Background: Within the cochlea, inner hair cells transduce pressure waves into neural signals while outer hair cells raise and lower the inner hair cells relative to the tectorial membrane. Prestin is the molecular motor protein expressed in outer hair cells that is responsible for this cochlear amplifier mechanism. In response to changing polarization of the cell, Prestin switches between its compact and expanded conformations, thereby stretching or shortening the length of the cell. During this process, the cell membrane exhibits nonlinear capacitance: the voltage-capacitance function looks like a bell curve. Nonlinear capacitance is the signature characteristic of Prestin function. It is now thought that Prestin functions as a partial anion (chloride) transporter and is structured with two sets of seven unique transmembrane domains. This theory supplanted the hypothesis that Prestin was an anion exchanger. A central difference between the two theories is that the chloride channel relies on cysteine-based disulfide bonding while the partial anion transporter does not. The new theory has been supported in rat and chicken Prestin, but it has not been tested in gerbil Prestin. Freeze fracture and electron microscopy have related the mathematical model of nonlinear capacitance to the actual Prestin in the membrane. Because the mathematical relationship has been demonstrated in gerbil Prestin, it is essential that the partial anion transporter hypothesis is supported in gerbil Prestin as well. This will enable future studies of Prestin's structure and mechanism that use both the biology and the mathematical model of nonlinear capacitance. Objective: The objective of this research is to support the partial anion transporter hypothesis in gerbil Prestin so that future experiments can rely on both this understanding of Prestin's mechanism and the model of nonlinear capacitance. Hypothesis: We hypothesize that the partial anion transporter hypothesis will be supported by demonstrating that cysteine-based disulfide bonds are not essential for gerbil Prestin function. Methods: For this experiment, we first mutated the nine cysteine residues of gerbil Prestin to alanine. Second, we expressed either the mutant protein or the wild-type protein in Chinese Hamster Ovary cells. Third, we performed whole-cell patch clamping under a ramp protocol that gave the membrane potentials ranging from - 160 mV to +160 mV. During the ramp protocol, we recorded the capacitance of the cell. Conclusions: Because both the mutant and wild-type Prestin constructs exhibited the bell-shaped nonlinear capacitance curves, we concluded that cysteine-based disulfide bonding is not essential for gerbil Prestin. For this reason, we believe that gerbil Prestin operates as a partial anion transporter.
"},{"label":"Degree","value":["Bachelor of Arts with Departmental Honors"]},{"label":"Author-supplied Keywords","value":["Prestin","Nonlinear capacitance","Outer hair cells"]},{"label":"Administrative Note","value":"Drupal Node ID: 24264