| Abstract #728, Date 2/17/99, Session V, Podium , 3:30p |
| Green card motors: OHC lateral membrane molecular motors may travel |
| *J. Santos-Sacchi, H.-B. Zhao (Yale University School of Medicine) |
The OHC, through its mechanical activity, enhances auditory sensitivity and frequency selectivity in the mammal. Voltage-dependent molecular motors restricted to the lateral plasma membrane drive the cell’s overall mechanical response. We are interested in characterizing lateral movements of these motors within the plane of the membrane, so that the identity of the motor and its interactions with other membrane components can be elucidated. In order to look at motor diffusion, we have developed a means to selectively ablate motors in a restricted region of the lateral plasma membrane. This is done by localized excitation of intracellular dyes (Lucifer Yellow, carboxyfluorescein) or plasma membrane restricted dyes (di-8-ANEPPS), possibly via free radical photo-inactivation. High-resolution, whole-cell capacitance measures (Santos-Sacchi et. al., J. Physiol. 510:225-235. 1998) are used to gauge motor destruction, since each motor contributes to the OHC’s nonlinear capacitance with its gating charge movement. Illumination is delivered to the cell under voltage clamp via a shuttered, quartz fiberoptic pulled to a few microns tip and placed within a few microns of the cell. Exposures irreversibly reduce whole cell capacitance by one to two pF of nonlinear capacitance. It is expected that repeated exposure to the same location will only inactivate more capacitance if functional motors diffuse into the exposed region. In fact, this is what we observe. That is, we are able to incrementally ablate nonlinear capacitance over the course of a few minutes. The voltage dependence of remaining OHC capacitance is not altered. Similar treatments in supporting cells show no effects on their linear capacitance. These data may indicate the occurrence of OHC molecular motor lateral mobility. Further studies are in progress to verify our hypothesis and quantify the effects. |