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REVIEW

Ca²⁺ Signaling in the Inner Ear

Fabio Mammano^1,2,3, Mario Bortolozzi¹, Saida Ortolano^1,3 and Fabio Anselmi²

¹ Istituto Veneto di Medicina Molecolare, Fondazione per la Ricerca Biomedica Avanzata, Padova;
² Centro Interdipartimentale per lo Studio dei Segnali Cellulari, and
³ Dipartimento di Fisica "G.Galilei", Università di Padova, Padova, Italy fabio.mammano{at}unipd.it

	Abstract

 
The inner ear contains delicate sensory receptors that have adapted to detect the minutest mechanical disturbances. Ca²⁺ ions are implicated in all steps of the transduction process, as well as in its regulation by an impressive ensemble of finely tuned feedback control mechanisms. Recent studies have unveiled some of the key players, but things do not sound quite right yet.

	Introduction

Top Introduction Ca2+ Modulation of... Ca2+ Control of Synaptic... The Cochlear Amplifier and... Other Ca2+ Signaling Pathways... Gap-Junction Coupling and Ca2+... Conclusions References

 
Sensory transduction in the inner ear (FIGURE 1) is the job of hair cells that are grouped into three types of sensory epithelia. The organ of Corti is the epithelium of the mammalian cochlea (hearing); the maculae and the cristae are the epithelia of the vestibular system (balance). The epithelia are polarized in that sensory hair cells as well as nonsensory (supporting) cells are divided into apical and basolateral domains. Diffusion of proteins between these subcellular domains is prevented by tight junctions between neighboring cells, which also serve to insulate endolymph, an extracellular fluid low in Na⁺ and Ca²⁺ but rich in K⁺, from perilymph, the normal extracellular fluid that bathes the basolateral membrane of the cells (FIGURE 1A).

View larger version (85K): [in this window] [in a new window]   FIGURE 1. Schematic view of selected Ca2+ signaling processes in the inner ear A: scheme of the inner ear (inset). Endolymphatic (blue) and stria vascularis (SV) compartments are delimited by a tight junction network. Perilymph bathes the rest of the cochlea. Scala media is separated from scala vestibuli by the Reissner’s membrane (RM). The organ of Corti rests on the basilar membrane (BM), surmounted by the tectorial membrane (TM). The SV borders the spiral ligament at the lateral wall of the cochlear duct. The organ of Corti hosts sensory inner (IHC) and outer (OHC) hair cells. IHCs mechanotransduction is activated by the fluid viscous drag applied to their stereocilia by the oscillation of the TM. B: P2R expression in hair cells. P2YR are associated with release of Ca2+ from intracellular stores [endoplasmic reticulum (ER) or Hensen’s body], whereas P2XR are localized on the stereocilia of the hair cells, in proximity of the MET channels, as well as in the apical cell surface. C: afferent (ribbon) synapse. [Ca2+]i concentration at the afferent ribbon synapse of the hair cell indirectly controls the activation of glutamate receptors (AMPARs) at the postsynaptic terminal. [Ca2+]i increase is amplified by CICR from tubular shaped ER extending to within 50 nm of the plasma membrane, close to the ribbon (see Ref. 100). D: efferent synapse and CICR. The inhibitory hyperpolarizing potassium current at the OHC efferent synapse is mediated by SK-type Ca2+-activated potassium channels. This mechanism is supplemented and modulated by CICR from a synaptic cistern facing plasma membrane acetylcholine receptors (nAChRs). Inset: ACh influences OHC electromotility also by activating Ca2+-dependent signaling pathways mediated by protein kinases that target cytoskeletal and prestin proteins. Function of prestin is influenced by intracellular anion concentrations, in particular Cl– controlled by Ca2+-dependent (CaCC) and voltage-dependent (e.g., ClC-2) chloride channels. OHC cell volume and turgor is regulated by Ca2+-dependent phosphorylation of an aquaporin-like protein and/or a GLUT-5 sugar carrier, both voltage dependent and located in the lateral plasma membrane.

View larger version (41K): [in this window] [in a new window]   FIGURE 2. Ca2+ dynamics at the apical pole of the hair cell A: mechanotransduction and calcium pumps. In this model, Myo1c of the adaptation-motor complex interacts, in a Ca2+-calmodulin-dependent manner, with the rigid cross-linked actin filaments at the core of the stereocilia to adjust the force delivered by the tip link to the MET channel. The elastic component of the motor is represented by an intracellular gating spring connected to the MET channel. Ca2+ levels are returned to baseline by the ejection activity of the calcium pump (PMCA2 w/a). B: time sequence of confocal images before and after UV photolysis of caged Ca2+ in hair cells from a mouse utricle culture at postnatal day 2 (P2). Timing relative to the onset of the 100-ms UV light pulse is shown on each numbered frame. Ca2+ concentration changes probed by Fluo-4 emission signal are encoded by the color scale bar beneath frame n.1. C: comparing time course of stereociliary fluorescence ratio changes evoked by Ca2+ photoliberation in a wild-type control (blue trace) and a PMCA2 KO mouse (red trace, corresponding to the frames in B); arrowheads below time axis show time of frame capture. D: methods. Fluo-4 fluorescence was excited by the 488-nm line of an argon laser, and fluorescence emission was selected around 520 nm. The UV illumination covered an area of ~5,000 µm2, comprising a few hair cells, and was generated by a 375-nm laser light beam, reflected off a 400 DCLP dichromatic beam splitter positioned at 45 degrees just above the microscope objective lens. See Ref. 45. Note: the otolith organs (utricle and saccule) respond to translational head movements (linear accelerations) or to the orientation of the head relative to gravity.

Though hair cells are the sensors for hearing and balance, several other cell types play essential roles for maintaining the environment necessary for optimal function of the inner ear. For example, vestibular and cochlear endolymph is secreted, respectively, by dark cells and by marginal cells of the stria vascularis. The stria is additionally responsible for maintaining cochlear endolymph at a potential of +80 to +100 mV (the so-called endochlear potential, or EP) relative to perilymph (FIGURE 1A), thus increasing the driving force for K⁺. MET channel activation causes the hair cell to release K⁺ ions that are either dispersed into perilymph or taken up by adjacent supporting cells and eventually returned to the stria vascularis. In this complex scenario of vertebrate inner ear, Ca²⁺ ions play numerous and fundamental roles. Here, we summarize the common aspects of transduction that are influenced by Ca²⁺ in vestibular and auditory hair cells, including MET function and neurotransmitter release. In the second part, we concentrate on the auditory periphery, reviewing the control exerted by Ca²⁺ on the cochlear amplifier and ion homeostasis in endolymph.

	Ca2+ Modulation of Mechanotransduction in Hair Cells

Top Introduction Ca2+ Modulation of... Ca2+ Control of Synaptic... The Cochlear Amplifier and... Other Ca2+ Signaling Pathways... Gap-Junction Coupling and Ca2+... Conclusions References

 
As mentioned in the introduction, hair bundle deflection opens MET channels (FIGURE 2A), one of the few ion channels not yet conclusively identified (6, 26). Stereocilia in hair bundles are organized into rows of graded heights, forming precisely uniform staircase patterns (103). Stimuli that are sensed by hair cells as excitatory deflect a hair bundle toward its tallest stereocilia, thus increasing tension in the tip link, an extracellular filament stretched between the tops of stereocilia in adjacent rows (28, 85). Application of the Ca²⁺ chelator BAPTA to reduce the extracellular calcium concentration ([Ca²⁺]_o) to submicromolar levels cuts the tip links and eliminates transduction (3). Tip-link tension is thought to be conveyed to MET channels through unidentified elastic elements, termed gating springs (78), mechanically in series with the tip links (28, 56). Values of up to two channels per tip link have been estimated for the mammalian cochlea (6), in accord with previous results in nonmammalian vertebrates (36, 76, 133).

Even though K⁺ ions carry most of the transduction current, MET channels are highly permeable to Ca²⁺ (43). At the tens of micromolar [Ca²⁺]_o found in endolymph (12, 27), Ca²⁺ ions carry ~10% of the total MET current (134). But, to complicate matters, it appears that Ca²⁺ ions can both permeate and block the transducer channels (134) by binding with millimolar affinity at a site presumably located halfway through the membrane electric field (40). These and several other observations led to the conclusion that endolymph composition is specialized to allow a compromise between the largest monovalent current and the largest Ca²⁺ influx (43). From a physiological point of view, the key question is the role of Ca²⁺ ions admitted into the stereocilia through MET channels. The most obvious answer is that elevated stereociliary Ca²⁺ levels promote MET channel closure through adaptation, a negative-feedback mechanism that involves a shift of the sensitive range of the MET process (42, 78) (see http://www.rockefeller.edu/labheads/hudspeth/graphicalSimulations.php). Because it is accompanied by the restoration of sensitivity to transient stimuli, adaptation has been postulated to depend on an active motor that readjusts tension in the gating springs (2, 77), whose stiffness may in turn be Ca²⁺ dependent (20). But adaptation is a complex phenomenon in which at least two phases, both Ca²⁺ dependent, can be distinguished on the basis of their different kinetics and mechanical correlates (44, 71, 163). In different species and different epithelia, slow adaptation has a time constant (_A) of 10–100 ms (55), whereas fast adaptation in turtle auditory hair cells has millisecond _A (at 2.8 mM [Ca²⁺]_o) (135), scaling down to sub-millisecond values in rodents (at 1.5 mM [Ca²⁺]_o) (6, 87, 89). It has been suggested that fast adaptation is mediated by Ca²⁺ entering through the MET channel to shut it by binding at a site on the channel itself or a closely associated intracellular component (20, 29, 136, 163) and that this may tune the channel for small displacements around a resting position that is continually readjusted by slow adaptation (42). However, the calcium-binding site for channel block is thought to be distinct from that mediating fast adaptation (40). The alternative proposal is that of a unified mechanism for both fast and slow adaptation (73, 151) that involves the operation of an adaptation-motor complex formed by myosin-1c (Myo1c) (55), one of the several unconventional myosin isoforms of the stereocilia (43, 65). Myo1c in this scheme interacts in a Ca²⁺-dependent manner with the rigid cross-linked actin filaments at the core of the stereocilia to adjust the force delivered by the tip link to the MET channel (FIGURE 2). In addition, phosphatidylinositol 4,5-bisphosphate (PIP2) binds directly to the IQ domains of Myo1c, regulating the motor activity, and consequently also transduction and adaptation (70). Ca²⁺ may also influence the activity of MET channels in an indirect way, i.e., via cAMP, because the rise in stereociliary [Ca²⁺] can activate a Ca²⁺-calmodulin-activated type I adenyl cyclase of the hair cells (37, 54, 135) followed by activation of protein kinase A and phosphorylation of relevant targets. In summary, adaptation 1) provides a transient response to slow or static stimuli, emphasizing changes in force; 2) positions the transduction element in the most sensitive part of its activation curve to maximize responsiveness, both during development and during physiological stimuli; and 3) prevents saturation of the transducer with larger stimuli (20).

Despite the staggering amount of heroically accumulated data on MET function, several aspects require further investigation and clarification. For instance, in virtually all types of hair cells studied so far, fast and slow adaptation are readily discriminated only for low-amplitude stimuli delivered (in the excitatory direction) under millimolar [Ca²⁺]_o. Indeed, fast adaptation seemingly requires a well-defined range of stereociliary Ca²⁺ levels, but whether this range occurs under physiological conditions in vivo remains an open question. Thus, at 100 µM [Ca²⁺]_o, MET currents of hair cells from mouse utricular macula or bullfrog saccular macula show negligible adaptation (20, 72), consistent with single MET channel recordings of frog auditory hair cells at 50 µM [Ca²⁺]_o or +80 mV holding potential (133). For larger displacements of the hair bundle, the time course of decline in the MET current slows down considerably, suggesting a prevalence of the slow component of adaptation (44). But MET current kinetics slow down also when the driving force for Ca²⁺ entry is reduced either by depolarizing the hair cell or by reducing [Ca²⁺]_o from nonphysiological millimolar levels to values between 20 and 100 µM that better approximate those found in endolymph (43). Furthermore, reducing [Ca²⁺]_o to near endolymphatic values in auditory hair cells of turtles and rodents results in doubling the unitary conductance, from 100 to 150 pS to 200 to 300 pS, and slowing of MET channel activation kinetics (6, 133). Interestingly, when turtle explants are exposed to 70 µM [Ca²⁺]_o, the typical (multi)exponential decay of the MET current evoked by step displacements of the hair bundle evolves into damped current oscillations at frequencies comprised between 58 and 230 Hz, in the lower fraction of the animal’s auditory range (134, 135). Under some conditions, the damping becomes negligible, and the turtle MET currents oscillate continuously in a limit-cycle mode (43), as found in other lower vertebrates in relation to low-frequency spontaneous hair bundle oscillations (110).

Clearly, fast adaptation ought to be most compelling for the mammalian cochlea, in which auditory information with frequency content in the kilohertz range is transmitted to the brain by the inner hair cells (IHCs) via synapses onto afferent terminals of the auditory nerve (41). It has been known for decades that when the mammalian ear is stimulated by sound with precisely defined frequency (pure tones, up to ~3.5 or 5.0 kHz depending on the species) the firing activity on the auditory nerve is cyclically and precisely concentrated only in one-half of the stimulating sinusoid, a phenomenon known as phase locking (1, 112). But rat IHCs, maintained at room temperature (19–22°C) at a potential of –80 mV, show level-dependent fast adaptation with shortest _A of ~0.5 ms (for the smallest stimuli in 1.5 mM [Ca²⁺]_o, both at the 4- and 14-kHz frequency points sampled in this study (6). _A should be considerably shorter, of the order of 60–70 µs, when corrected for the environmental conditions in vivo (as done in Ref. 89 for OHCs). Therefore, if the properties of IHC MET channels are indeed invariant along the cochlea (6), during each cycle of, say, a 1-kHz tone, adaptation would be expected to impart a significant and Ca²⁺-dependent (i.e., level-dependent) distortion to the transduction current and, consequently, receptor potential and afferent firing. Instead, published period histograms of phase-locked firing activity, evoked by pure tones around 1 kHz, track remarkably well the (half-wave rectified) stimulus waveform. In particular, they are symmetric around the peak firing probability over a wide range of input sound levels (1), in accord with the undistorted shape of the phasic (a.c.) receptor potential component (30, 124). In conclusion, it seems difficult to escape the questions of 1) how fast is fast adaptation under physiological (endolymphatic) [Ca²⁺]_o in vivo and 2) what of it is not an artifact due to the highly artificial recording conditions that are commonly employed?

Whatever the answer to these pressing questions, we now have a fairly clear picture of the fate undergone by Ca²⁺ entering through MET channels. It is rapidly bound by endogenous buffers within the stereocilia (63, 137) to be eventually shuttled back to endolymph by the plasma membrane Ca²⁺ ATPases (PMCAs) that are expressed at high density in the stereocilia (~2,000/µm²) (134, 162, 164, 165). Ablation of the PMCA2 gene causes deafness and balance disorders in mice (93), associated to reduction of the Ca²⁺ extrusion rate (FIGURE 2, B–D). The extrusion task is performed by the w/a splicing isoform of PMCA2 (60, 61, 69). The choice of this unusual variant of the otherwise very efficient PMCA2 is surprising, given that this isoform is probably less responsive to the two natural PMCA activators, calmodulin and acidic phospholipids, and should be unable to rapidly enhance its activity in response to a sudden Ca²⁺ increase (14). It is also thought that PMCAs create an ambient of higher extracellular Ca²⁺ in the immediate proximity of the hair bundle (165), possibly with the complicity of the acellular structures overlying the hair cells in the cochlea (FIGURE 1A) (147) and, particularly, in the vestibular system (109) where PMCAs would also contribute to the formation and maintenance of the otoliths in the otolithic membranes (93) (structures in the saccule or utricle comprised of a combination of a gelatinous matrix and calcium carbonate crystals; the inertia of these small particles causes them to stimulate hair cells when the head accelerates). Furthermore, a mutation in the PMCA2 pump in the deaf waddler (dfw) mouse (G283S, downstream of the phospholipids binding domain in the loop connecting transmembrane domains 2 and 3) has been found to cause recessively inherited deafness (152). A mutation next to the active site of the pump (V586M) was later found to depress activity of the pump and to increase loss of hearing in heterozygous human patients that also carried a homozygous mutation in cadherin 23 (CDH23) (146), a member of the rapidly growing complement of stereocilia proteins (56). An unrelated T1999S substitution in the CDH23 gene has been recognized as a precondition for the development of a deafness phenotype in a human family when associated to a missense mutation of PMCA2, G293S, close to the dfw site (45). As discussed in detail in Ref. 127, an increasing number of other proteins have been found to be associated with hereditary hearing loss, the most common sensory deficit in humans (see the Hereditary Hearing Loss Homepage, http://webh01.ua.ac.be/hhh/), and their analysis is proving essential for the understanding of sensory transduction in the normal ear.

	Ca2+ Control of Synaptic Function

Top Introduction Ca2+ Modulation of... Ca2+ Control of Synaptic... The Cochlear Amplifier and... Other Ca2+ Signaling Pathways... Gap-Junction Coupling and Ca2+... Conclusions References

 
As mentioned above, MET current flowing into the apex of hair cells (and shaped by adaptation?) produces a graded receptor potential at the cell basolateral membrane. Therefore, the hair cell can be functionally subdivided into apical and basal compartments, devoted to mechanoelectrical transduction and synaptic dialogue with afferent (and efferent) nerve fibers, respectively (52). At the afferent synapses of all hair cells, glutamate release is triggered by Ca²⁺ influx through L-type voltage-gated calcium channels (Ca_V1.3) clustered at such active zones ("hot spots") together with BK-type (Ca²⁺-sensitive) voltage gated K⁺ channels (116). Estimates of the total number of Ca²⁺ channels of these ~300-nm diameter micro- or nano-domains have been obtained in frog saccular hair cells [~90 channels for each of the ~20 active zones (138)] and mouse inner hair cells (IHCs) [~80 for 10–20 active zones (13)]. Domains of presynaptic Ca²⁺ are spatiotemporally restricted by the presence of mobile, proteinaceous Ca²⁺ buffers, calretinin, calbindin, and parvalbumin, which have been found in a variety of cochlear and vestibular hair cells with concentration in the millimolar range (63). However, discrepancies regarding both the amount and kinetic properties of such buffers in different hair cells suggest that their exact role and scope of function need to be analyzed further.

The afferent synapse of both vestibular and auditory hair cells is of the ribbon type (51, 120) (FIGURE 1C) and operates under stringent Ca²⁺ control conditions (8), and even more so in the auditory system where the establishment and maintenance of accurate phase relationship with sound stimuli relay on fast and tonic simultaneous multi-vesicular release (57, 86). Accurate and reliable timing of afferent discharge underlies the phenomenon of phase locking, discussed above, as well as the localization of sound sources in space, which requires discriminating time differences in the arrival of sound down to tens of microseconds (113). At any instant, only about 100 vesicles are tethered to the synaptic "ribbon" (or "dense body") of IHCs, and about 14 of these are docked to the plasma membrane (101, 142), constituting the "readily releasable" pool (115). The presynaptic Ca²⁺ current that closely follows the membrane potential (79) is directly proportional to vesicle release (83, 145). This linear relationship extends across the synapse to the postsynaptic current; therefore, the postsynaptic glutamate AMPA receptors faithfully transmit the information conveyed by the hair cell receptor potential (86). In higher frequency cells, overall higher frequencies of vesicle release are obtained primarily as a result of a greater number of synapses, whereas the lower Ca²⁺ entry per synapse may prevent postsynaptic receptor saturation (154). The Ca²⁺-dependence of release is non-saturating and independent of hair cell frequency, suggesting that release, not refilling, is the rate-limiting factor (145). This conclusion is supported by the rapid, 1.9 vesicle per millisecond resupply that enables temporally precise and sustained release rates, two orders of magnitude higher than those of conventional synapses (62). How the ribbon replenishes its pool of vesicles, concentrating them at the site of Ca²⁺ influx, remains unclear (52, 120). Although the microtubule motor protein KIF3A was identified on the ribbon (117), evidence accumulates against an active "conveyor belt" for the resupply mechanism (67, 125). Interestingly, vesicles of the ribbon synapse lack synapsins as well as synaptotagmin 1 and 2, which are considered the Ca²⁺ sensors for vesicle fusion in neurons and neuroendocrine cells (140), whereas the core fusion complex contains some components in common with other synapses: SNARE proteins (syntaxin 1, synaptobrevin 1, and SNAP-25); the ribbon structural component RIBEYE; Bassoon and Piccolo, contributing to the anchoring of the ribbon at the active zone (120).

In addition to Ca²⁺ flowing through plasma membrane voltage-gated channels, it has been proposed that the increase of cytoplasmic free calcium concentration ([Ca²⁺]_i) can also depend (or be amplified) by Ca²⁺ released from a presynaptic endoplasmic reticulum network of tubules and cysternae lying only a few tens of nanometers from the hair cell plasma membrane (25, 100, 141, 149) (FIGURE 1C). The mechanism of release depends on a caffeine and ryanodine sensitive Ca²⁺ channel through the mechanism of calcium-induced calcium release, CICR (FIGURE 3). An antagonistic concentration of ryanodine reduced afferent nerve fiber activity both in frog vestibular hair cells (99) and in mammalian IHC (7). In rat cochlear hair cells, CICR may be mediated by type I and II ryanodine receptors (RyR1–2) (59, 114). In IHCs, the highest levels of RyR1 labeling were observed over an area of rough endoplasmic reticulum that sits in the cytoplasmic region beneath the nucleus (59), supporting the conclusion that CICR enhances neurotransmitter release at the afferent region (57, 90). It has also been evidenced that RyRs and BK-type (Ca²⁺-sensitive) voltage-gated potassium channels, co-localized mainly at the extrasynaptic apical neck of the IHCs, filter the receptor potential, and in turn depress neurotransmission in extreme conditions of enhanced Ca²⁺ accumulation that may occur during sound overstimulation or ischemia (7).

View larger version (53K): [in this window] [in a new window]   FIGURE 3. Ca2+ stores at the synaptic pole of the hair cells A: time course of Ca2+ responses evoked by caffeine application (5 mM, 10 s) on hair cells in a slice of frog Crista Ampullaris loaded with fura 2-AM. B: pseudo-line-scan representation of fluorescence intensity changes R.Abscissas represent time, and ordinates are distance along the line (a–b)superimposed on the cell in A. C: response analyzed at the point marked by the black arrow in B.See Ref. 99. D: schematic representation of the semicircular canal and crista ampullaris. Top: within each ampulla, the hair cells and their supporting cells lie embedded in a saddle-shaped neuroepithelial ridge, the crista, which extends across the base of the ampulla. Type I hair cells are concentrated in central regions of the crista, and type II hair cells are more numerous in peripheral areas. Arising from the crista and completely enveloping the stereocilia of the hair cells is a gelatinous structure, the cupula. The latter attaches to the roof and walls of the ampulla, forming a fluid-tight partition that has the same specific density as that of endolymph. Bottom: rotational head movements produce angular accelerations that cause the endolymph in the membranous ducts to be displaced so that the cupula is pushed to one side or the other. These cupular movements displace the stereocilia (and kinocilium) of the hair cells in the same direction.

	The Cochlear Amplifier and Its Regulation by Ca2+

Top Introduction Ca2+ Modulation of... Ca2+ Control of Synaptic... The Cochlear Amplifier and... Other Ca2+ Signaling Pathways... Gap-Junction Coupling and Ca2+... Conclusions References

 
As early as 1948, there were suggestions that an active mechanism might be necessary to explain the exquisite sensitivity and frequency resolving powers of the cochlea (58). It is now amply documented that a vulnerable active process known as cochlear amplifier (34) enhances up to a thousand-fold the vibrations of the basilar membrane (139) and is thus key to the perception of low-level sounds (107), as well as to other facets of sound processing performed by the cochlea (118, 119) (see The Cochlea Homepage, http://vimm.it/cochlea). This is the job of the outer hair cells (OHCs) (41), in which synaptic function is eclipsed by electromotility, a feature of these receptor/actuators that has no analog in any other cell of the body (15). The source of the electromotile cell length variations in OHCs is prestin (FIGURE 1D, INSET), an 81-kDa protein similar to pendrin and other members of the sulfate-anion transporter family but uniquely packed in the OHC lateral plasma membrane at a density of several thousand per square micron (31). Prestin, in contrast to enzymatic-activity-based motors, is a direct voltage-to-force converter that uses cytoplasmic Cl^– and HCO₃^– anions as extrinsic sensors for the receptor potential (122) and can operate at microsecond rates (up to a frequency of, at least, 70 kHz) (46). The cell membrane fraction containing the prestin motor is a significant voltage-dependent contributor to the cell axial stiffness (66), together with the cytoskeletal cortical lattice and subsurface cisterns (121, 158). The OHC motor function can be modulated by numerous signaling pathways (47), some of which are Ca²⁺ dependent (48, 49), and, due to the strategic location of OHCs in the organ of Corti (FIGURE 1A), any change of OHC mechanical properties, particularly somatic length and axial stiffness, is bound to profoundly affect cochlear mechanics. On a slow time scale, OHC stiffness is controlled by phosphorylation of both cytoskeletal and prestin proteins. The polymerization state of cortical cytoskeleton filaments might be affected by activation of RhoA kinases under the control of intracellular Ca²⁺, resulting in a stiffness increase (64, 166). Furthermore, OHC electromotility can be modulated by cGMP-dependent protein kinase acting at two prestin phosphorylation sites, as indicated by experiments performed in prestin-transfected cells (35). As mentioned above, prestin’s function is influenced by intracellular anion concentrations, in particular by [Cl^–], which may be regulated by Ca²⁺-dependent and voltage-dependent chloride channels. Finally, water or sugar balance, influencing cell volume and turgor, can be regulated by a Ca²⁺-dependent phosphorylation of an aquaporin-like protein and/or a GLUT-5 sugar carrier, both voltage dependent and located in the lateral plasma membrane (47) (FIGURE 1D, INSET).

A Ca²⁺-dependent decrease of OHC axial stiffness was observed following the application of acetylcholine (ACh) to the cell basal pole (32). This is related to OHCs being the target of olivocochlear fibers that carry efferent inhibitory feedback from the brain (50) (FIGURE 1D). The efferent synapse uses a Ca²⁺-permeable excitatory ACh receptor (75) to evoke both fast ionotropic and slow metabotropic responses (48). The fast response is a hyperpolarizing potassium current mediated by small-conductance Ca²⁺-activated potassium channels (SK2) (123). The slow component, occurring with a delay of ~10 s, mediates an increase in the motile response of isolated unloaded OHCs, attributed to the activation of Ca²⁺-/calmodulin-dependent phosphorylation of unspecified cytoskeletal proteins (153). OHC efferent synapses rely on the CICR mechanism (39), which is mediated by RyR1 receptors of a "synaptoplasmic" cistern at the postsynaptic site (104) (FIGURE 1D). It could be speculated that ACh triggers some sort of intracellular Ca²⁺ wave propagating in the proximity of the plasma membrane from the base of the cell to the lateral wall, via CICR from subsurface cisternae located beneath the cortical cytoskeleton. However, this phenomenon has not yet been experimentally demonstrated (47). The ensuing decrease in the global axial stiffness of the cell should exert an inhibitory effect on the OHC amplification mechanism in the organ of Corti (32). The biochemical pathway targeting OHC stiffness either after ACh application or following the increase of [Ca²⁺]_i appears to share a currently uncharacterized common mechanism (49). OHC axial stiffness decreases in response to increases of [Ca²⁺]_i (49) that also trigger cell elongation with consequent circumferential contraction, known as "Ca²⁺-dependent slow motility" (24, 130). In summary, current data strongly argue for a crucial role of prestin-dependent amplification of mechanical stimuli in the mammalian cochlea (18, 19, 33, 102, 105, 143). The relevance for amplification of Ca²⁺-dependent hair bundle movements, identified in rat OHCs as mechanical correlates of fast adaptation (17, 87) similar to those of nonmammalian vertebrates that lack prestin (110, 111), remains highly controversial (82, 88).

	Other Ca2+ Signaling Pathways that Impact Inner Ear Function and Sound Perception

Top Introduction Ca2+ Modulation of... Ca2+ Control of Synaptic... The Cochlear Amplifier and... Other Ca2+ Signaling Pathways... Gap-Junction Coupling and Ca2+... Conclusions References

 
Extracellular signaling by nucleotides has long been associated with sensory systems, where ATP acts as a co-transmitter and/or neuromodulator (156). In both endolymphatic and perilymphatic compartments, basal level of extracellular ATP is maintained in the low nanomolar range by the action of ectonucleotidases (159). Even minimal departures from this level signal important changes to purinergic receptors (P2R) that play multiple important roles in sensory transduction and neurotransmission, consistent with the widespread expression of ATP gated ion channels (P2XR) and G-protein coupled receptors (P2YR) on inner ear cells, including cochlear hair cells (74). In most cases, Ca²⁺ ions are key intermediates in the ATP-evoked signaling cascades. Thus application of ATP to guinea pig IHC results in a rapid production of NO, presumably via ATP increase in [Ca²⁺]_i. This in turn leads to a cGMP-PKG-dependent inhibition of the ATP-induced Ca²⁺ elevation in these cells (148). In OHCs, focal application of ATP to the hair bundle determines a biphasic increase in [Ca²⁺]_i (106). Initially, Ca²⁺ enters the cell apex through P2X ionotropic channels. Because it opens a pathway for K⁺ ions (FIGURE 1B) in parallel to MET channels (FIGURE 2A), this process has been proposed to mediate a humoral modulation of mechanotransduction (75). But other mechanisms may be activated by the second phase of the ATP-evoked Ca²⁺ response, due to IP₃-induced Ca ²⁺ release from internal stores mediated by PLC-coupled P2YR. In guinea pig OHCs, the release site coincides with the system of endoplasmic reticulum membranes and mitochondria known as Hensen’s body, located at the base of the hair bundle. Ca²⁺ released in this region, beneath the cuticular plate that supports the hair bundle, effectively bypasses the clearance mechanism of the stereocilia and potentially influences mechanotransduction by modulation of unconventional myosin isoenzymes and actin-binding proteins involved in maintaining bundle integrity (106). Besides Myo1c, at least two other myosin isoforms, myosin VIIa and XVa, are found in the stereocilia and may alter bundle mechanics by acting on the attachment sites of the later links that hold the bundle together. Tropomyosin in the stereociliary rootlets, at the level of the cuticular plate, may also be involved in this process (38). In summary, myosin, tropomyosin, and actin together regulate the stiffness of bundle and cuticular plate under the control of Ca²⁺ binding proteins whose action is in turn regulated by Ca²⁺ pumps and buffers that control the concentration of intracellular Ca²⁺ at the apical pole of the hair cell, as explained in the relevant section above. Importantly, cochlear models indicate that even minimal disturbance to the mechanotransduction process may significantly alter the motor output of the cochlear amplifier, which operates at the threshold of spontaneous oscillations (107, 118, 119). Indeed, the estimated P2X-mediated conductance of hair cells must be minimal with respect to the one calculated for the Reissner’s membrane (FIGURE 1A) because 1) measurements of cochlear partition resistance (CoPR) in guinea pig endolymphatic compartment show that activation of hair cell P2XR has negligible influence on EP modulation and 2) the EP value following ATP administration in the endolymph is not significantly altered in kanamicin treated animals lacking OHCs (157). Finally, mechanotransduction may be also affected by reduction of EP brought about by ATP stimulation of marginal cells in the gerbil stria vascularis, which determines a metabotropic Ca²⁺-dependent inhibition of K⁺ channel KCNE1 via P2Y₄R (108).

	Gap-Junction Coupling and Ca2+ Signaling in the Cochlear Duct

Top Introduction Ca2+ Modulation of... Ca2+ Control of Synaptic... The Cochlear Amplifier and... Other Ca2+ Signaling Pathways... Gap-Junction Coupling and Ca2+... Conclusions References

 
The homeostatic regulation of EP and endolymphatic K⁺ levels, key prerequisites for the maintenance of MET function, require K⁺ recirculation through the stria vascularis (FIGURE 1A) that houses a dense capillary network, supporting the high metabolic rate of this compartment (131). K⁺ recirculation in the inner ear has been recently reviewed in this Journal (68). Briefly, K⁺ released from cochlear hair cells following mechanotransduction (84) has been initially proposed to diffuse into scala tympani perilymph before entering the lateral wall of the cochlea (168). Here, it is taken up by fibrocytes in the spiral ligament, a densely vascularized endosteum at the outer limits of the cochlear canals, which is covered by the stria vascularis toward the cochlear duct. The stria vascularis in turn comprises basal, intermediate, and marginal cells. Gap-junction channels interconnect the fibrocytes of the spiral ligament and also medial and intermediate cells of the stria vascularis (22). Therefore, it has been proposed that intercellular transport across the gap junction network of this connective tissue participates in K⁺ recirculation. After reaching the stria vascularis, K⁺ ions are released by intermediate cells into the strial luminal site and are finally taken up by strial marginal cells to be secreted back into endolymph (81, 160, 161).

The fraction of K⁺ being lost to perilymph due to MET channel activation is unknown, but disruption of the K-Cl cotransporters Kcc4 and Kcc3, which causes deafness in mice (10, 11), indicates that active K⁺ uptake by the Deiter’s cells and other supporting cells of the organ of Corti is essential to cochlear ion homeostasis. These cells belong in distinct gap junction networks formed by nonsensory epithelial cells (22, 80), including interdental cells of the spiral limbus, inner sulcus cells, organ of Corti supporting cells, outer sulcus cells, and cells within the root processes of the spiral ligament (92). Therefore, immediately after being released from the hair cells, a substantial proportion of the K⁺ ions is likely to be taken up into these syncytia that may be an integral part of the recycle scheme (91).

In the organ of Corti, most gap-junction channels are assembled from connexin (Cx) protein subunits, primarily connexin 26 (Cx26, GJB2 gene) and co-localized Cx30 (GJB6) (92, 98). Mouse models confirmed that both connexins are essential for cochlear function and cell survival (23, 94, 155), and the complex deafness DFNB1 locus (13q11-q12) has been shown to contain both gap-junction genes. Deafness-causing mutations have been reported in two other connexin genes, Cx31 (GJB3) and Cx43 (GJA1) (22); however, DFNB1 mutations are responsible for about half of all cases of human deafness in countries surrounding the Mediterranean Sea. In particular, over 50 causative mutations in GJB2 have been shown to account for the vast majority of prelingual hearing impairment in most populations (see The Connexin-deafness homepage, http://davinci.crg.es/deafness/). DFNB1 is thus almost as frequent as cystic fibrosis (128). Yet deafness associated with mutations in the Cx26 gene may not solely depend on the loss of K⁺ coupling and K⁺ recirculation. Collectively, secondary messengers and small metabolites are deemed to be the molecular constituents that are directly passed from one cell to another: important molecules include cAMP, cGMP, IP₃, adenosine, ADP, and ATP, to name only a few (97). Indeed, Cx26 channels carrying the deafness-related V84L substitution present normal localization and similar conductance to wild-type protein (16), but significantly impaired permeability to IP₃ (4). Furthermore, direct injection of IP₃ in cochlear supporting cells of neonatal rat organotypic cultures generates a wave of calcium that spreads throughout the tissue and is suppressed when junctional channels (or unapposed Cx hemichannels facing scala media) are blocked by acidification with CO₂ (4). IP₃ permeability defects (167) may interfere with K⁺ recycle function, because K⁺/Cl^– efflux from supporting cells (10) may be, directly or indirectly, controlled by Ca²⁺.

Pharmacological dissection indicates that ATP, acting through a highly sensitive purinergic-/IP₃-mediated signaling pathway, is the principal paracrine mediator implicated in the propagation of waves through supporting and epithelial cells (129) (FIGURE 4). Interestingly, Ca²⁺ waves generated by brief (50–200 ms) application of low doses of ATP (4–1 µM) from a puff pipette are identical to Ca²⁺ waves evoked by mechanical stimulation of hair cells, mimicking in vitro the effects of sound over stimulation (53). A fundamental step is ATP release at the endolymphatic surface of the epithelium, which triggers adjacent cell responses (FIGURE 4E). Based on connexin expression models and inhibition by blockers, connexin hemichannels have been proposed as a diffusion pathway for the release of extracellular messengers, including ATP (5). This is the case of retinal pigment epithelium where ATP is released in the extracellular space through Cx43 hemichannels, with pore opening controlled by spontaneous elevations of intracellular Ca²⁺ (126). But transmission of Ca²⁺-related signals may exploit alternative nonvesicular release mechanisms, along with IP₃ diffusion through gap junctions (4, 144) (FIGURE 4F). Thus anion channels, pannexins, and P2X₇ receptors have all been proposed as conduits for ATP to the extracellular milieu in other systems (150). The significance of Ca²⁺ wave propagation through the cochlear tissue, as well as that of oscillations in intracellular Ca²⁺ concentration levels (95, 96), are intimately connected to the role of Ca²⁺ in the control of key cellular processes. The extraordinary sensitivity to ATP of cochlear neonatal culture suggests that spreading of intracellular calcium through the syncitium is an essential component for the perception of sound and is potentially involved in sound-induced gene expression (53).

View larger version (61K): [in this window] [in a new window]   FIGURE 4. Ca2+ waves in the organ Corti A: one representative experimental record, showing Ca2+ transient responses in fura 2-loaded supporting cells following focal ATP application (50 ms, 4 µM) to OHCs. Shown are pseudocolor images of the Ca2+ wave spreading at three different time points. B:[Ca2+]i profiles from the regions of interest (ROIs) superimposed over six selected supporting cells in A (top). C: distance of wave-front points from the point of stimulus application (white asterisk) vs. time; wave speed is estimated by the slope of the least square linear fit (solid line). D: the amplitude of mean fura 2 ratio signal vs. distance shows no significant attenuation of the [Ca2+]i response with distance from the point source, implicating an active mechanism of propagation. E: proposed mechanism of Ca2+ wave propagation. ATP release following hair cell stimulation induces an auto-sustained spreading of the signal in adjacent supporting cells, determined by further release of ATP into endolymph through an unidentified channel and concurrent IP3 diffusion through gap-junction channels. F : IP3 diffusion through gap junctions. The differently colored ribbons represent alpha-helices in the transmembrane portion of the six connexin proteins forming the connexon. See Ref. 9.

	Conclusions

Top Introduction Ca2+ Modulation of... Ca2+ Control of Synaptic... The Cochlear Amplifier and... Other Ca2+ Signaling Pathways... Gap-Junction Coupling and Ca2+... Conclusions References

	Acknowledgments

 
We thank Tullio Pozzan for critically reading the manuscript and Pascal Martin for helpful discussions.

This review was supported by the FP6 program of the European Union (FP6 Integrated Project EUROHEAR, LSHG-CT-20054-512063) and the Telethon Foundation (Projects GGP04169 and GGP02043).

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