Calcium ions serve as intracellular messengers in 2 activities of hair cells: in conjunction with Ca2(+)-activated K+ channels, they produce the electrical resonance that tunes each cell to a specific frequency of stimulation, and they trigger the release of a chemical synaptic transmitter. Our experiments indicate that both of these functions are conducted within a region that extends a few hundred nanometers around each presynaptic active zone. In focal electrical recordings from the plasma membranes of isolated anuran hair cells, we found nearly all of a cell's Ca2+ channels and Ca2(+)-activated K+ channels clumped at a fixed ratio in an average of 20 clusters on the basolateral membrane surface. Because serial-section electron microscopy indicated that each hair cell has approximately 19 afferent synaptic contacts with a similar distribution upon its basolateral surface, we conclude that the channel clusters coincide with synaptic active zones. Ensemble-variance analysis of current fluctuations indicated that each cell has a total of approximately 1800 Ca2+ channels and approximately 700 Ca2(+)-activated K+ channels; if these are uniformly divided, we estimate that each channel cluster contains approximately 90 Ca2+ and approximately 40 Ca2(+)-activated K+ channels. Freeze-fracture electron microscopy demonstrated an average of 133 large intramembrane particles in the presynaptic membrane at each active zone, an observation that suggests that the particles are the clustered channels. We used the K+ channel's sensitivity to intracellular Ca2+ to assay the concentration of free Ca2+ in the presynaptic cytoplasm, which we found to vary between 10 microM and 1 mM over the physiological range of membrane potentials. The inferred concentrations agreed with the values predicted for free diffusion of Ca2+ away from Ca2+ channels scattered randomly within a 300-nm-diameter synaptic active zone. The close association among Ca2+ channels, Ca2(+)-activated K+ channels, and synaptic active zones is necessary both for the rapid activation of K+ currents required in electrical resonance and for the transmission at afferent synapses of information about the phases of high-frequency stimuli.

The vertebrate hair cell is a sensory receptor that responds to mechanical stimulation of its hair bundle, which usually consists of numerous large microvilli (stereocilia) and a singe true cilium (the kinocilium). We Mechanical stimuli reach hair cells, the sensory receptors of the vertebrate inner ear and lateral line system, through hair bundles at their apices. These bundles generally consist of numerous, closely packed microvilli, termed stereocilia, and a single true cilium, the kinocilium. The association of ciliary derivatives with sensory transduction in various other sensory systems (1, 2), including the hair cells of invertebrates (3), has led to the suggestion that the kinocilium is the essential component of the transduction apparatus in vertebrate hair cells (4), and to uncertainty as to how transduction occurs in the mammalian cochlea, whose hair cells lack kinocilia (5). By microdissection of hair bundles in living vestibular hair cells from the bullfrog, we have found that transduction does not require a normal relationship of the kinocilium to the stereocilia; indeed, stereocilia by themselves suffice to mediate a response.MATERIALS AND METHODS Sacculi were dissected from the vestibular apparatus of the inner ear of the bullfrog (Rana catesbeiana) and maintained in vitro for electrophysiological recording as described (6). The otolithic membrane' a sheet of proteinaceous material to which the distal tips of the hair bundles normally adhere, was dissected away after a 30-min digestion in collagenase (3 mg/ml, 220 C, type I, Sigma). This proteolytic procedure loosens the attachments of hair bundles to the otolithic membrane and reduces trauma to the cells when the membrane is removed.The preparation was placed in a 0.5-ml experimental chamber and superfused with a saline solution containing 113 mM Na+, 2 mM K+, 4 mM Ca2+, 123 mM Cl-, and 3 mM Dglucose, buffered to pH 7.3 with 1 mM Hepes. Individual hair cells were then impaled with fine glass microelectrodes bent to allow vertical penetrations during visual observation through a X40 water-immersion objective lens and Nomarski differential interference optics (7).Each impaled cell was mechanically stimulated with a glass probe, about 2.5 ,um in diameter at its end, which engaged the distal tip of the hair bundle. In some experiments, the stimulus probe terminated as a hollow tube that engulfed the end of the hair bundle (6). In other cases a blunt stimulus probe, rendered "sticky" by either of two procedures, adhered to the hair bundle. In one procedure, probes were covalently derivatized with charged amino groups by refluxing for 8 hr at 1110C in 10% -y-aminopropyltriethoxysilane (Pierce) in toluene. Such probes presumably bond to negative surface charges on the hair cell membrane. Alternatively, stimulus probes were made adherent by treatment with 1 mg/ml solutions of lectins (concanavalin A, grade IV, or castor bean lectin, type II; Sigma), which evidently bind to sugars on the cell surface: Probes of either type adhere well to kinoci...

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