Calcium current in type I hair cells isolated from the semicircular canal crista ampullaris of the rat

doi:10.1016/j.brainres.2003.09.033

Brain Research

Volume 994, Issue 2, 24 December 2003, Pages 175-180

https://doi.org/10.1016/j.brainres.2003.09.033 Get rights and content

Abstract

The low voltage gain in type I hair cells implies that neurotransmitter release at their afferent synapse should be mediated by low voltage activated calcium channels, or that some peculiar mechanism should be operating in this synapse. With the patch clamp technique, we studied the characteristics of the Ca²⁺ current in type I hair cells enzymatically dissociated from rat semicircular canal crista ampullaris. Calcium current in type I hair cells exhibited a slow inactivation (during 2-s depolarizing steps), was sensitive to nimodipine and was blocked by Cd²⁺ and Ni²⁺. This current was activated at potentials above −60 mV, had a mean half maximal activation of −36 mV, and exhibited no steady-state inactivation at holding potentials between −100 and −60 mV. This data led us to conclude that hair cell Ca²⁺ current is most likely of the L type. Thus, other mechanisms participating in neurotransmitter release such as K⁺ accumulation in the synaptic cleft, modulation of K⁺ currents by nitric oxide, participation of a Na⁺ current and possible metabotropic cascades activated by depolarization should be considered.

Introduction

Ionic conductance of vestibular hair cells varies depending on the animal species, the region where the cells originate, the developmental stage of the animal, and the hair cell type. However, in types I and II hair cells studied up to date, potassium currents dominate the basolateral membrane response [8], [17], [18], [29].

Type I hair cells have a very peculiar afferent synapse which forms a calyceal structure surrounding the basolateral surface of the cell; many authors have wondered what the possible function of this calyx might be [9], [30], [31], [32]. Due to the difussional properties of the microspace formed between the calyx and the hair cell, it has been suggested that changes in this microambient may be relevant for synaptic transmission in type I hair cells. In contrast, type II hair cells have bouton afferent endings which have been thoroughly studied [10]. They correspond to a typical chemical synapse, and transmitter release has been shown to be calcium dependent [13], [20], [33].

The most significant electrophysiological difference between types I and II vestibular hair cells is the expression of a low voltage-activated, non-inactivating outward K⁺ current (g_K,L) in type I hair cells [8], [26], [28]. g_K,L is substantially activated at the resting potential, the half activation voltage (V_1/2) value of g_K,L can be as negative as −90 mV in physiological conditions, greatly reducing cell input resistance [28]. In fact, the low input resistance of type I hair cells determines that mechanoelectrical transducer currents seem not to be large enough to depolarize these cells to produce neurotransmitter release. Therefore, for these cells to activate the neurotransmitter release machinery, voltage-dependent Ca²⁺ channels should have a very negative activating voltage, or there should be some other mechanisms contributing to further depolarize the cell [7], [9], [32].

In this study, we addressed the problem of determining the characteristics and particularly the half activation voltage of the Ca²⁺ current in type I hair cells.

Section snippets

Materials and methods

Experiments were performed in young Long-Evans rats (postnatal days 14–17) supplied by the bioterium “Claude Bernard” of the University of Puebla. All efforts were made to minimize animal suffering and to reduce the number of animals used, as outlined in the “Guide to the Care and Use of Laboratory Animals” issued by the National Academy of Science, USA.

Hair cells were enzymatically dissociated from the semicircular canal crista ampullaris of the rat. Tissue pieces containing the vestibular

Results

Stable recordings in normal extra and intracellular solutions (Table 1) were readily obtained. Typically, type I hair cells displayed a g_K,L current, thus having a small membrane resistance (R_m) value compared with type II hair cells (Table 2). In current clamp with a membrane potential of about −73±4 mV, type I hair cells showed a low voltage gain with a near linear slope of 2.4±0.2 mV/100 pA (data not shown). A current injection of as much as 600 pA depolarized the cells up to −61±6 mV (n=4).

Discussion

Our results are consistent with the idea that a single Ca²⁺ channel subtype may account for the major part of the macroscopic Ca²⁺ current. We found a small inward current with a peak amplitude of about 50 pA at −17 mV. Current amplitude increased by using Ba²⁺ as a current carrier, was blocked by Cd²⁺ and Ni²⁺, and was sensitive to nimodipine. This current, activated above −60 mV with V_1/2=−36 mV, showed a slowly developing inactivation while using Ca²⁺ as a current carrier. Variations in the

Acknowledgements

The authors wish to thank Marcela Sánchez Álvarez for proof reading the English manuscript. This material is based upon work supported by the Consejo Nacional de Ciencia y Tecnologı́a, México (CONACyT) grant 40672 to RV and by Secretaria de Educación Superior, DGES-VIEP grant II-84G02. AA was supported by a CONACyT fellowship.

References (33)

P.H. Barry
JPCalc, a software package for calculating liquid junction potential corrections in patch-clamp, intracellular, epithelial and bilayer measurements and for correcting junction potential measurements
J. Neurosci. Methods
(1994)
M.J. Correia et al.
An electrophysiological comparison of solitary type I and type II vestibular hair cells
Neurosci. Lett.
(1990)
P.S. Guth et al.
The vestibular hair cells: post-transductional signal processing
Prog. Neurobiol.
(1998)
D. Lenzi et al.
Calcium signalling in hair cells: multiple roles in a compact cell
Curr. Opin. Neurobiol.
(1994)
I. López et al.
Differential subcellular immunolocalization of voltage-gated calcium channel alpha1 subunits in the chinchilla cristae ampullaris
Neurosci.
(1999)
M. Martini et al.
Calcium currents in hair cells isolated from semicircular canals of the frog
Biophys. J.
(2000)
P. Perin et al.
Regional distribution of calcium currents in frog semicircular canal hair cells
Hear. Res.
(2001)
J. Rae et al.
Low access resistance perforated patch recording using amphotericin B
J. Neurosci. Methods
(1991)
D.A. Schessel et al.
Morphophysiology of synaptic transmission between type I hair cells and vestibular primary afferents. An intracellular study employing horseradish peroxidase in the lizard, Calotes versicolor
Brain Res.
(1991)
E. Soto et al.
The receptor potential in type I and type II vestibular system hair cells: a model analysis
Hear. Res.
(2002)

H. Bao et al.

Voltage-gated calcium channel currents in type I and type II hair cells isolated from the rat crista

J. Neurophysiol.

(2003)

O. Behrend et al.

Cyclic GMP inhibits and shifts the activation curve of the delayed-rectifier (I_K1) of type I mammalian vestibular hair cells

NeuroReport

(1997)

M. Bosica et al.

Na⁺ and Ca²⁺ currents in chick embryo developing type I and type II hair cells

C. Boyer et al.

Potassium depolarization of mammalian vestibular sensory cells increases [Ca²⁺] through voltage-sensitive calcium channels

Eur. J. Neurosci.

(1998)

A.M. Brichta et al.

Regional analysis of whole cell currents from hair cells of the turtle posterior crista

J. Neurophysiol.

(2002)

J.W.Y. Chen et al.

Major potassium conductance in type I hair cells from rat semicircular canals: characterization and modulation by nitric oxide

J. Neurophysiol.

(2000)

Cited by (18)

K+ Accumulation and Clearance in the Calyx Synaptic Cleft of Type I Mouse Vestibular Hair Cells
2020, Neuroscience
Citation Excerpt :
While the basolateral membrane of Type I hair cells is enclosed by a single giant afferent nerve terminal, called a calyx, each Type II hair cell is contacted by several (10 to 20) small bouton afferent endings and makes synapses with the outer faces of calyx endings (Lysakowski and Goldberg, 1997). Upon stimulation of vestibular hair cells, the opening of voltage-gated L-Type Ca2+ channels (Bao et al., 2003; Almanza et al., 2003; Zampini et al., 2006) triggers the exocytosis of glutamate, which depolarizes the afferent terminal by binding to AMPA receptors (Bonsacquet et al., 2006; Dulon et al., 2009; Songer and Eatock, 2013; Sadeghi et al., 2014; Kirk et al., 2017). However, a non-quantal mode of transmission has also been reported to occur at the Type I hair cell-calyx synapse (Yamashita and Ohmori, 1990; Holt et al., 2007; Songer and Eatock, 2013), though the molecular mechanism is not fully understood.
Vestibular organs of Amniotes contain two types of sensory cells, named Type I and Type II hair cells. While Type II hair cells are contacted by several small bouton nerve terminals, Type I hair cells receive a giant terminal, called a calyx, which encloses their basolateral membrane almost completely. Both hair cell types release glutamate, which depolarizes the afferent terminal by binding to AMPA post-synaptic receptors. However, there is evidence that non-vesicular signal transmission also occurs at the Type I hair cell-calyx synapse, possibly involving direct depolarization of the calyx by K⁺ exiting the hair cell. To better investigate this aspect, we performed whole-cell patch-clamp recordings from mouse Type I hair cells or their associated calyx. We found that [K⁺] in the calyceal synaptic cleft is elevated at rest relative to the interstitial (extracellular) solution and can increase or decrease during hair cell depolarization or repolarization, respectively. The change in [K⁺] was primarily driven by G_K,L, the low-voltage-activated, non-inactivating K⁺ conductance specifically expressed by Type I hair cells. Simple diffusion of K⁺ between the cleft and the extracellular compartment appeared substantially restricted by the calyx inner membrane, with the ion channels and active transporters playing a crucial role in regulating intercellular [K⁺]. Calyx recordings were consistent with K⁺ leaving the synaptic cleft through postsynaptic voltage-gated K⁺ channels involving K_V1 and K_V7 subunits. The above scenario is consistent with direct depolarization and hyperpolarization of the calyx membrane potential by intercellular K⁺.
Intercellular K+ accumulation depolarizes Type I vestibular hair cells and their associated afferent nerve calyx
2012, Neuroscience
Citation Excerpt :
The Ba2+ current (IBa) flowing through Ca2+ channels activated at around –60 mV and reached a peak at –30 mV (Fig. 6D, red symbols), showed rapid (<1 ms) activation kinetics (Fig. 6C), and little if any inactivation (Fig. 6B). These properties recall those found for ICa in rat vestibular Type I cells (Almanza et al., 2003; Bao et al., 2003). Immunocytochemical studies showed a dense expression of K+ channels in the calyceal inner face (Lysakowski et al., 2011), which could be involved in intercellular K+ accumulation as well.
Mammalian vestibular organs contain two types of sensory receptors, named Type I and Type II hair cells. While Type II hair cells are contacted by several small afferent nerve terminals, the basolateral surface of Type I hair cells is almost entirely enveloped by a single large afferent nerve terminal, called calyx. Moreover Type I, but not Type II hair cells, express a low-voltage-activated outward K⁺ current, I_K,L, which is responsible for their much lower input resistance (Rm) at rest as compared to Type II hair cells. The functional meaning of I_K,L and associated calyx is still enigmatic. By combining the patch-clamp whole-cell technique with the mouse whole crista preparation, we have recorded the current- and voltage responses of in situ hair cells. Outward K⁺ current activation resulted in K⁺ accumulation around Type I hair cells, since it induced a rightward shift of the K⁺ reversal potential the magnitude of which depended on the amplitude and duration of K⁺ current flow. Since this phenomenon was never observed for Type II hair cells, we ascribed it to the presence of a residual calyx limiting K⁺ efflux from the synaptic cleft. Intercellular K⁺ accumulation added a slow (τ > 100 ms) depolarizing component to the cell voltage response. In a few cases we were able to record from the calyx and found evidence for intercellular K⁺ accumulation as well. The resulting depolarization could trigger a discharge of action potentials in the afferent nerve fiber. Present results support a model where pre- and postsynaptic depolarization produced by intercellular K⁺ accumulation cooperates with neurotransmitter exocytosis in sustaining afferent transmission arising from Type I hair cells. While vesicular transmission together with the low Rm of Type I hair cells appears best suited for signaling fast head movements, depolarization produced by intercellular K⁺ accumulation could enhance signal transmission during slow head movements.
Pimozide Increases a Delayed Rectifier K+ Conductance in Chicken Embryo Vestibular Hair Cells
2023, Biomedicines
Signal transmission in mature mammalian vestibular hair cells
2022, Frontiers in Cellular Neuroscience
An allosteric gating model recapitulates the biophysical properties of I<inf>K,L</inf> expressed in mouse vestibular type I hair cells
2017, Journal of Physiology
Sensory transduction at the frog semicircular canal: How hair cell membrane potential controls junctional transmission
2015, Frontiers in Cellular Neuroscience

View all citing articles on Scopus

View full text

Research reportCalcium current in type I hair cells isolated from the semicircular canal crista ampullaris of the rat

Abstract

Introduction

Section snippets

Materials and methods

Results

Discussion

Acknowledgements

J. Neurosci. Methods

Neurosci. Lett.

Prog. Neurobiol.

Curr. Opin. Neurobiol.

Neurosci.

Biophys. J.

Hear. Res.

J. Neurosci. Methods

Brain Res.

Hear. Res.

Voltage-gated calcium channel currents in type I and type II hair cells isolated from the rat crista

J. Neurophysiol.

Cyclic GMP inhibits and shifts the activation curve of the delayed-rectifier (IK1) of type I mammalian vestibular hair cells

NeuroReport

Na+ and Ca2+ currents in chick embryo developing type I and type II hair cells

Potassium depolarization of mammalian vestibular sensory cells increases [Ca2+] through voltage-sensitive calcium channels

Eur. J. Neurosci.

Regional analysis of whole cell currents from hair cells of the turtle posterior crista

J. Neurophysiol.

Major potassium conductance in type I hair cells from rat semicircular canals: characterization and modulation by nitric oxide

J. Neurophysiol.

Research report
Calcium current in type I hair cells isolated from the semicircular canal crista ampullaris of the rat

Cyclic GMP inhibits and shifts the activation curve of the delayed-rectifier (I_K1) of type I mammalian vestibular hair cells

Na⁺ and Ca²⁺ currents in chick embryo developing type I and type II hair cells

Potassium depolarization of mammalian vestibular sensory cells increases [Ca²⁺] through voltage-sensitive calcium channels