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G protein-coupled inward rectifiers (GIRKs) are activated directly by G protein [beta][gamma], 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 [beta][gamma] 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., lannaccone, S. T., Brunt, E., Barohn, R., etal. (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.

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 CaV1.3 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. 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 KCa1.1 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. 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 CaV1.3 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 FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Examines the role of glutamate residue of G protein-coupled inward rectifiers (GIRK) in the regulation of potassium channels. Localization of GIRK2 in the domain of C terminus; Mechanisms of GIRK and inward rectifiers trafficking to cell membranes; Interaction between GIRK2 and G protein ΒΓ.

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., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)