell wall sysnthesis as it does in the amiloride-added cells.
From the kinetic analysis of the inhibition of motility of alkalophilic Bacillus spp. by naphthamil shown in FIG.7 revealed that naphthamil inhibited the motility of Na+-driven flagellar motors in a noncompetitive manner with Na+ in the medium. Recent studies show that unlike amiloride, a major interaction site of phenamil on the motor is not identical to the Na+ interaction site of the motor. Besides, a mutant of an alkalophilic Bacills sp. that was isolated as a phenamil-resistant swarmer showed no change in the sensitivity of its motility to amiloride, prompting speculation that the interaction site for phenamil on the motor is different from the site for amiloride since the latter is strictly located at the Na+ interaction site of the motor. In our research, naphthamil was found to cause inhibition on the Na+-driven flagellar motors by behaving in a noncompetitive manner with Na+ in the medium. Thus it is likely to predict that the interaction site of naphthamil on the motor differs from that of amiloride too. The results suggest that the site of the action of naphthamil on the motor, as phenamil, is the Na+ interaction site located at the inner side of the force-generating unit. However, napthamil and phenamil could not have interacted on the same site since a mutant of an alkalophilic Bacills sp. that was isolated as a phenamil-resistant swarmer showed no change in the sensitivity of its motility to naphthamil. Hence, naphthamil seemingly interacts with Na+ interaction site, which is different from that of phenamil at the inner side of the motor. Anyway, there is another hypothesis suggests that both phenamil and napthamil interact on the same site within the motor in the consideration of both structures. There is a possibility that some regions were mutated in phenamil-resistant cells and thus allow phenamil only getting through because pheyl-group is smaller than naphthyl-group, though both phenamil and naphthamil may react on the same site.
According to previous reports, besides amiloride, benzamil and phenamil were very potent Na+ channel inhibitors with little or no effect on the Na+/H+ antiporter. The relative potencies of amiloride, benzamil, and phenamil on the Na+ channels are parallel to their relative potencies on the flagellar motors of RA1 cells. Thus the structure of the Na+-interacting site of the Na+-driven flagellar motors may have some similarity with that of Na+ channels but not with that of Na+/H+ antiporters. The structure of the newly synthesized naphthamil for this research is similar to phenamil and is thus expected to have a strong effect on the Na+ channels but little effect on the Na+/ H+ antiporters and Na+/Ca+ exchange systems.
Other amiloride analogs such as benzamil HCl, dichlorophenamil, iodophenamil, hydrophenamil, phenamil isomer, and phenylguanizine were studied. Iodophenamil and benzamil HCl were found to have a stronger effect than amiloride on the Na+ channels. The order of potency is shown as below.