Mechanoelectrical transducer currents in turtle auditory hair cells adapted to maintained stimuli via a Ca(2+)-dependent mechanism characterized by two time constants of approximately 1 and 15 ms. The time course of adaptation slowed as the stimulus intensity was raised because of an increased prominence of the second component. The fast component of adaptation had a similar time constant for both positive and negative displacements and was unaffected by the myosin ATPase inhibitors, vanadate and butanedione monoxime. Adaptation was modeled by a scheme in which Ca(2+) ions, entering through open transducer channels, bind at two intracellular sites to trigger independent processes leading to channel closure. It was assumed that the second site activates a modulator with 10-fold slower kinetics than the first site. The model was implemented by computing Ca(2+) diffusion within a single stereocilium, incorporating intracellular calcium buffers and extrusion via a plasma membrane CaATPase. The theoretical results reproduced several features of the experimental responses, including sensitivity to the concentration of external Ca(2+) and intracellular calcium buffer and a dependence on the onset speed of the stimulus. The model also generated damped oscillatory transducer responses at a frequency dependent on the rate constant for the fast adaptive process. The properties of fast adaptation make it unlikely to be mediated by a myosin motor, and we suggest that it may result from Ca(2+) binding to the transducer channel or a nearby cytoskeletal element.

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Mechanoelectrical transducer currents in turtle auditory hair cells adapt to maintained stimuli via a Ca2+-dependent mechanism that is sensitive to the level of internal calcium buffer. We have used the properties of transducer adaptation to compare the effects of exogenous calcium buffers in the patch electrode solution with those of the endogenous buffer assayed with perforated-patch recording. The endogenous buffer of the hair bundle was equivalent to 0.1-0.4 mM BAPTA and, in a majority of cells, supported adaptation in an external Ca2+ concentration of 70 microM similar to that in turtle endolymph. The endogenous buffer had a higher effective concentration, and the adaptation time constant was faster in cells at the high-frequency end than at the low-frequency end of the cochlea. Experiments using buffers with different Ca2+-binding rates or dissociation constants indicated that the speed of adaptation and the resting open probability of the transducer channels could be differentially regulated and imply that the endogenous buffer must be a fast, high-affinity buffer. In some hair cells, the transducer current did not decay exponentially during a sustained stimulus but displayed damped oscillations at a frequency (58-230 Hz) that depended on external Ca2+ concentration. The gradient in adaptation time constant and the tuned transducer current at physiological levels of calcium buffer and external Ca2+ suggest that transducer adaptation may contribute to hair cell frequency selectivity. The results are discussed in terms of feedback regulation of transducer channels mediated by Ca2+ binding at two intracellular sites.

A B S T R A C T A major factor determining the electrical resonant frequency of turtle cochlear hMr cells is the time course of the Ca-activated K current (Art, J. J., and R. Fettiplace. 1987.Journal of Physiology. 385:207-242). We have examined the notion that this time course is dictated by the K channel kinetics by recording single Ca-activated K channels in inside-out patches from isolated cells. A hair cell's resonant frequency was estimated from its known correlation with the dimensions of the hair bundle. All cells possess BK channels with a similar unit conductance of ~ 320 pS but with different mean open times of 0.25-12 ms. The time constant of relaxation of the average single-channel current at -50 mV in 4 p.M Ca varied between cells from 0.4 to 13 ms and was correlated with the hair bundle height. The magnitude and voltage dependence of the time constant agree with the expected behavior of the macroscopic K(Ca) current, whose speed may thus be limited by the channel kinetics. All BK channels had similar sensitivities to Ca which produced half-maximal activation for a concentration of ~2 p.M at +50 mV and 12 p.M at -50 mV. We estimate from the voltage dependence of the whole-cell K(Ca) current that the BK channels may be fully activated at -35 mV by a rise in intracellular Ca to 50 p.M. BK channels were occasionally observed to switch between slow and fast gating modes which raises the possibility that the range of kinetics of BK channels observed in different hair cells reflects a common channel protein whose kinetics are regulated by an unidentified intracellular factor. Membrane patches also contained 30 pS SK channels which were ~5 times more Ca-sensitive than BK channels at -50 inV. The SK channels may underlie the inhibitory synaptic potential produced in hair cells by efferent stimulation.

SUMMARY1. The voltage-dependent conductances of turtle cochlear hair cells of known resonant frequency were characterized by tight-seal, whole-cell recording during superfusion with solutions containing normal (28 mM) and reduced (01-10 /OM) Ca2+.2. In 1 /SM Ca2+, the current flowing through the voltage-dependent Ca2+ channels was increased roughly fivefold and had a reversal potential near 0 mV. This observation may be explained by the Ca2+ channels becoming non-selectively permeable to monovalent cations in low-Ca2+ solutions. Lowering the Ca2+ further to 0-1 /IM produced little increase in the current.3. The size of the non-selective current increased systematically with the resonant frequency of the hair cell over the range from 10 to 320 Hz. This suggests that hair cells tuned to higher frequencies contain more voltage-dependent Ca2+ channels.4. There was a good correlation between the amplitudes of the non-selective current and the K+ current which underlies electrical tuning of these hair cells. The amplitude of the K+ current also increased systematically with resonant frequency.5. In cells with resonant frequencies between 120 and 320 Hz, the K+ current was completely abolished in 1 ,UM Ca2 , consistent with prior evidence that this current flows through Ca2+ activated K+ channels. In a majority of cells tuned between 50 and 120 Hz, the K+ current was incompletely blocked in 1 ,UM Ca2+, but was eliminated in 041 /M Ca2 . In all hair cells the K+ current was abolished by 25 mM tetraethylammonium chloride.6. In cells tuned to 10-20 Hz, the K+ current was not substantially diminished even in 0-1 /LM Ca2 , which argues that it may not be Ca2+ activated.7. In cells tuned to frequencies above 100 Hz, the K+ current could still be evoked by depolarization during superfusion with 10 /1M Ca2+. However, its half-activation voltage was shifted to more depolarized levels and its maximum amplitude was systematically reduced with increasing resonant frequency. 8. These observations are consistent with the notion that in cells tuned to more than 50 Hz, there is a fixed ratio of the number of voltage-dependent Ca2+ channels t To whom reprint requests should be addressed. MS 1889J. J. ART, R. FETTIPLACE AND Y.-C. WU to Ca2+-activated K+ channels, the numbers of each increasing in proportion to resonant frequency. The results also provide indirect evidence that the Ca2+-activated K+ channels in cells tuned to higher frequencies may be less sensitive to intracellular Ca2+.