Katherine J. Rennie

The delayed rectifier, IKI, is the major conductance in type I vestibular hair cells across vestibular end organs

Hair cells were dissociated from the semicircular canal, utricle, lagena and saccule of white king pigeons. Type I hair cells were identified morphologically based on the ratios of neck width to cuticular plate width (NPR < 0.72) as well as neck width to cell body width (NBR < 0.64). The perforated patch variant of the whole-cell recording technique was used to measure electrical properties from type I hair cells. In voltage-clamp, the membrane properties of all identified type I cells were dominated by a predominantly outward potassium current, previously characterized in semicircular canal as IKI. Zero-current potential, activation, deactivation, slope conductance, pharmacologic and steady-state properties of the complex currents were not statistically different between type I hair cells of different vestibular end organs. The voltage dependence causes a significant proportion of this conductance to be active about the cell′s zero-current potential. The first report of the whole-cell activation kinetics of the conductance is presented, showing a voltage dependence that could be best fit by an equation for a single exponential. Results presented here are the first data from pigeon dissociated type I hair cells from utricle, saccule and lagena suggesting that the basolateral conductances of a morphologically identified population of type I hair cells are conserved between functionally different vestibular end organs; the major conductance being a delayed rectifier characterized previously in semicircular canal hair cells as IKI.

Effects of Cationic Substitutions on Delayed Rectifier Current in Type I Vestibular Hair Cells

Abstract. The resting potassium current (IKI) in gerbil dissociated type I vestibular hair cells has been characterized under various ionic conditions in whole cell voltage-clamp. When all K+ in the patch electrode solution was replaced with Na+, (Na+)in or Cs+, (Cs+)in, large inward currents were evoked in response to voltage steps between −90 and −50 mV. Activation of these currents could be described by a Hodgkin-Huxley-type kinetic scheme, the order of best fit increasing with depolarization. Above ∼−40 mV currents became outward and inactivated with a monoexponential time course. Membrane resistance was inversely correlated with external K+ concentration. With (Na+)in, currents were eliminated when K+ was removed from the external solution or following extracellular perfusion of 4-aminopyridine, indicating that currents flowed through IKI channels. Also, reduction of K+ entry through manipulation of membrane potential reduced the magnitude of the outward current. Under symmetrical Cs+, 0 K+ conditions IKI is highly permeable to Cs+. However, inward currents were reduced when small amounts of external K+ were added. Higher concentrations of K+ resulted in larger currents indicating an anomalous mole fraction effect in mixtures of external Cs+ and K+.

Development of K(+) and Na(+) conductances in rodent postnatal semicircular canal type I hair cells.

The rodent vestibular system is immature at birth. During the first postnatal week, vestibular type I and type II hair cells start to acquire their characteristic morphology and afferent innervation. We have studied postnatal changes in the membrane properties of type I hair cells acutely isolated from the semicircular canals (SCC) of gerbils and rats using whole cell patch clamp and report for the first time developmental changes in ionic conductances in these cells. At postnatal day (P) 5 immature hair cells expressed a delayed rectifier K(+) conductance (G(DR)) which activated at potentials above approximately -50 mV in both species. Hair cells also expressed a transient Na(+) conductance (G(Na)) with a mean half-inactivation of approximately -90 mV. At P6 in rat and P7 in gerbil, a low-voltage activated K(+) conductance (G(K,L)) was first observed and conferred a low-input resistance, typical of adult type I hair cells, on SCC type I hair cells. G(K,L) expression in hair cells increased markedly during the second postnatal week and was present in all rat type I hair cells by P14. In gerbil hair cells, G(K,L) appeared later and was present in all type I hair cells by P19. During the third postnatal week, G(Na) expression declined and was absent by the fourth postnatal week in rat and the sixth postnatal week in gerbils. Understanding the ionic changes associated with hair cell maturation could help elucidate development and regeneration mechanisms in the inner ear.

Return of Potassium Ion Channels in Regenerated Hair Cells

Abstract: Recent electrophysiological studies in pigeon have demonstrated that potassium channels are completely functional in regenerated type II hair cells at 21 days post‐treatment (PT) with ototoxic doses of streptomycin. The currents return in the order they appear during development. The mixture of ionic currents in a regenerated type II hair cell in a particular region of the neuroepithelium is the same as in its ancestor in that region. The return of currents in regenerated type I hair cells is more complicated. The dominant conductance gKI is not present until after 70 days PT. Before 70 days, the ionic currents in type I hair cells resemble those of regenerated type II hair cells, suggesting that the ionic currents in type II hair cells might be precursors of the ionic currents in regenerated type I hair cells. New data show that at one year PT, the kinetics and drug sensitivity of the dominant K+ conductance in type I hair cells are identical to gKI. Supporting cells, believed to be the precursors of regenerated type II hair cells, have effectively no voltage‐gated outward potassium channels, suggesting that regenerated type II hair cells must develop these channels de novo. The next step is to understand the mechanisms by which the potassium channel protein is synthesized, migrates through the cytosol, and is inserted into the plasmalemma of regenerating hair cells. These mechanisms are unknown. We propose that intracellular calcium is involved in this process, as well as in the differentiation, proliferation, and gene regulation of precursor cells fated to become hair cells.

Zonal variations in K+ currents in vestibular crista calyx terminals.

We developed a rodent crista slice to investigate regional variations in electrophysiological properties of vestibular afferent terminals. Thin transverse slices of the gerbil crista ampullaris were made and electrical properties of calyx terminals in central zones (CZ) and peripheral zones (PZ) compared with whole cell patch clamp. Spontaneous action potential firing was observed in 25% of current-clamp recordings and was either regular or irregular in both zones. Firing was abolished when extracellular choline replaced Na(+) but persisted when hair cell mechanotransduction channels or calyx AMPA receptors were blocked. This suggests that ion channels intrinsic to the calyx can generate spontaneous firing. In response to depolarizing voltage steps, outward K(+) currents were observed at potentials above -60 mV. K(+) currents in PZ calyces showed significantly more inactivation than currents in CZ calyces. Underlying K(+) channel populations contributing to these differences were investigated. The KCNQ channel blocker XE991 dihydrochloride blocked a slowly activating, sustained outward current in both PZ and CZ calyces, indicating the presence of KCNQ channels. Mean reduction was greatest in PZ calyces. XE991 also reduced action potential firing frequency in CZ and PZ calyces and broadened mean action potential width. The K(+) channel blocker 4-aminopyridine (10-50 μM) blocked rapidly activating, moderately inactivating currents that were more prevalent in PZ calyces. α-Dendrotoxin, a selective blocker of KV1 channels, reduced outward currents in CZ calyces but not in PZ calyces. Regional variations in K(+) conductances may contribute to different firing responses in calyx afferents.

Channeling your inner ear potassium: K(+) channels in vestibular hair cells.

During development of vestibular hair cells, K(+) conductances are acquired in a specific pattern. Functionally mature vestibular hair cells express different complements of K(+) channels which uniquely shape the hair cell receptor potential and filtering properties. In amniote species, type I hair cells (HCI) have a large input conductance due to a ubiquitous low-voltage-activated K(+) current that activates with slow sigmoidal kinetics at voltages negative to the membrane resting potential. In contrast type II hair cells (HCII) from mammalian and non-mammalian species have voltage-dependent outward K(+) currents that activate rapidly at or above the resting membrane potential and show significant inactivation. A-type, delayed rectifier and calcium-activated K(+) channels contribute to the outward K(+) conductance and are present in varying proportions in HCII. In many species, K(+) currents in HCII in peripheral locations of vestibular epithelia inactivate more than HCII in more central locations. Two types of inward rectifier currents have been described in both HCI and HCII. A rapidly activating K(+)-selective inward rectifier current (IK1, mediated by Kir2.1 channels) predominates in HCII in peripheral zones, whereas a slower mixed cation inward rectifier current (Ih), shows greater expression in HCII in central zones of vestibular epithelia. The implications for sensory coding of vestibular signals by different types of hair cells are discussed. This article is part of a Special Issue entitled .

Kv1 channels and neural processing in vestibular calyx afferents.

Potassium-selective ion channels are important for accurate transmission of signals from auditory and vestibular sensory end organs to their targets in the central nervous system. During different gravity conditions, astronauts experience altered input signals from the peripheral vestibular system resulting in sensorimotor dysfunction. Adaptation to altered sensory input occurs, but it is not explicitly known whether this involves synaptic modifications within the vestibular epithelia. Future investigations of such potential plasticity require a better understanding of the electrophysiological mechanisms underlying the known heterogeneity of afferent discharge under normal conditions. This study advances this understanding by examining the role of the Kv1 potassium channel family in mediating action potentials in specialized vestibular afferent calyx endings in the gerbil crista and utricle. Pharmacological agents selective for different sub-types of Kv1 channels were tested on membrane responses in whole cell recordings in the crista. Kv1 channels sensitive to α-dendrotoxin and dendrotoxin-K were found to prevail in the central regions, whereas K+ channels sensitive to margatoxin, which blocks Kv1.3 and 1.6 channels, were more prominent in peripheral regions. Margatoxin-sensitive currents showed voltage-dependent inactivation. Dendrotoxin-sensitive currents showed no inactivation and dampened excitability in calyces in central neuroepithelial regions. The differential distribution of Kv1 potassium channels in vestibular afferents supports their importance in accurately relaying gravitational and head movement signals through specialized lines to the central nervous system. Pharmacological modulation of specific groups of K+ channels could help alleviate vestibular dysfunction on earth and in space.

Inhibition of K+ Currents in Type I Vestibular Hair Cells by Gentamicin and Neomycin

Significant ototoxicity limits the use of aminoglycoside (AG) antibiotics. Several mechanisms may contribute to the death of both auditory and vestibular hair cells. In this study the effects of gentamicin and neomycin on K⁺ currents in mature and early postnatal type I vestibular hair cells (HCI) were tested directly. The whole-cell patch clamp technique was used to assess the effects of AG and KCNQ channel modulators on K⁺ currents (I_K) in HCI acutely isolated from gerbil semicircular canals. Extracellular neomycin (1 m<smlcap>M</smlcap>) rapidly reduced peak outward I_K by 16 ± 4% (n = 9) in mature HCI (postnatal days, P, 25-66). Gentamicin (5 m<smlcap>M</smlcap>) reduced outward I_K by 16 ± 3% (n = 8). A similar reduction in outward current was seen in immature HCI (P5-9) that lacked the low-voltage-activated component of I_K observed in mature cells. Intracellular application of gentamicin and neomycin also reduced I_K in mature HCI. Modulators of KCNQ channels were used to probe KCNQ channel involvement. The selective KCNQ antagonist XE991 did not reduce I_K and the neomycin-induced reduction in I_K was not reversed by the KCNQ agonist flupirtine. Application of intracellular poly-<smlcap>D</smlcap>-lysine to sequester PIP₂ did not reduce I_K. Application of the K⁺ channel blocker 4-aminopyridine (4-AP) strongly reduced I_K, and extracellular AG in the presence of 4-AP gave no further inhibition of I_K. In summary, AG significantly reduce the 4-AP-sensitive I_K in early postnatal and mature HCI. K⁺ current inhibition differs from that seen in outer hair cells, since it does not appear to involve PIP₂ sequestration or KCNQ channels.

AMPA receptor-mediated rapid EPSCs in vestibular calyx afferents

In the vestibular periphery neurotransmission between hair cells and primary afferent nerves occurs via specialized ribbon synapses. Type I vestibular hair cells (HCIs) make synaptic contacts with calyx terminals, which enclose most of the HCI basolateral surface. To probe synaptic transmission, whole cell patch-clamp recordings were made from calyx afferent terminals isolated together with their mature HCIs from gerbil crista. Neurotransmitter release was measured as excitatory postsynaptic currents (EPSCs) in voltage clamp. Spontaneous EPSCs were classified as simple or complex. Simple events exhibited a rapid rise time and a fast monoexponential decay (time constant < 1 ms). The remaining events, constituting ~40% of EPSCs, showed more complex characteristics. Extracellular Sr2+ greatly increased EPSC frequency, and EPSCs were blocked by the AMPA receptor blocker NBQX. The role of presynaptic Ca2+ channels was assessed by application of the L-type Ca2+ channel blocker nifedipine (20 µM), which reduced EPSC frequency. In contrast, the L-type Ca2+ channel opener BAY K 8644 increased EPSC frequency. Cyclothiazide increased the decay time constant of averaged simple EPSCs by approximately twofold. The low-affinity AMPA receptor antagonist γ-d-glutamylglycine (2 mM) reduced the proportion of simple EPSCs relative to complex events, indicating glutamate accumulation in the restricted cleft between HCI and calyx. In crista slices EPSC frequency was greater in central compared with peripheral calyces, which may be due to greater numbers of presynaptic ribbons in central hair cells. Our data support a role for L-type Ca2+ channels in spontaneous release and demonstrate regional variations in AMPA-mediated quantal transmission at the calyx synapse.NEW & NOTEWORTHY In vestibular calyx terminals of mature cristae we find that the majority of excitatory postsynaptic currents (EPSCs) are rapid monophasic events mediated by AMPA receptors. Spontaneous EPSCs are reduced by an L-type Ca2+ channel blocker and notably enhanced in extracellular Sr2+ EPSC frequency is greater in central areas of the crista compared with peripheral areas and may be associated with more numerous presynaptic ribbons in central hair cells.

Modeling Ion Channel Kinetics with HPC

Performance improvements for computational sciences such as biology, physics, and chemistry are critically dependent on advances in multicore and manycore hardware. However, these emerging systems require substantial investment in software development time to migrate, optimize, and validate existing science models. The focus of our study is to examine the step???by???step process of adapting new and existing computational biology models to multicore and distributed memory architectures. We analyze different strategies that may be more efficient in multicore vs. manycore environments. Our target application, Kingen, was developed to simulate AMPAR ion channel activity and to optimize kinetic model rate constants to biological data. Kingen uses a genetic algorithm to stochastically search parameter space to find global optima. As each individual in the population describes a rate constant parameter set in the kinetic model and the model is evaluated for each individual, there is significant computational complexity and parallelism in even a simple model run.