Simultaneous Dual Recordings From Vestibular Hair Cells and Their Calyx Afferents Demonstrate Multiple Modes of Transmission at These Specialized Endings.

In the vestibular periphery, transmission via conventional synaptic boutons is supplemented by post-synaptic calyceal endings surrounding Type I hair cells. This review focusses on the multiple modes of communication between these receptors and their enveloping calyces as revealed by simultaneous dual-electrode recordings. Classic orthodromic transmission is accompanied by two forms of bidirectional communication enabled by the extensive cleft between the Type I hair cell and its calyx. The slowest cellular communication low-pass filters the transduction current with a time constant of 10-100 ms: potassium ions accumulate in the synaptic cleft, depolarizing both the hair cell and afferent to potentials greater than necessary for rapid vesicle fusion in the receptor and potentially triggering action potentials in the afferent. On the millisecond timescale, conventional glutamatergic quantal transmission occurs when hair cells are depolarized to potentials sufficient for calcium influx and vesicle fusion. Depolarization also permits a third form of transmission that occurs over tens of microseconds, resulting from the large voltage- and ion-sensitive cleft-facing conductances in both the hair cell and the calyx that are open at their resting potentials. Current flowing out of either the hair cell or the afferent divides into the fraction flowing across the cleft into its cellular partner, and the remainder flowing out of the cleft and into the surrounding fluid compartment. These findings suggest multiple biophysical bases for the extensive repertoire of response dynamics seen in the population of primary vestibular afferent fibers. The results further suggest that evolutionary pressures drive selection for the calyx afferent.

Synaptic cleft microenvironment influences potassium permeation and synaptic transmission in hair cells surrounded by calyx afferents in the turtle.

KEY POINTS: In central regions of vestibular semicircular canal epithelia, the [K+ ] in the synaptic cleft ([K+ ]c ) contributes to setting the hair cell and afferent membrane potentials; the potassium efflux from type I hair cells results from the interdependent gating of three conductances. Elevation of [K+ ]c occurs through a calcium-activated potassium conductance, GBK , and a low-voltage-activating delayed rectifier, GK(LV) , that activates upon elevation of [K+ ]c . Calcium influx that enables quantal transmission also activates IBK , an effect that can be blocked internally by BAPTA, and externally by a CaV 1.3 antagonist or iberiotoxin. Elevation of [K+ ]c or chelation of [Ca2+ ]c linearizes the GK(LV) steady-state I-V curve, suggesting that the outward rectification observed for GK(LV) may result largely from a potassium-sensitive relief of Ca2+ inactivation of the channel pore selectivity filter. Potassium sensitivity of hair cell and afferent conductances allows three modes of transmission: quantal, ion accumulation and resistive coupling to be multiplexed across the synapse. ABSTRACT: In the vertebrate nervous system, ions accumulate in diffusion-limited synaptic clefts during ongoing activity. Such accumulation can be demonstrated at large appositions such as the hair cell-calyx afferent synapses present in central regions of the turtle vestibular semicircular canal epithelia. Type I hair cells influence discharge rates in their calyx afferents by modulating the potassium concentration in the synaptic cleft, [K+ ]c , which regulates potassium-sensitive conductances in both hair cell and afferent. Dual recordings from synaptic pairs have demonstrated that, despite a decreased driving force due to potassium accumulation, hair cell depolarization elicits sustained outward currents in the hair cell, and a maintained inward current in the afferent. We used kinetic and pharmacological dissection of the hair cell conductances to understand the interdependence of channel gating and permeation in the context of such restricted extracellular spaces. Hair cell depolarization leads to calcium influx and activation of a large calcium-activated potassium conductance, GBK , that can be blocked by agents that disrupt calcium influx or buffer the elevation of [Ca2+ ]i , as well as by the specific KCa 1.1 blocker iberiotoxin. Efflux of K+ through GBK can rapidly elevate [K+ ]c , which speeds the activation and slows the inactivation and deactivation of a second potassium conductance, GK(LV) . Elevation of [K+ ]c or chelation of [Ca2+ ]c linearizes the GK(LV) steady-state I-V curve, consistent with a K+ -dependent relief of Ca2+ inactivation of GK(LV) . As a result, this potassium-sensitive hair cell conductance pairs with the potassium-sensitive hyperpolarization-activated cyclic nucleotide-gated channel (HCN) conductance in the afferent and creates resistive coupling at the synaptic cleft.

Accumulation of K+ in the synaptic cleft modulates activity by influencing both vestibular hair cell and calyx afferent in the turtle.

KEY POINTS: In the synaptic cleft between type I hair cells and calyceal afferents, K+ ions accumulate as a function of activity, dynamically altering the driving force and permeation through ion channels facing the synaptic cleft. High-fidelity synaptic transmission is possible due to large conductances that minimize hair cell and afferent time constants in the presence of significant membrane capacitance. Elevated potassium maintains hair cells near a potential where transduction currents are sufficient to depolarize them to voltages necessary for calcium influx and synaptic vesicle fusion. Elevated potassium depolarizes the postsynaptic afferent by altering ion permeation through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, and contributes to depolarizing the afferent to potentials where a single EPSP (quantum) can generate an action potential. With increased stimulation, hair cell depolarization increases the frequency of quanta released, elevates [K+ ]cleft and depolarizes the afferent to potentials at which smaller and smaller EPSPs would be sufficient to trigger APs. ABSTRACT: Fast neurotransmitters act in conjunction with slower modulatory effectors that accumulate in restricted synaptic spaces found at giant synapses such as the calyceal endings in the auditory and vestibular systems. Here, we used dual patch-clamp recordings from turtle vestibular hair cells and their afferent neurons to show that potassium ions accumulating in the synaptic cleft modulated membrane potentials and extended the range of information transfer. High-fidelity synaptic transmission was possible due to large conductances that minimized hair cell and afferent time constants in the presence of significant membrane capacitance. Increased potassium concentration in the cleft maintained the hair cell near potentials that promoted the influx of calcium necessary for synaptic vesicle fusion. The elevated potassium concentration also depolarized the postsynaptic neuron by altering ion permeation through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. This depolarization enabled the afferent to reliably generate action potentials evoked by single AMPA-dependent EPSPs. Depolarization of the postsynaptic afferent could also elevate potassium in the synaptic cleft, and would depolarize other hair cells enveloped by the same neuritic process increasing the fidelity of neurotransmission at those synapses as well. Collectively, these data demonstrate that neuronal activity gives rise to potassium accumulation, and suggest that potassium ion action on HCN channels can modulate neurotransmission, preserving the fidelity of high-speed synaptic transmission by dynamically shifting the resting potentials of both presynaptic and postsynaptic cells.

Evaluation of hydrogen ion concentrations in prostates from rats and dogs using fluorescent confocal microscopy.

The knowledge of intracellular spatial distribution of pH in prostates in animal models reflective of human prostate may have implications for drug development upon pH dependent drug delivery and activity. Freshly dissected prostate tissues (in vitro) or the entire prostate gland (in vivo) were loaded with fluorescent dyes and viewed using confocal microscopy. Images were initially taken in tissues perfused with RPMI-1640 medium. Calibration in situ was performed with high potassium buffers of known pH containing nigericin. Acetoxymethyl ester carboxy-SNARF-1 was visible in epithelial cells (but not stroma) in rat and dog prostates. The pH of lysosomes in prostate epithelial cells was 5.2 as determined by fluorescence of Lyso Sensor Green DND-189. A method of in situ confirmation of tissue viability was developed by a secondary loading and visualization of the BCECF fluorescent dye. Besides the direct measurement of the pH in rat and dog tissues (pH approximately 7.0), a method of pH measurement in prostate tissue (rather than in cell culture) was developed.

A glutamate residue at the C terminus regulates activity of inward rectifier K+ channels: implication for Andersen's syndrome.

G protein-coupled inward rectifiers (GIRKs) are activated directly by G protein betagamma subunits, whereas classical inward rectifiers (IRKs) are constitutively active. We found that a glutamate residue of GIRK2 (E315), located on a hydrophobic domain of the C terminus, is crucial for the channel activation. This glutamate (or aspartate) residue is conserved in all members of the Kir family. Substitution of alanine for the glutamate on GIRK1, GIRK2, and IRK2, expressed in HEK293 cells, greatly reduced the whole-cell currents. The whole-cell current of GIRK channels with a constitutively active gate, GIRK2(V188A), [Yi, B. A., Lin, Y. F., Jan, Y. N. & Jan, L. Y. (2001) Neuron 29, 657-667] was also reduced by the same glutamate mutation. Mean open time and conductance of single channels in GIRK2 and IRK2 were not affected by the mutation, indicating that the reduced whole-cell current resulted from a lowered probability of channel activation. The mutated GIRK and IRK showed normal trafficking to the cell membrane. The mutated GIRK2 retained the ability to interact with G protein betagamma subunits, and it showed almost the same inwardly rectifying property as the wild type. The mutated GIRK1 and GIRK2 retained ion selectivity to K(+) ions. This glutamate residue corresponds to one of the residues causing Andersen's syndrome [Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canun, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., Iannaccone, S. T., Brunt, E., Barohn, R., et al. (2001) Cell 105, 511-519]. Our interpretation is that this region of the glutamate residue is crucial in relaying the activating message from the ligand sensor region to the gate.