Electrical and mechanical properties of active hair bundle motion in bullfrog saccule hair cells

Calcium influx through the transduction channels after a mechanical stimulus produces an adaptation of the current in two phases, occurring in tens of milliseconds and milliseconds, which can be attributed to a myosin-1c motor and to channel closure after Ca2+ binding, respectively. The negatively-directed twitch, the mechanical correlate of fast adaptation, produces force, and is considered a candidate for a bundle-based cochlea amplifier. Qualitatively similar backwards movements of the bundle could result from Ca2+ binding to several intracellular elements, such the channel or an associated protein which changes the open probability of the channel; an internal elastic element, decreasing its stiffness; or an associated inelastic release element which extends by a fixed distance. To move the bundle, I employed a gradient force light trap ("optical tweezers") which used a Nd:YAG laser refracted by a 2 mum polystyrene bead attached to the kinociliary bulb of a frog saccule hair cell to exert a force of several hundred pN. The movement of the bead was separately detected by a HeNe laser and a quadrant photodiode, with the cell patch clamped. Hyperpolarizing steps after a depolarization, starting calcium influx, produced a twitch, which was measured at a series of bundle holding forces and was compared to predicted bundle movements fitted with one-parameter fits for each binding site. Only a change in open probability after calcium binding could account for the twitch. This model accurately reproduced the time-dependent bundle movements, including the twitch, for mechanical bundle deflections and voltage changes. A depolarization produces an additional initial negative movement of the bundle called the "flick". This amplitude was roughly linear with voltage between -160 and +60 mV, but saturated at larger depolarizations, with a time constant of ∼0.2 ms and up to 6nm for a 160 mV depolarization. Neither calcium entry nor transduction was required, but reduction of tip link tension either by elimination with BAPTA or slackening with large negative deflections abolished the flick. These data suggest that it is associated with the transduction apparatus, and may reflect a voltage-dependent conformational change of the channel itself.