The goal of the proposed research is to characterize the cellular mechanisms that contribute to tuning in turtle cochlear hair cells. By understanding how these cellular mechanisms contribute to frequency selectivity, we will be able to assess their limitations and applicability to hearing in higher vertebrates including man. A combination of whole-cell and single-channel patch-clamp techniques will be used on solitary cells isolated from identified regions of the turtle cochlea.
The first aim i s to analyze the mechanical response of the hair-cell ciliary bundle, and to determine the role of this response in the overall transduction mechanism. The source, strength and voltage-sensitivity of the ciliary motion will be determined. The ability of the transducer channel to detect this motion will be ascertained, and any variation in the size and voltage-sensitivity with characteristic frequency will be assessed.
The second aim i s to characterize further the conductances in the basolateral hair-cell membrane implicated in electrical resonance. Changes in the size and kinetics of the membrane currents in cells of known characteristic frequency will be analyzed using intracellular perfusion. Specific protocols address the isolation inward currents due to the apical transducer and the basolateral calcium current, the characterization of possible electrogenic transport of calcium out of the cell, and a separation of the calcium from the calcium-activated potassium currents in a single cell.
The third aim i s to analyze the kinetic behavior of the calcium-activated potassium channel and to characterize the calcium- and voltage- sensitivity. Since the kinetic behavior of this current is highly correlated with the characteristic frequency to which a hair cell is tuned, it is presumed to play a major role in frequency selectivity. Simultaneous cell-attached single-channel and whole- cell recording will be used to compare the behavior of the single channel and the macroscopic calcium-activated potassium current. The proposed research will analyze these ionic currents to assess whether hair-cell tuning due to basolateral ionic conductances coexists with, or is enhanced by, mechanisms acting via the transduction process in the ciliary bundle. The ionic currents measured under voltage-clamp will be used to reconstruct the membrane resonance seen in current-clamp.
