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1Turtle auditory hair cells contain multiple isoforms of the pore‐forming α‐subunit of the large‐conductance Ca2+‐activated K+(KCa) channel due to alternative splicing at two sites. Six splice variants were studied by expression in Xenopusoocytes.2The isoforms possessed differences in apparent Ca2+sensitivity and kinetics. The lowest Ca2+sensitivity was observed in a novel variant resulting from a 26 amino acid deletion around one of the splice sites.3Co‐expression of a bovine β‐subunit slowed the current relaxation 10‐fold compared with channels formed from α‐subunits alone but preserved the original order of kinetic differences. The β‐subunit also increased the Ca2+sensitivity of isoforms to bring them nearer the range of sensitivity of the native KCachannels of the hair cell.4With channels formed from α‐subunits or α+β‐subunits, the half‐activation voltage in a fixed Ca2+concentration, and the time constant of the current relaxation, varied linearly with the combined size of the insertions/deletions at the splice sites.5Experiments in which the β/α concentration ratio was varied indicated that the β‐subunit exerts an all‐or‐none effect on the Ca2+sensitivity and kinetics of the channel.6Co‐expression of an avian β2‐subunit had effects on kinetics and Ca2+sensitivity of several α‐isoforms which were qualitatively similar to those produced by the bovine β‐subunit.7We conclude that differential expression of alternatively spliced α‐subunit variants and a non‐uniform distribution of a β‐subunit can produce a range of KCachannel properties needed to explain the tonotopic organization of the turtle cochlea.

Turtle auditory hair cells are frequency tuned by the activity of large-conductance calcium-activated potassium (KCa) channels, the frequency range being dictated primarily by the channel kinetics. Seven alternatively spliced isoforms of the KCa channel alpha-subunit, resulting from exon insertion at two splice sites, were isolated from turtle hair cells. These, when expressed in Xenopusoocytes, produced KCa channels with a range of apparent calcium sensitivities and channel kinetics. However, most expressed channels were less calcium sensitive than the hair cells' native KCa channels. Coexpression of alpha-subunit with a bovine beta-subunit substantially increased the channel's calcium sensitivity while markedly slowing its kinetics, but kinetic differences between isoforms were preserved. These data suggest a molecular mechanism for hair cell frequency tuning involving differential expression of different KCa channel alpha-subunits in conjunction with an expression gradient of a regulatory beta-subunit.

1. Transducer currents were recorded in turtle cochlear hair cells during mechanical stimulation of the hair bundle. The currents were measured under whole‐cell voltage clamp in isolated cells that were firmly stuck to the floor of the recording chamber. 2. Stimuli were calibrated by projecting the image of the hair bundle onto a rapidly scanned 128 photodiode array. This technique showed that, while the cell body was immobilized, the tip of the bundle would follow faithfully the motion of an attached glass probe up to frequencies of more than 1 kHz. 3. The relationship between inward transducer current and bundle displacement was sigmoidal. Maximum currents of 200‐400 pA were observed for deflections of the tip of the bundle of 0.5 microns, equivalent to rotating the bundle by about 5 deg. 4. In response to a step deflection of the bundle, the current developed with a time constant (about 0.4 ms for small stimuli) that decreased with the size of displacement. This suggests that the onset of the current was limited by the gating kinetics of the transduction channel. The onset time course was slowed about fourfold for a 20 degrees C drop in temperature. 5. For small maintained displacements, the current relaxed to about a quarter of the peak level with a time constant of 3‐5 ms. This adaptation was associated with a shift of the current‐displacement relationship in the direction of the stimulus. The rate and extent of adaptation were decreased by lowering external Ca2+. 6. Adaptation was strongly voltage sensitive, and was abolished at holding potentials positive to the reversal potential of the transducer current of about 0 mV. It was also diminished by loading cells with 10 mM of the Ca2+ chelator BAPTA. These observations suggest that adaptation may be partly controlled by influx of Ca2+ through the transducer channels. 7. Removal of adaptation produced asymmetric responses, with fast onsets but slow decays following return of the bundle to its resting position; the offset time course depended on both the magnitude and duration of the prior displacement. 8. In some experiments, hair bundles were deflected with a flexible glass fibre whose motion was monitored using a dual photodiode arrangement. Positive holding potentials abolished adaptation of the transducer currents, but had no influence on the time course of motion of the fibre. We have no evidence therefore that adaptation is caused by a mechanical reorganization within the bundle.

Turtle auditory–hair cells are frequency–tuned by the activity of calcium–activated potassium (KCa) channels, a cell's characteristic frequency being determined by the KCachannel density and kinetics which both vary systematically along the cochlea. As a first step towards identifying the source of KCachannel variation, we have isolated, by reverse–transcription polymerase chain reaction on dissociated hair cells, the main cDNAs homologous to the slogene which encodes the channel's αsubunit. A total of six alternatively spliced variants were identified, the smallest of which is 94identical to a mouse Slosequence. Variation occurs by insertion of exons at only two splice sites, two of these exons encoding novel 31– and 61–amino acid sequences. As we were unable to detect splicing at other potential sites, we infer that the six variants correspond to naturally occurring combinations. The spatial distribution of the variants, defined by isolating hair cells from different regions of the cochlea, indicated that some isoforms were non–uniformly distributed. Those containing large inserts in the first splice site were notably absent from the highest–frequency region. We suggest that alternative splicing of the slogene may contribute to variation in KCachannel properties.

1. The electrical responses of single auditory nerve fibres or cochlear hair cells were recorded in the isolated half‐head of the turtle Pseudemys scripta elegans. Responses to sound stimuli presented to the tympanum could be recorded for at least 4 hr after isolation.