Probing the pore of the auditory hair cell mechanotransducer channel in turtle

Hair cell mechano-electric transducer (MET) channels play a pivotal role. in auditory and vestibular signal detection, yet few data exist regarding their molecular nature. Present work characterizes the MET channel pore, a region whose properties are thought to be intrinsically determined. Two approaches were used. First, the channel was probed with antagonists of candidate channel subtypes including: cyclic nucleotide-gated channels, transient receptor potential channels and gap-junctional channels. Eight new antagonists were identified. Most of the effective antagonists had a partially charged amine group predicted to penetrate the channelpore, antagonizing current flow, while the remainder of the molecule prevented further permeation of the compound through the pore. This blocking mechanism was tested using curare to demonstrate the open channel nature of the block and by identifying methylene blue as a permeant channel blocker. The second approach estimated dimensions of the channel pore with simple amine compounds. The narrowest diameter of the pore was calculated as 12.5 +/- 0.8 Angstrom and the location of a binding site similar to45% of the way through the membrane electric field was calculated. Channel length was estimated as similar to31Angstrom and the width of the pore mouth at < 17Angstrom. Each effective antagonist had a minimal diameter, measured about the penetrating amine, of less than the pore diameter, with a direct correlation between IC50 and minimal diameter. The IC50 was also directly related to the length of the amine side chains, further validating the proposed pore blocking mechanism. Data provided by these two approaches support a hypothesis regarding channel permeation and block that incorporates molecular dimensions and ion interactions within the pore.