Excitable cells use ion channels to tailor their biophysical properties to the functional demands made upon them. During development, these demands may alter considerably, often associated with a change in the cells' complement of ion channels. Here we present evidence for such a change in inner hair cells, the primary sensory receptors in the mammalian cochlea. In mice, responses to sound can first be recorded from the auditory nerve and observed behaviourally from 10-12 days after birth; these responses mature rapidly over the next 4 days. Before this time, mouse inner hair cells have slow voltage responses and fire spontaneous and evoked action potentials. During development of auditory responsiveness a large, fast potassium conductance is expressed, greatly speeding up the membrane time constant and preventing action potentials. This change in potassium channel expression turns the inner hair cell from a regenerative, spiking pacemaker into a high-frequency signal transducer.

Journal of PhysiologyImmature IHCs from the mouse express, in addition to an inward L-type Ca 2+ current containing the a1D (Ca v 1.3) subunit (Platzer et al. 2000), a large Na + current (I Na ; Kros et al. 1993;Kros, 1996). Although Na + currents have been reported in vestibular and cochlear hair cells of other vertebrates including mammals (Evans & Fuchs 1987;Sugihara & Furukawa, 1989;Sokolowski et al. 1993; Witt et al. 1994;Oliver et al. 1997;Lennan et al. 1999;Masetto et al. 2003), little is known about whether and how they contribute to the physiological responses of the hair cells. To gain more insight into the biophysical basis of the action potential, we set out to investigate the relative contribution of both I Ca and I Na to action potentials at body temperature and using a physiological extracellular Ca 2+ concentration. METHODS Tissue preparationApical-coil IHCs (n = 325) of CD-1 mice (Swiss CD-1, Charles Rivers, Margate, UK) were studied in acutely dissected organs of Corti from E16.5 to P20, where the day of birth (P0) corresponds to E19.5. For embryonic experiments only, mice were paired overnight and checked for vaginal plugs the following morning. Assuming ovulation occurs midway through the dark cycle, the midpoint of the light cycle of the day following mating is considered to be E0.5. Adult and neonatal mice were killed by cervical dislocation and embryos by decapitation, in accordance with UK Home Office regulations. The cochleae were dissected in extracellular solution composed of (mM): 135 NaCl, 5.8 KCl, 1.3 CaCl 2 , 0.9 MgCl 2 , 0.7 NaH 2 PO 4 , 5.6 D-glucose, 10 Hepes-NaOH, 2 sodium pyruvate. Amino acids and vitamins for Eagle's minimum essential medium (MEM) were added from concentrates (Invitrogen, Paisley, UK). The pH was adjusted to 7.5 and the osmolality was about 308 mmol kg _1 . The organs of Corti were transferred to a microscope chamber containing extracellular solution, in which they were immobilized with a nylon mesh fixed to a stainless steel ring. The chamber (volume 2 ml) was perfused at a flow rate of about 10 ml h _1 from a peristaltic pump and mounted on the stage of an upright microscope (Zeiss ACM, Germany or Olympus, Japan). The organs of Corti were observed with Nomarski differential interference contrast optics (w 40 water-immersion objectives). To expose the basolateral surfaces of the cells, a small tear was made in the epithelium with a suction pipette (tip diameter about 2-4 mm) filled with extracellular solution. The cells were then cleaned by a stream of fluid from this pipette before patching. Only cells of healthy appearance were selected for electrophysiological recordings. Criteria included an intact hair bundle, cell membranes with a smooth surface, absence of vacuoles in the cytoplasm and lack of Brownian motion of mitochondria. The position of cells along the cochlea was recorded as fractional distance from the extreme apex. In the immature cochlea, cells were positioned at a fractional distance of between 0.16 and 0.24. Mature IHCs were positioned bet...

The type I and type II hair cells of mature amniote vestibular organs have been classified according to their afferent nerve terminals: calyx and bouton, respectively. Mature type I and type II cells also have different complements of voltage-gated channels. Type I cells alone express a delayed rectifier, gK,L, that is activated at resting potential. We report that in mouse utricles this electrophysiological differentiation occurs during the first postnatal week. Whole-cell currents were recorded from hair cells in denervated organotypic cultures and in acutely excised epithelia. From postnatal day 1 (P1) to P3, most hair cells expressed a delayed rectifier that activated positive to resting potential and a fast inward rectifier, gK1. Between P4 and P8, many cells acquired the type I-specific conductance gK,L and/or a slow inward rectifier, gh. By P8, the percentages of cells expressing gK,L and gh were at mature levels. To investigate whether the electrophysiological differentiation correlated with morphological changes, we fixed utricles at different times between P0 and P28. Ultrastructural criteria were developed to classify cells when calyces were not present, as in cultures and neonatal organs. The morphological and electrophysiological differentiation followed different time courses, converging by P28. At P0, when no hair cells expressed gK,L, 33% were classified as type I by ultrastructural criteria. By P28, approximately 60% of hair cells in acute preparations received calyx terminals and expressed gK,L. Data from the denervated cultures showed that neither electrophysiological nor morphological differentiation depended on ongoing innervation.

The molecular basis of sensory hair cell mechanotransduction is largely unknown. In order to identify genes that are essential for mechanosensory hair cell function, we characterized a group of recently isolated zebrafish motility mutants. These mutants are defective in balance and swim in circles but have no obvious morphological defects. We examined the mutants using calcium imaging of acoustic-vibrational and tactile escape responses, high resolution microscopy of sensory neuroepithelia in live larvae, and recordings of extracellular hair cell potentials (microphonics). Based on the analyses, we have identified several classes of genes. Mutations in sputnik and mariner affect hair bundle integrity. Mutant astronaut and cosmonaut hair cells have relatively normal microphonics and thus appear to affect events downstream of mechanotransduction. Mutant orbiter, mercury, and gemini larvae have normal hair cell morphology and yet do not respond to acoustic-vibrational stimuli. The microphonics of lateral line hair cells of orbiter, mercury, and gemini larvae are absent or strongly reduced. Therefore, these genes may encode components of the transduction apparatus.

The large conductance voltage-and Ca 2؉ -activated potassium (BK) channel has been suggested to play an important role in the signal transduction process of cochlear inner hair cells. BK channels have been shown to be composed of the pore-forming ␣-subunit coexpressed with the auxiliary ␤1-subunit. Analyzing the hearing function and cochlear phenotype of BK channel ␣-(BK␣ ؊/؊ ) and ␤1-subunit (BK␤1 ؊/؊ ) knockout mice, we demonstrate normal hearing function and cochlear structure of BK␤1 ؊/؊ mice. During the first 4 postnatal weeks also, BK␣ ؊/؊ mice most surprisingly did not show any obvious hearing deficits. High-frequency hearing loss developed in BK␣ ؊/؊ mice only from Ϸ8 weeks postnatally onward and was accompanied by a lack of distortion product otoacoustic emissions, suggesting outer hair cell (OHC) dysfunction. Hearing loss was linked to a loss of the KCNQ4 potassium channel in membranes of OHCs in the basal and midbasal cochlear turn, preceding hair cell degeneration and leading to a similar phenotype as elicited by pharmacologic blockade of KCNQ4 channels. Although the actual link between BK gene deletion, loss of KCNQ4 in OHCs, and OHC degeneration requires further investigation, data already suggest human BK-coding slo1 gene mutation as a susceptibility factor for progressive deafness, similar to KCNQ4 potassium channel mutations.cochlea ͉ KCNQ4 C a 2ϩ -activated potassium (BK) channels are heterooctamers of four ␣-and four ␤-subunits. The pore-forming ␣-subunit (KCNMA1) is a member of the slo family of potassium channels (1), originally identified in Drosophila (2). Studies of BK channels from smooth muscle have identified an auxiliary ␤1-subunit (KCNMB1) whose presence in the channel complex confers an increased voltage and calcium sensitivity toward the poreforming ␣-subunit (3).In turtle and chick, there is evidence that differential splicing of the BK channel ␣-subunit in conjunction with a graded expression of the auxiliary ␤-subunit along the tonotopic axis provides the functional heterogeneity of BK channels that underlies electrical tuning (for review, see ref. 4).In inner hair cells (IHCs) of the mammalian organ of Corti, the predominant K ϩ conductance is a voltage-and Ca 2ϩ -activated K ϩ channel termed I K,f (5, 6). BK channel mRNA (7,8) and protein expression (8) were shown in IHCs, indicating that I K,f flows through BK channels. The presumed physiological roles of BK channels are (i) a decrease of the membrane time constant even at the resting potential and (ii) fast repolarization of the receptor potential. Both contribute to phase-locked receptor potentials up to high sound frequencies (6). In addition to IHCs, BK type Ca 2ϩ -activated K ϩ conductances have been measured in OHCs (9) and in efferent fibers onto outer hair cells (OHCs) (10). The role of BKs in either OHCs or efferents is still controversially discussed (9).Studying the expression of BK channel ␣-splice variants and ␤-isoforms in rat cochlea using in situ hybridization and PCR techniques revealed the strict coexpressio...