Unexpected impairment of INa underpins reentrant arrhythmias in a knock-in swine model of Timothy syndrome

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Precision medicine: a new era for inner ear diseases

The inner ear is the organ responsible for hearing and balance. Inner ear dysfunction can be the result of infection, trauma, ototoxic drugs, genetic mutation or predisposition. Often, like for Ménière disease, the cause is unknown. Due to the complex access to the inner ear as a fluid-filled cavity within the temporal bone of the skull, effective diagnosis of inner ear pathologies and targeted drug delivery pose significant challenges. Samples of inner ear fluids can only be collected during surgery because the available procedures damage the tiny and fragile structures of the inner ear. Concerning drug administration, the final dose, kinetics, and targets cannot be controlled. Overcoming these limitations is crucial for successful inner ear precision medicine. Recently, notable advancements in microneedle technologies offer the potential for safe sampling of inner ear fluids and local treatment. Ultrasharp microneedles can reach the inner ear fluids with minimal damage to the organ, collect μl amounts of perilymph, and deliver therapeutic agents in loco. This review highlights the potential of ultrasharp microneedles, combined with nano vectors and gene therapy, to effectively treat inner ear diseases of different etiology on an individual basis. Though further research is necessary to translate these innovative approaches into clinical practice, these technologies may represent a true breakthrough in the clinical approach to inner ear diseases, ushering in a new era of personalized medicine

IK,L properties of vestibular Type I hair cells are affected by the nerve calyx ending

Mammalian vestibular epithelia have a distinctive sensory cell , called Type I hair cell, that is contacted by an afferent calyx enveloping the entire cell basolateral membrane. Type I cells express a signature low-voltage-activated outward rectifying K+ current, IK,L, which is responsible for their low input resistance at rest . Despite its functional importance, however, IK,L biophysical properties and molecular profile have not yet been defined. Its voltage- and time-dependent properties have been reported to vary at different developmental stages, among cells at a same age, and also over time in the same cell. By using patch-clamp recording from in situ and dissociated mouse crista Type I cells, we found that the observed variability in IK,L properties may be accounted for by different degrees of K+ accumulation in the narrow space of the synaptic cleft between the hair cell and the residual nerve calyx. After complete calyx removal, IK,L properties in adult animals were in fact consistent among cells and did not change during the recording. IK,L in these cells showed a quite slow deactivation kinetics (time constant ~ 1 s at –80 mV), a complex activation kinetics best described by a three exponential function, a half-activation voltage of –69 mV, and a steep voltage dependence (S = 3.68). This study provides the first complete biophysical description of the genuine properties of IK,L, and suggests that in vivo IK,L properties are dependent on K+ accumulation into the synaptic cleft. Intercellular K+ accumulation might represent a direct way to change both the hair cell and the calyx membrane potential, thus allowing an additional form of communication that cooperates with the conventional glutamatergic synaptic transmission

Electrophysiological evidence for potassium accumulation between type I hair cells and calyx terminal in mammalian crista ampullaris

Two types of hair cells are present in mammalian vestibular sensory epithelia, called Type I and Type II hair cells, which differ in electrophysiological properties and innervation. Type II hair cells are contacted by several bouton nerve terminals, while Type I hair cells are contacted by a calyx nerve terminal that envelopes the entire basolateral membrane. Only Type I hair cells, moreover, express a low-voltage activated outward K+ current, called IK,L, which confers upon them a much lower input resistance at rest compared to Type II hair cells. As a consequence, in Type I hair cells large transducer currents would be necessary to change the cell membrane potential and to depolarize the cell enough to activate voltage-gated Ca2+ channels and related neurotransmitter release. How the calyx synapse operates remains in fact enigmatic. It has been speculated that K+ accumulation in the synaptic cleft cooperates with conventional (vesicular) synaptic transmission in sustaining afferent transmission by Type I hair cells. By combining the patch-clamp whole-cell configuration with the whole crista preparation, we have recorded the current and voltage responses of mouse semicircular canal Type I and Type II hair cells in situ. Depolarizing voltage steps elicited in Type II hair cells a large outward K+ current characterized by a substantial time-dependent inactivation, while the same voltage-protocol elicited in most Type I hair cells a large and sustained outward K+ current. However, in a notable percentage (51%) of Type I hair cells investigated, the outward K+ current showed a substantial time-dependent relaxation. In these cells, moreover, upon repolarization to –40 mV the instantaneous current was inward, reversing to outward slowly with time. A reasonable explanation for the above results is that during large outward K+ currents, K+ accumulates around Type I hair cells, thus shifting the K+ reversal potential (VrevK+) toward more positive values. The rightward shift of VrevK+ would produce both the outward current relaxation during depolarizing voltage steps and the instantaneous inward current upon repolarization at –40 mV. Since we never observed such effects when recording from Type II hair cells, we hypothesized that the presence of a residual nerve calyx was responsible for K+ accumulation around Type I hair cells. We also found that by using voltage protocols that increased extracellular K+ accumulation, IK,L deactivation was slowed down. Similar results, i.e. VrevK+ rightward shift and IK,L deactivation slowdown, were obtained by local perfusion of the preparation with an extracellular solution enriched in K+, thus corroborating our hypothesis about K+ accumulation. In conclusion, our results provide electrophysiological evidence for an increased K+ concentration in the synaptic cleft between Type I hair cell and its calyx ending during outward K+ current activation. The resulting depolarization might be aimed at reinforcing and prolonging Ca2+ channels activation and thus afferent transmission during slow head movements detected by vestibular organs

Non conventional signal transmission at the mouse vestibular Type I hair cell - calyx synapse

Vestibular sensory epithelia of Amniotes contain two types of hair cells, Type I and Type II, which differ in electrophysiological properties and synaptic contacts. Type I hair cells alone express a low-voltage activated outward rectifying K+ conductance, named GK,L. Moreover, each Type II hair cell is contacted by several bouton afferent endings, while a single large calyceal afferent terminal encloses the basolateral membrane of Type I hair cells, where voltage-gated Ca2+ and K+ channels and the presynaptic sites for glutamate release are located. Besides vesicular transmission, a nonquantal transmission has been hypothesized to occur at the calyx synapse, whereby K+ exiting the hair cell directly depolarizes the calyx terminal. To investigate K+ involvement in signal transmission, we whole-cell recorded from in vitro mouse Type I hair cells or their associated calyx. We found that intercellular (in the synaptic cleft) K+ increased or decreased depending upon hair cell membrane potential as a consequence of GK,L negative voltage-range of activation . Moreover, we found evidence for the calyx inner membrane facing the synaptic cleft expressing voltage-gated K+ channels of the KV1 and KV7 type. Present results suggests a scenario where hair bundle deflection produces calyx depolarization or hyperpolarization by modulating K+ flux across the hair cell and through postsynaptic voltage-gated K+ channels

Allosteric gating of K,L channels expressed in mouse vestibular Type I hair cells

Vestibular signals are relayed to the CNS by Type I and Type II hair cells. While Type II hair cells are contacted by several “bouton-like” afferent nerve terminals, Type I hair cells are characterized by their basolateral membrane being enveloped in a single large afferent nerve terminal, named calyx, whose functional meaning is still unknown. Furthermore, Type I hair cells express an outward rectifying K+ current, IK,L, which is active at unusually negative membrane voltages. The molecular nature of IK,L still escapes. By using the patch-clamp whole-cell technique, we examined the voltage- and time-dependent properties of IK,L in Type I hair cells of the mouse semicircular canal. We found that the biophysical properties of IK,L were affected by an unstable K+ equilibrium potential (VeqK+). Both the outward and inward K+ currents shifted VeqK+ consistent with K+ accumulation or depletion, respectively, in the extracellular space. We attributed this phenomenon to a residual calyx attached to the basolateral membrane of the hair cell. We therefore optimized the hair cell dissociation protocol in order to isolate mature Type I hair cells without their calyx. In these cells, the uncontaminated IK,L showed a half-activation at –73.5 mV and a steep voltage dependence (3.1 mV). IK,L also showed complex activation and deactivation kinetics, which we faithfully reproduced by an allosteric channel gating scheme where the channel is able to open from all (five) closed states. The “side” open states substantially contribute to IK,L activation at negative voltages. This study provides the first complete description of the “native” biophysical properties of IK,L in adult mouse vestibular Type I hair cells

An allosteric gating model recapitulates the biophysical properties of I_K,L expressed in mouse vestibular type I hair cells

Type I and type II hair cells are the sensory receptors of the mammalian vestibular epithelia. Type I hair cells are characterized by their basolateral membrane being enveloped in a single large afferent nerve terminal, named the calyx, and by the expression of a low-voltage-activated outward rectifying K+ current, IK,L. The biophysical properties and molecular profile of IK,L are still largely unknown. By using the patch-clamp whole-cell technique, we examined the voltage- and time-dependent properties of IK,L in type I hair cells of the mouse semicircular canal. We found that the biophysical properties of IK,L were affected by an unstable K+ equilibrium potential (VeqK+). Both the outward and inward K+ currents shifted VeqK+ consistent with K+ accumulation or depletion, respectively, in the extracellular space, which we attributed to a residual calyx attached to the basolateral membrane of the hair cells. We therefore optimized the hair cell dissociation protocol in order to isolate mature type I hair cells without their calyx. In these cells, the uncontaminated IK,L showed a half-activation at –79.6 mV and a steep voltage dependence (2.8 mV). IK,L also showed complex activation and deactivation kinetics, which we faithfully reproduced by an allosteric channel gating scheme where the channel is able to open from all (five) closed states. The ‘early’ open states substantially contribute to IK,L activation at negative voltages. This study provides the first complete description of the ‘native’ biophysical properties of IK,L in adult mouse vestibular type I hair cells.Instituto de Estudios Inmunológicos y FisiopatológicosFacultad de Ciencias Exacta

An allosteric gating model recapitulates the biophysical properties of I_K,L expressed in mouse vestibular type I hair cells

Type I and type II hair cells are the sensory receptors of the mammalian vestibular epithelia. Type I hair cells are characterized by their basolateral membrane being enveloped in a single large afferent nerve terminal, named the calyx, and by the expression of a low-voltage-activated outward rectifying K+ current, IK,L. The biophysical properties and molecular profile of IK,L are still largely unknown. By using the patch-clamp whole-cell technique, we examined the voltage- and time-dependent properties of IK,L in type I hair cells of the mouse semicircular canal. We found that the biophysical properties of IK,L were affected by an unstable K+ equilibrium potential (VeqK+). Both the outward and inward K+ currents shifted VeqK+ consistent with K+ accumulation or depletion, respectively, in the extracellular space, which we attributed to a residual calyx attached to the basolateral membrane of the hair cells. We therefore optimized the hair cell dissociation protocol in order to isolate mature type I hair cells without their calyx. In these cells, the uncontaminated IK,L showed a half-activation at –79.6 mV and a steep voltage dependence (2.8 mV). IK,L also showed complex activation and deactivation kinetics, which we faithfully reproduced by an allosteric channel gating scheme where the channel is able to open from all (five) closed states. The ‘early’ open states substantially contribute to IK,L activation at negative voltages. This study provides the first complete description of the ‘native’ biophysical properties of IK,L in adult mouse vestibular type I hair cells.Instituto de Estudios Inmunológicos y FisiopatológicosFacultad de Ciencias Exacta

An allosteric gating model recapitulates the biophysical properties of I_K,L expressed in mouse vestibular type I hair cells

Type I and type II hair cells are the sensory receptors of the mammalian vestibular epithelia. Type I hair cells are characterized by their basolateral membrane being enveloped in a single large afferent nerve terminal, named the calyx, and by the expression of a low-voltage-activated outward rectifying K+ current, IK,L. The biophysical properties and molecular profile of IK,L are still largely unknown. By using the patch-clamp whole-cell technique, we examined the voltage- and time-dependent properties of IK,L in type I hair cells of the mouse semicircular canal. We found that the biophysical properties of IK,L were affected by an unstable K+ equilibrium potential (VeqK+). Both the outward and inward K+ currents shifted VeqK+ consistent with K+ accumulation or depletion, respectively, in the extracellular space, which we attributed to a residual calyx attached to the basolateral membrane of the hair cells. We therefore optimized the hair cell dissociation protocol in order to isolate mature type I hair cells without their calyx. In these cells, the uncontaminated IK,L showed a half-activation at –79.6 mV and a steep voltage dependence (2.8 mV). IK,L also showed complex activation and deactivation kinetics, which we faithfully reproduced by an allosteric channel gating scheme where the channel is able to open from all (five) closed states. The ‘early’ open states substantially contribute to IK,L activation at negative voltages. This study provides the first complete description of the ‘native’ biophysical properties of IK,L in adult mouse vestibular type I hair cells.Instituto de Estudios Inmunológicos y FisiopatológicosFacultad de Ciencias Exacta