Channels In Inner Hair Cells Research Articles

The mechanism of Ca2+ independent activation of BKCa channels in mouse inner hair cells and the crucial role of the BK channels in auditory perception

BK channels are expressed in mouse cochlear inner hair cells (IHCs) and exhibit Ca2+-independent activation at negative potentials. However, the mechanism underlying Ca2+-independent activation of the BK channels in mouse IHCs remains unknown. In this study, we found the BK channel expressed in IHCs contains both the STREX-2 (stress axis regulated exon) variant and an alternative splice of exon9 (alt9), which significantly shift the voltage dependence of the BK channels when co-expressed with LRRC52 in 0 [Ca2+]i. Furthermore, we discovered that mechanical force also induces negative shifts in the voltage dependence of IHC-expressed BK channels. Thus, we propose that the additive effects of mechanical force, special isoforms, and LRRC52 co-expression on voltage dependence shifts may account for the Ca2+-independent activation of the BK channel in IHC. Additionally, we found that the IHCs-specific deletion of the BK channels causes hearing damage in mice. Our study suggests a mechanism for Ca2+-independent activation in IHCs and highlights the crucial role of the BK channel in auditory perception.

Pou4f1 Defines a Subgroup of Type I Spiral Ganglion Neurons and Is Necessary for Normal Inner Hair Cell Presynaptic Ca2+ Signaling.

Acoustic signals are relayed from the ear to the brain via spiral ganglion neurons (SGNs) that receive auditory information from the cochlear inner hair cells (IHCs) and transmit that information to the cochlear nucleus of the brainstem. Physiologically distinct classes of SGNs have been characterized by their spontaneous firing rate and responses to sound and those physiological distinctions are thought to correspond to stereotyped synaptic positions on the IHC. More recently, single-cell profiling has identified multiple groups of SGNs based on transcriptional profiling; however, correlations between any of these groups and distinct neuronal physiology have not been determined. In this study, we show that expression of the POU (Pit-Oct-Unc) transcription factor Pou4f1 in type I SGNs in mice of both sexes correlates with a synaptic location on the modiolar side of IHCs. Conditional deletion of Pou4f1 in SGNs beginning in mice at embryonic day 13 rescues the early path-finding and apoptotic phenotypes reported for germline deletion of Pou4f1, resulting in a phenotypically normal development of SGN patterning. However, conditional deletion of Pou4f1 in SGNs alters the activation of Ca2+ channels in IHCs primarily by increasing their voltage sensitivity. Moreover, the modiolar to pillar gradient of active zone Ca2+ influx strength is eliminated. These results demonstrate that a subset of modiolar-targeted SGNs retain expression of Pou4f1 beyond the onset of hearing and suggest that this transcription factor plays an instructive role in presynaptic Ca2+ signaling in IHCs.SIGNIFICANCE STATEMENT Physiologically distinct classes of type I spiral ganglion neurons (SGNs) are necessary to encode sound intensities spanning the audible range. Although anatomical studies have demonstrated structural correlates for some physiologically defined classes of type I SGNs, an understanding of the molecular pathways that specify each type is only now emerging. Here, we demonstrate that expression of the transcription factor Pou4f1 corresponds to a distinct subgroup of type I SGNs that synapse on the modiolar side of inner hair cells. The conditional deletion of Pou4f1 after SGN formation does not disrupt ganglion size or morphology, change the distribution of IHC synaptic locations, or affect the creation of synapses, but it does influence the voltage dependence and strength of Ca2+ influx at presynaptic active zones in inner hair cells.

Voltage-Gated Cav1 Channels in Disorders of Vision and Hearing.

Cav1 channels mediate L-type Ca(2+) currents that trigger the exocytotic release of glutamate from the specialized "ribbon" synapse of retinal photoreceptors (PRs) and cochlear inner hair cells (IHCs). Genetic evidence from animal models and humans support a role for Cav1.3 and Cav1.4 as the primary Cav channels in IHCs and PRs, respectively. Because of the unique features of transmission at ribbon synapses, Cav1.3 and Cav1.4 exhibit unusual properties that are well-suited for their physiological roles. These properties may be intrinsic to the channel subunit(s) and/or may be conferred by regulatory interactions with synaptic signaling molecules. This review will cover advances in our understanding of the function of Cav1 channels at sensory ribbon synapses, and how dysregulation of these channels leads to disorders of vision and hearing.

Conductance and block of hair-cell mechanotransducer channels in transmembrane channel-like protein mutants.

Transmembrane channel-like (TMC) proteins TMC1 and TMC2 are crucial to the function of the mechanotransducer (MT) channel of inner ear hair cells, but their precise function has been controversial. To provide more insight, we characterized single MT channels in cochlear hair cells from wild-type mice and mice with mutations in Tmc1, Tmc2, or both. Channels were recorded in whole-cell mode after tip link destruction with BAPTA or after attenuating the MT current with GsMTx-4, a peptide toxin we found to block the channels with high affinity. In both cases, the MT channels in outer hair cells (OHCs) of wild-type mice displayed a tonotopic gradient in conductance, with channels from the cochlear base having a conductance (110 pS) nearly twice that of those at the apex (62 pS). This gradient was absent, with channels at both cochlear locations having similar small conductances, with two different Tmc1 mutations. The conductance of MT channels in inner hair cells was invariant with cochlear location but, as in OHCs, was reduced in either Tmc1 mutant. The gradient of OHC conductance also disappeared in Tmc1/Tmc2 double mutants, in which a mechanically sensitive current could be activated by anomalous negative displacements of the hair bundle. This "reversed stimulus-polarity" current was seen with two different Tmc1/Tmc2 double mutants, and with Tmc1/Tmc2/Tmc3 triple mutants, and had a pharmacological sensitivity comparable to that of native MT currents for most antagonists, except dihydrostreptomycin, for which the affinity was less, and for curare, which exhibited incomplete block. The existence in the Tmc1/Tmc2 double mutants of MT channels with most properties resembling those of wild-type channels indicates that proteins other than TMCs must be part of the channel pore. We suggest that an external vestibule of the MT channel may partly account for the channel's large unitary conductance, high Ca(2+) permeability, and pharmacological profile, and that this vestibule is disrupted in Tmc mutants.

C-Terminal Modulation of Cav1.3 L-Type Calcium Channels Modifies their Gating Properties in Cochlear Inner Hair Cells

The Ca2+ currents in inner hair cells (IHCs) are crucial for synaptic transmission and flow through voltage-gated calcium channels (VGCCs) formed by the α1 subunit Cav1.3. VGCCs exhibit a calmodulin (CaM) mediated calcium-dependent inactivation (CDI), via binding CAM to the channel's C-terminus. In IHCs, Cav1.3 exhibits unusually weak CDI, probably caused by calcium binding proteins (CaBP) competing with CaM. IHCs express long and short Cav1.3 splice variants either including (long variant, Cav1.3L) or excluding (short variants) a C-terminal modulatory domain. In expression systems - lacking CaBPs - the C-terminal modulatory mechanism (CTM) functions via intramolecular interaction of a proximal (PCRD) and a distal C-terminal regulatory domain (DCRD) by inhibiting CaM binding near the PCRD, thereby inhibiting CaM-mediated CDI (Bock et al., JBC 2011). Here, the role of the CTM for IHC VGCCs was investigated in Cav1.3L-DCRDHA/HA mice in which CTM was disrupted by partial replacement of the DCRD with an HA tag.Localization of HA-tagged Cav1.3 channels in IHCs was determined by immunohistochemistry. Channel properties were investigated by whole-cell patch-clamp recordings. Hearing was assessed using auditory brainstem responses (ABR) and distortion products of otoacoustic emissions (DPOAE).Anti-HA immunolabeling was present at all IHC ribbons. Patch-clamp recordings revealed significantly reduced CDI and increased amplitudes of Ca2+ and Ba2+ currents in Cav1.3L-DCRDHA/HA IHCs. Non-stationary fluctuation analysis showed unchanged numbers of Cav1.3 channels and single channel currents. Voltage dependence and activation kinetics of ICa and IBa, ABR thresholds and DPOAEs were unaffected.Our data demonstrate that the long Cav1.3 isoform is an intrinsic component of Cav1.3 clusters at all IHC ribbon synapses and that its DCRD is required for normal CDI and ICa amplitude.Funding:Austrian Science Fund (SFB-F4402), EC-project MRTN-CT-2006-35367 (“CavNet”), Innsbruck University, DFG (SFB-894)

Temperature Enhances Activation and Inactivation Kinetics of Potassium Currents in Inner Hair Cells Isolated from Guinea-Pig Cochlea

ObjectivesUntil recently, most patch-clamp recordings in inner hair cells (IHCs) have been performed at room temperature. The results acquired at room temperature should be corrected if they are to be related to in vivo findings. However, the temperature dependency to ion channels in IHCs is unknown. The aim of this study was to investigate the effect of temperature on the potassium currents in IHCs.MethodsIHCs were isolated from a mature guinea-pig cochlea and potassium currents were recorded at room temperature (around 25℃) and physiological temperatures (35℃-37℃).ResultsIHCs showed outwardly rectifying currents in response to depolarizing voltage pulses, with only a slight inward current when hyperpolarized. The amplitude of both outward and inward currents demonstrated no temperature dependency, however, activation and inactivation rates were faster at 36℃ than at room temperature. Half-time for activation was shorter at 36℃ than at room temperature at membrane potentials of -10, +10, +20, +30, and +40 mV. Q10 for the activation rate was 1.83. The inactivation time constant in outward tetraethylammonium-sensitive potassium currents was much smaller at 36℃ than at room temperature between the membrane potentials of -20 and +60 mV. Q10 for the inactivation time constant was 3.19.ConclusionThe results of this study suggest that the amplitude of potassium currents in IHCs showed no temperature dependence either in outward or inward-going currents, however, activation and inactivation accelerated at physiological temperatures.

Critical role for cochlear hair cell BK channels for coding the temporal structure and dynamic range of auditory information for central auditory processing

Large conductance, voltage- and Ca2+-activated K+ (BK) channels in inner hair cells (IHCs) of the cochlea are essential for hearing. However, germline deletion of BKα, the pore-forming subunit KCNMA1 of the BK channel, surprisingly did not affect hearing thresholds in the first postnatal weeks, even though altered IHC membrane time constants, decreased IHC receptor potential alternating current/direct current ratio, and impaired spike timing of auditory fibers were reported in these mice. To investigate the role of IHC BK channels for central auditory processing, we generated a conditional mouse model with hair cell-specific deletion of BKα from postnatal day 10 onward. This had an unexpected effect on temporal coding in the central auditory system: neuronal single and multiunit responses in the inferior colliculus showed higher excitability and greater precision of temporal coding that may be linked to the improved discrimination of temporally modulated sounds observed in behavioral training. The higher precision of temporal coding, however, was restricted to slower modulations of sound and reduced stimulus-driven activity. This suggests a diminished dynamic range of stimulus coding that is expected to impair signal detection in noise. Thus, BK channels in IHCs are crucial for central coding of the temporal fine structure of sound and for detection of signals in a noisy environment.—Kurt, S., Sausbier, M., Rüttiger, L., Brandt, N., Moeller, C. K., Kindler, J., Sausbier, U., Zimmermann, U., van Straaten, H., Neuhuber, W., Engel, J., Knipper, M., Ruth, P., Schulze, H. Critical role for cochlear hair cell BK channels for coding the temporal structure and dynamic range of auditory information for central auditory processing.

The Ca2+Channel Subunit β2 Regulates Ca2+Channel Abundance and Function in Inner Hair Cells and Is Required for Hearing

Hearing relies on Ca(2+) influx-triggered exocytosis in cochlear inner hair cells (IHCs). Here we studied the role of the Ca(2+) channel subunit Ca(V)beta(2) in hearing. Of the Ca(V)beta(1-4) mRNAs, IHCs predominantly contained Ca(V)beta(2). Hearing was severely impaired in mice lacking Ca(V)beta(2) in extracardiac tissues (Ca(V)beta(2)(-/-)). This involved deficits in cochlear amplification and sound encoding. Otoacoustic emissions were reduced or absent in Ca(V)beta(2)(-/-) mice, which showed strongly elevated auditory thresholds in single neuron recordings and auditory brainstem response measurements. Ca(V)beta(2)(-/-) IHCs showed greatly reduced exocytosis (by 68%). This was mostly attributable to a decreased number of membrane-standing Ca(V)1.3 channels. Confocal Ca(2+) imaging revealed presynaptic Ca(2+) microdomains albeit with much lower amplitudes, indicating synaptic clustering of fewer Ca(V)1.3 channels. The coupling of the remaining Ca(2+) influx to IHC exocytosis appeared unaffected. Extracellular recordings of sound-evoked spiking in the cochlear nucleus and auditory nerve revealed reduced spike rates in the Ca(V)beta(2)(-/-) mice. Still, sizable onset and adapted spike rates were found during suprathreshold stimulation in Ca(V)beta(2)(-/-) mice. This indicated that residual synaptic sound encoding occurred, although the number of presynaptic Ca(V)1.3 channels and exocytosis were reduced to one-third. The normal developmental upregulation, clustering, and gating of large-conductance Ca(2+) activated potassium channels in IHCs were impaired in the absence of Ca(V)beta(2). Moreover, we found the developmental efferent innervation to persist in Ca(V)beta(2)-deficient IHCs. In summary, Ca(V)beta(2) has an essential role in regulating the abundance and properties of Ca(V)1.3 channels in IHCs and, thereby, is critical for IHC development and synaptic encoding of sound.

Calcium- and Calmodulin-Dependent Inactivation of Calcium Channels in Inner Hair Cells of the Rat Cochlea

Modulation of voltage-gated calcium channels was studied in inner hair cells (IHCs) in an ex vivo preparation of the apical turn of the rat organ of Corti. Whole cell voltage clamp in the presence of potassium channel blockers showed inward calcium currents with millisecond activation and deactivation kinetics. When temperature was raised from 22 to 37 degrees C, the calcium currents of immature IHCs [<12 days postnatal (P12)] increased threefold in amplitude, and developed more pronounced inactivation. This was determined to be calcium-dependent inactivation (CDI) on the basis of its reliance on external calcium (substitution with barium), sensitivity to internal calcium-buffering, and voltage dependence (reflecting the calcium driving force). After the onset of hearing at P12, IHC calcium current amplitude and the extent of inactivation were greatly reduced. Although smaller than in prehearing IHCs, CDI remained significant in the mature IHC near the resting membrane potential. CDI in mature IHCs was enhanced by application of the endoplasmic calcium pump blocker, benzo-hydroquinone. Conversely, CDI in immature IHCs was reduced by calmodulin inhibitors. Thus voltage-gated calcium channels in mammalian IHCs are subject to a calmodulin-mediated process of CDI. The extent of CDI depends on the balance of calcium buffering mechanisms and may be regulated by calmodulin-specific processes. CDI provides a means for the rate of spontaneous transmitter release to be adjusted to variations in hair cell resting potential and steady state calcium influx.

Ca2+‐binding proteins tune Ca2+‐feedback to Cav1.3 channels in mouse auditory hair cells

Sound coding at the auditory inner hair cell synapse requires graded changes in neurotransmitter release, triggered by sustained activation of presynaptic Ca(v)1.3 voltage-gated Ca(2+) channels. Central to their role in this regard, Ca(v)1.3 channels in inner hair cells show little Ca(2+)-dependent inactivation, a fast negative feedback regulation by incoming Ca(2+) ions, which depends on calmodulin association with the Ca(2+) channel alpha(1) subunit. Ca(2+)-dependent inactivation characterizes nearly all voltage-gated Ca(2+) channels including Ca(v)1.3 in other excitable cells. The mechanism underlying the limited autoregulation of Ca(v)1.3 in inner hair cells remains a mystery. Previously, we established calmodulin-like Ca(2+)-binding proteins in the brain and retina (CaBPs) as essential modulators of voltage-gated Ca(2+) channels. Here, we demonstrate that CaBPs differentially modify Ca(2+) feedback to Ca(v)1.3 channels in transfected cells and explore their significance for Ca(v)1.3 regulation in inner hair cells. Of multiple CaBPs detected in inner hair cells (CaBP1, CaBP2, CaBP4 and CaBP5), CaBP1 most efficiently blunts Ca(2+)-dependent inactivation of Ca(v)1.3. CaBP1 and CaBP4 both interact with calmodulin-binding sequences in Ca(v)1.3, but CaBP4 more weakly inhibits Ca(2+)-dependent inactivation than CaBP1. Ca(2+)-dependent inactivation is marginally greater in inner hair cells from CaBP4(-/-) than from wild-type mice, yet CaBP4(-/-) mice are not hearing-impaired. In contrast to CaBP4, CaBP1 is strongly localized at the presynaptic ribbon synapse of adult inner hair cells both in wild-type and CaBP4(-/-) mice and therefore is positioned to modulate native Ca(v)1.3 channels. Our results reveal unexpected diversity in the strengths of CaBPs as Ca(2+) channel modulators, and implicate CaBP1 rather than CaBP4 in conferring the anomalous slow inactivation of Ca(v)1.3 Ca(2+) currents required for auditory transmission.

Synaptic organization in cochlear inner hair cells deficient for the CaV1.3 (α1D) subunit of L-type Ca2+ channels

Cochlear inner hair cells (IHCs) release neurotransmitter onto afferent auditory nerve fibers in response to sound stimulation. Normal development and function of inner hair cells require the expression of α subunit 1.3 forming L-type voltage-gated Ca2+ channel (CaV1.3).Here, we used immunohistochemistry and reverse transcription-polymerase chain reaction to study the synaptic organization and expression of large conductance Ca2+-activated potassium channels in IHCs of mice lacking the CaV1.3 Ca2+ channel (CaV1.3−/−). Despite the near complete block of evoked afferent synaptic transmission, hair cell ribbon synapses were formed and remained preserved for at least 4 weeks after birth. Moreover, these “silent” afferent synapses held major components of the synaptic machinery such as Bassoon, Piccolo, and CSP. Hence, the block of exocytosis might be solely attributed to the lack of Ca2+ influx through CaV1.3 channels. Later on, CaV1.3 deficient IHCs subsequently lost their afferent synapses. This was probably due to a secondary degeneration of the postsynaptic spiral ganglion neurons.In line with a prolonged efferent synaptic transmission onto CaV1.3 deficient IHCs, which normally ceases around onset of hearing, we found juxtaposed immunoreactive spots of efferent presynaptic synaptophysin and postsynaptic (IHCs) small conductance Ca2+-activated potassium channels (SK channels) up to six weeks after birth. Finally, we show a substantial reduction of mRNA for the α subunit of the large conductance Ca2+-activated potassium channel (BK) in the apical cochlea, suggesting a reduced transcription of its gene in CaV1.3 deficient IHCs. CaV1.3 deficient IHCs lacked the apical spot-like immunoreactivity of clustered BK channels, which normally contribute to the temporal precision of hair cell afferent synaptic transmission.In summary, these data indicate that the CaV1.3 channels are crucially involved in regulation of the expression of BK and SK channels. CaV1.3 channels seem not to be essential for ribbon synapse formation, but are required for the maintenance of ribbon synapses and spiral ganglion neurons.

Increase in efficiency and reduction in Ca2+ dependence of exocytosis during development of mouse inner hair cells

Developmental changes in the coupling between Ca2+ entry and exocytosis were studied in mouse inner hair cells (IHCs) which, together with the afferent endings, form the primary synapse of the mammalian auditory system. Ca2+ currents (ICa) and changes in membrane capacitance (DeltaCm) were recorded using whole-cell voltage clamp from cells maintained at body temperature, using physiological (1.3 mM) extracellular Ca2+. The magnitudes of both ICa and DeltaCm increased with maturation from embryonic stages until postnatal day 6 (P6). Subsequently, ICa gradually declined to a steady level of about -100 pA from P13 while the Ca2+-induced DeltaCm remained relatively constant, indicating a developmental increase in the Ca2+ efficiency of exocytosis. Although the size of ICa changed during development, its activation properties did not, suggesting the presence of a homogeneous population of Ca2+ channels in IHCs throughout development. The Ca2+ dependence of exocytosis changed with maturation from a fourth power relation in immature cells to an approximately linear relation in mature cells. This change applies to the release of both a readily releasable pool (RRP) and a slower secondary pool of vesicles, implying a common release mechanism for these two kinetically distinct pools that becomes modified during development. The increased Ca2+ efficiency and linear Ca2+ dependence of mature IHC exocytosis, especially over the physiological range of intracellular Ca2+, could improve the high-fidelity transmission of both brief and long-lasting stimulation. These properties make the mature cell ideally suited for fine intensity discrimination over a wide dynamic range.

Contribution of BK Ca2+-activated K+ channels to auditory neurotransmission in the Guinea pig cochlea.

Large-conductance calcium-activated potassium (BK) channels are known to play a prominent role in the hair cell function of lower vertebrates where these channels determine electrical tuning and regulation of neurotransmitter release. Very little is known, by contrast, about the role of BK channels in the mammalian cochlea. In the current study, we perfused specific toxins in the guinea pig cochlea to characterize the role of BK channels in cochlear neurotransmission. Intracochlear perfusion of charybdotoxin (ChTX) or iberiotoxin (IbTX) reversibly reduced the compound action potential (CAP) of the auditory nerve within minutes. The cochlear microphonics (CM at f1 = 8 kHz and f2 = 9.68 kHz) and their distortion product (DPCM at 2f1-f2) were essentially not affected, suggesting that the BK specific toxins do not alter the active cochlear amplification at the outer hair cells (OHCs). We also tested the effects of these toxins on the whole cell voltage-dependent membrane current of isolated guinea pig inner hair cells (IHCs). ChTX and IbTX reversibly reduced a fast outward current (activating above -40 mV, peaking at 0 mV with a mean activation time constant tau ranging between 0.5 and 1 ms). A similar block of a fast outward current was also observed with the extracellular application of barium ions, which we believe permeate through Ca2+ channels and block BK channels. In situ hybridization of Slo antisense riboprobes and immunocytochemistry demonstrated a strong expression of BK channels in IHCs and spiral ganglion and to a lesser extent in OHCs. Overall, our results clearly revealed the importance of BK channels in mammalian cochlear neurotransmission and demonstrated that at the presynaptic level, fast BK channels are a significant component of the repolarizing current of IHCs.