Transient increase in cytosolic free calcium evoked by repolarization in type I vestibular hair cells of rats.

Simultaneous whole-cell patch clamp and Fura-2 microfluorimetric recordings of membrane currents and intracellular free calcium concentration ([Ca2+]i) were made from type I vestibular hair cells isolated from cristae ampullares of adult rats. Cells held between -110 or -70 mV and depolarized up to -20 mV did not evoke any [Ca2+]i changes for any duration of the membrane depolarization (up to 3 s). Returning the membrane to repolarizing potential induced a transient [Ca2+]i increase. At the pulse break, an inward current was evoked. The [Ca2+]i increase and inward current amplitude were dependent on the duration and the amplitude of the previous depolarization. A liminar value of membrane depolarization of -55 +/- 3 mV (mean resting potential -62 +/- 7 mv) had to be applied to induce [Ca2+]i increase upon subsequent repolarization. [Ca2+]i response and inward current could not be evoked in calcium-free solution. Both responses were restored when calcium was added to the medium.

CSlo encodes calcium-activated potassium channels in the chick's cochlea.

Large conductance, calcium-activated (BK) potassium channels play a central role in the excitability of cochlear hair cells. In mammalian brains, one class of these channels, termed Slo, is encoded by homologues of the Drosophila 'slowpoke' gene. By homology screening with mouse Sla cDNA, we have isolated a full-length clone (cSlo1) from a chick's cochlear cDNA library, rSlol had greater than 90% identity with mouse Slo at the amino acid level, and was even better matched to a human brain Slo at the amino and carboxy termini. cSlol had none of the additional exons found in splice variants from mammalian brain. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to show expression of cSlal in the microdissected hair cell epithelium basilar papilla. Transient transfection of HIEK 293 cells demonstrated that cSlol encoded a potassium channel whose conductance averaged 224 pS at +60 mV in symmetrical 140 mM K. Macroscopic currents through cSlol channels were blocked by scorpion toxin or tetraethyl ammonium, and were voltage and calcium dependent. cSlol is likely to encode BK-type calcium-activated potassium channels in cochlear hair cells.

Inward potassium rectifier current in type I vestibular hair cells isolated from guinea pig.

Large inward current activated by hyperpolarization was studied using whole cell patch clamp technique in type I vestibular hair cells of guinea pig. Near the resting membrane potential, at an holding potential of -60 mV (HP -60), this current increased with hyperpolarizing steps and showed time-dependent decay for steps below -80 mV. This current was progressively inactivated at more negative holding potential and was totally abolished at HP -90 mV. The underlying conductance was a K+ conductance as indicated by: (i) its dependence on the external potassium concentration; (ii) its tail currents, which reversed at about -90 mV in solutions with a normal gradient for K+ ions. Pharmacological studies revealed that external application of 4-aminopyridine (5 mM) reversibly blocked (95%) the total inward current, while external application of tetraethylammonium (10 mM) or cesium (2 mM) did not significantly affect the amplitude of this current. This potassium inward rectifier current could contribute to restoration of the resting membrane potential during negative stimulations.

Motile responses of isolated guinea pig vestibular hair cells.

Vestibular hair cells were isolated from the guinea pig vestibule by a micromechanical non-enzymatic procedure. Perfusion with 125 mM K+ solution induced irreversible slow shortening of the necks in 42.8% of the hair cells tested. Mechanical stimulation, creating a displacement of the hair bundle towards the kinocilium, induced either irreversible coiling or tilting of the neck of the cells, or reversible fast tilting of the cuticular plate (44.5% of tested cells). The response to the Ca2+ antagonist, Flunarizine, suggested that these movements were calcium-dependent. We propose several explanations of the physiological role of these mechanisms and discuss the possibility that fast tilting of the cuticular plate is a physiological movement involving the hair cells at the periphery of the vestibular receptors. The regulation of the vestibular message at the apex of type I hair cells is also considered.

Voltage dependent reversible movements of the apex in isolated guinea pig vestibular hair cells.

Type I vestibular hair cells isolated from guinea pig were placed in the whole cell clamp configuration, and electrically stimulated by depolarizing voltage pulses. The voltage dependent reversible movements of the cell apex affected the length of the cell neck, the position of the cuticular plate, and the tilting and bending of the stereocilia. The cell neck shortened when the membrane was depolarized by 10 mV while cuticular plate and the stereocilia tilting did not begin until 20 mV. The shortening was 0.5 to 1 micron, and the cuticular plate tilting was up to 15 degrees for depolarization amplitudes of 20-40 mV. These movements were reversed within a few seconds. More complex, larger movements were induced by stronger depolarizations. The cuticular plate tilting and the hair bundle bending were always in the opposite direction to the kinocilium position. The small reversible movements of the mammalian type I vestibular hair cells are discussed in terms of mechanical adaptation processes and morphological features. It is suggested that such active movements of the vestibular hair cells occur in vivo.

Non-typical K(+)-current in cesium-loaded guinea pig type I vestibular hair cell.

Isolated guinea pig type I vestibular hair cells were voltage clamped at HP-110 mV in whole cell clamp configuration and depolarized up to +20 mV. Increasing depolarizations elicited large outward currents. These currents were replaced, in cesium-loaded cells, by inward/outward currents that reversed at membrane potentials between -55 and -30 mV. The reversal potential varied from cell to cell, and appeared to depend on the intracellular potassium cesium ratio. The current remaining in the presence of intracellular cesium was essentially due to a non-typical potassium conductance, which decreased in the presence of 4-AP and was blocked by 4-AP plus TEA. This current appeared as soon as the membrane was depolarized, showing the high potassium permeability of type I vestibular hair cells. A small part of this current was a strictly calcium inward current, sensitive to flunarizine, with a leakage component in the hyperpolarized state and a voltage component when the cell was depolarized.

Voltage-dependent potassium currents in cochlear hair cells of the embryonic chick.

1. Hair cells were isolated from apical and basal regions of the embryonic chick's cochlea. Outward potassium currents were recorded using whole cell tight-seal voltage clamp. 2. Outward currents in basal hair cells activated and inactivated rapidly. The average time to half-maximum at 0 mV was 2.9 ms. The time constant of inactivation at 0 mV was 71 ms. Boltzmann fits to conductance-voltage curves gave an average half-activation voltage of -36 mV, and steady-state inactivation was half-maximal at -62 mV. 3. Potassium currents in apical hair cells had slower kinetics, with a time to half-maximum of 6.7 ms and an inactivation time constant of 242 ms at + 10 mV. The half-activation voltage derived from Boltzmann fits was -16 mV and that for inactivation was -43 mV. 4. With respect to kinetic and voltage-dependent properties, the rapidly and slowly activating potassium currents of embryonic cells were similar to the rapidly inactivating "A" current of mature short hair cells and to the delayed rectifier of mature tall hair cells. However, unlike the adult currents, the embryonic currents did not show differential sensitivities to tetraethylammonium chloride and 4-aminopyridine. As early as the tenth day of embryogenesis, hair cells at the apical and basal extremes of the cochlea produced functionally distinct voltage-gated potassium currents.

The vestibular type I hair cells: a self-regulated system?

In this study the functional role of afferent nerve calyx surrounding the type I vestibular hair cells was investigated. Synaptic microvesicles were present at the apex of the calyx in the vestibular epithelium of human foetuses at 9 weeks from gestation. Whole cell clamped type I hair cells isolated from guinea pig epithelium presented active movements as shortening of the neck and tilting of the cuticular plate at the cessation of the depolarising step. These movements were calcium dependent. With the aim of establishing the kinetics of calcium influx during the cell depolarisation, intracellular free calcium rate variations were investigated by coupling cytofluorimetry technique with whole cell patch clamp. An increase of intracellular calcium was only observed at the repolarisation of type I hair cells. Thus, a regulatory short-loop is thought to exist to control adaptation phenomena at the upper part of the type I hair cell. It is suggested that this occurs through the release of a neurotransmitter from the apex of the afferent calyx.

Potassium currents in type II vestibular hair cells isolated from the guinea-pig's crista ampullaris.

Type II vestibular hair cells were isolated from cristae ampullares of guinea-pig and maintained in vitro for 2-3 h. Outward membrane currents were studied under whole-cell voltage-clamp conditions. Type II hair cells had resting potentials of about -45 mV. Depolarizing voltage steps from a holding potential of -80 or -90 mV induced time- and voltage-dependent outward currents which slowly decayed to a sustained level. Tail currents reversed at about -70 mV, indicating that the outward currents were mainly carried by potassium ions. The currents had an activation threshold around -50 mV. The transient component was completely removed by a depolarizing pre-pulse positive to -10 mV. While bath application of 4-aminopyridine (5 mM) reduced both components, extracellular tetraethylammonium (10 mM) or zero calcium preferentially diminished the sustained current. We conclude that at least two potassium conductances are present, a delayed rectifier with a relatively fast inactivation and a calcium-dependent potassium current. Depolarizing current injections induced an electrical resonance in the voltage responses, with a frequency of 25-100 Hz, larger currents causing higher frequencies.