Static length changes of cochlear outer hair cells can tune low-frequency hearing.

The cochlea not only transduces sound-induced vibration into neural spikes, it also amplifies weak sound to boost its detection. Actuators of this active process are sensory outer hair cells in the organ of Corti, whereas the inner hair cells transduce the resulting motion into electric signals that propagate via the auditory nerve to the brain. However, how the outer hair cells modulate the stimulus to the inner hair cells remains unclear. Here, we combine theoretical modeling and experimental measurements near the cochlear apex to study the way in which length changes of the outer hair cells deform the organ of Corti. We develop a geometry-based kinematic model of the apical organ of Corti that reproduces salient, yet counter-intuitive features of the organ's motion. Our analysis further uncovers a mechanism by which a static length change of the outer hair cells can sensitively tune the signal transmitted to the sensory inner hair cells. When the outer hair cells are in an elongated state, stimulation of inner hair cells is largely inhibited, whereas outer hair cell contraction leads to a substantial enhancement of sound-evoked motion near the hair bundles. This novel mechanism for regulating the sensitivity of the hearing organ applies to the low frequencies that are most important for the perception of speech and music. We suggest that the proposed mechanism might underlie frequency discrimination at low auditory frequencies, as well as our ability to selectively attend auditory signals in noisy surroundings.

Voltage- and ATP-dependent structural rearrangements of the P2X2 receptor associated with the gating of the pore.

P2X2 is an extracellular ATP-gated cation channel which has a voltage-dependent gating property even though it lacks a canonical voltage sensor. It is a trimer in which each subunit has two transmembrane helices and a large extracellular domain. The three inter-subunit ATP binding sites are linked to the pore forming transmembrane (TM) domains by ß-strands. We analysed structural rearrangements of the linker strands between the ATP binding site and TM domains upon ligand binding and voltage change, electrophysiologically in Xenopus oocytes, using mutants carrying engineered thiol-modifiable cysteine residues. (1) We demonstrated that the double mutant D315C&I67C (at ß-14 and ß-1, respectively) shows a 2- to 4-fold increase in current amplitude after treatment with a reducing reagent, dithiothreitol (DTT). Application of the thiol-reactive metal Cd(2+) induced current decline due to bond formation between D315C and I67C. This effect was not observed in wild type (WT) or in single point mutants. (2) Cd(2+)-induced current decline was analysed in hyperpolarized and depolarized conditions with different pulse protocols, and also in the presence and absence of ATP. (3) Current decline induced by Cd(2+) could be clearly observed in the presence of ATP, but was not clear in the absence of ATP, showing a state-dependent modification. (4) In the presence of ATP, Cd(2+) modification was significantly faster in hyperpolarized than in depolarized conditions, showing voltage-dependent structural rearrangements of the linker strands. (5) Experiments using tandem trimeric constructs (TTCs) with controlled number and position of mutations in the trimer showed that the bridging by Cd(2+) between 315 and 67 was not intra- but inter-subunit. (6) Finally, we performed similar analyses of a pore mutant T339S, which makes the channel activation voltage insensitive. Cd(2+) modification rates of T339S were similar in hyperpolarized and depolarized conditions. Taking these results together, we demonstrated that structural rearrangements of the linker region of the P2X2 receptor channel are induced not only by ligand binding but also by membrane potential change.

Signal transmission within the P2X2 trimeric receptor.

P2X2 receptor channel, a homotrimer activated by the binding of extracellular adenosine triphosphate (ATP) to three intersubunit ATP-binding sites (each located ∼50 Å from the ion permeation pore), also shows voltage-dependent activation upon hyperpolarization. Here, we used tandem trimeric constructs (TTCs) harboring critical mutations at the ATP-binding, linker, and pore regions to investigate how the ATP activation signal is transmitted within the trimer and how signals generated by ATP and hyperpolarization converge. Analysis of voltage- and [ATP]-dependent gating in these TTCs showed that: (a) Voltage- and [ATP]-dependent gating of P2X2 requires binding of at least two ATP molecules. (b) D315A mutation in the ß-14 strand of the linker region connecting the ATP-binding domains to the pore-forming helices induces two different gating modes; this requires the presence of the D315A mutation in at least two subunits. (c) The T339S mutation in the pore domains of all three subunits abolishes the voltage dependence of P2X2 gating in saturating [ATP], making P2X2 equally active at all membrane potentials. Increasing the number of T339S mutations in the TTC results in gradual changes in the voltage dependence of gating from that of the wild-type channel, suggesting equal and independent contributions of the subunits at the pore level. (d) Voltage- and [ATP]-dependent gating in TTCs differs depending on the location of one D315A relative to one K308A that blocks the ATP binding and downstream signal transmission. (e) Voltage- and [ATP]-dependent gating does not depend on where one T339S is located relative to K308A (or D315A). Our results suggest that each intersubunit ATP-binding signal is directly transmitted on the same subunit to the level of D315 via the domain that contributes K308 to the ß-14 strand. The signal subsequently spreads equally to all three subunits at the level of the pore, resulting in symmetric and independent contributions of the three subunits to pore opening.

[Structual and functional dynamics of the ATP receptor channel P2X(2)].

ATP is known to function as a neurotransmitter. There are 2 major families of ATP receptors-the ion channel-type P2X receptors and metabotropic P2Y receptors. P2X receptors are known to possess unique properties of pore dilation that depends on the time lapse after ATP application; further, they exert their functions by directly interacting with nicotinic ACh receptors. These properties suggest the flexibility of the pore formed by these receptors. We studied the biophysical properties of P2X(2) receptor by using in vitro expression systems and focused on various dynamic regulations and structural rearrangements. Firstly, the pore property clearly depended on the expression levels of the P2X(2) receptors on the membrane. When the expression level was high, inward rectification was weak, and pore dilation was clearly observed. We also clarified that the key feature of the pore property is not the number of channels expressed but the number of open channels on the membrane. Secondly, we focused on the regulation of these channels by phosphoinositides (PIP(ns)). PIP(ns) are known to regulate the activity of various ion channels, but in the case of P2X(2), we observed that PIP(ns) regulate not only the activity but also the processes of pore dilation and desensitization. The binding of PIP(ns) to P2X(2) in the pore dilated state was observed to be less stable. Application of a reagent, which decreases the levels of PIP(ns), and mutation of the binding site facilitated desensitization of P2X(2) in the pore dilated state. Thirdly, we analyzed the voltage-dependent gating of these channels. Although P2X(2) lacks a canonical voltage-sensor domain, it undergoes voltage-dependent activation upon hyperpolarization. Further, we observed that the voltage-dependent gating depends on ATP concentration; conductance-voltage relationship curve shifted toward depolarization potential with increase in ATP concentration. We found that the ATP-binding site and the extracellular sideus of the transmembrane region were critical for the voltage-dependent gating. These results show that P2X(2) channel pore is exceptionally flexible, and that the channel activity is dynamically regulated by various factors, including not only ATP but also PIP(ns) and membrane potential.

Functional and structural identification of amino acid residues of the P2X2 receptor channel critical for the voltage- and [ATP]-dependent gating.

The extracellular ATP-gated cation channel P2X(2) is known to show voltage-dependent gating in spite of the absence of a canonical voltage sensor domain. We previously observed that the hyperpolarization-evoked activation of P2X(2) at the steady state in the presence of ATP varied depending on [ATP]. With increasing [ATP], the conductance-voltage (G-V) relationship shifted to more depolarized potentials and the activation kinetics were accelerated. Using a three-state model consisting of an ATP binding step and a rate limiting gating step, we successfully reproduced the voltage-dependent gating including its [ATP] dependence. In this study, in order to identify the structural basis of voltage and ATP dependence, we analysed various mutants in the Xenopus oocyte expression system under two-electrode voltage clamp. In the ATP binding region mutant K308R, the G-V relationship was shifted towards more hyperpolarized potentials, there was no clear [ATP] dependence, and activation was faster than in wild-type (WT). These results could be simulated by assuming an increase in the off rate of the gating step, in addition to changes in the ATP binding step. With F44C mutation in the 1st transmembrane (TM) region (TM1) or T339S in TM2, activation in low [ATP] was slow and the channel was constitutively active at all membrane potentials in high [ATP]. These results could be simulated by reducing the off rate of the gating step. Phenotypes of the double mutants, K308R/F44C and K308R/T339S, were similar to WT, suggesting that TM and ATP binding region mutants offset the effect of each other. Analysis of the effects on WT of two other agonists, ADP and AP(4)A, revealed that the electrostatic charge is not the sole critical factor. Taking these results together with the recently reported crystal structure, it was suggested that upon binding of ATP, the occupied binding site indirectly interacts with the extracellular end of the TM regions to trigger conformational changes for gating in a voltage-dependent manner.

Dynamic aspects of functional regulation of the ATP receptor channel P2X2.

The P2X(2) channel is a ligand-gated channel activated by ATP. Functional features that reflect the dynamic flexibility of the channel include time-dependent pore dilatation following ATP application and direct inhibitory interaction with activated nicotinic acetylcholine receptors on the membrane. We have been studying the mechanisms by which P2X(2) channel functionality is dynamically regulated. Using a Xenopus oocyte expression system, we observed that the pore properties, including ion selectivity and rectification, depend on the open channel density on the membrane. Pore dilatation was apparent when the open channel density was high and inward rectification was modest. We also observed that P2X(2) channels show voltage dependence, despite the absence of a canonical voltage sensor. At a semi-steady state after ATP application, P2X(2) channels were activated upon membrane hyperpolarization. This voltage-dependent activation was also [ATP] dependent. With increases in [ATP], the speed of hyperpolarization-induced activation was increased and the conductance-voltage relationship was shifted towards depolarized potentials. Based on analyses of experimental data and various simulations, we propose that these phenomena can be explained by assuming a fast ATP binding step and a rate-limiting voltage-dependent gating step. Complete elucidation of these regulatory mechanisms awaits dynamic imaging of functioning P2X(2) channels.

Voltage- and [ATP]-dependent gating of the P2X(2) ATP receptor channel.

P2X receptors are ligand-gated cation channels activated by extracellular adenosine triphosphate (ATP). Nonetheless, P2X(2) channel currents observed during the steady-state after ATP application are known to exhibit voltage dependence; there is a gradual increase in the inward current upon hyperpolarization. We used a Xenopus oocyte expression system and two-electrode voltage clamp to analyze this "activation" phase quantitatively. We characterized the conductance-voltage relationship in the presence of various [ATP], and observed that it shifted toward more depolarized potentials with increases in [ATP]. By analyzing the rate constants for the channel's transition between a closed and an open state, we showed that the gating of P2X(2) is determined in a complex way that involves both membrane voltage and ATP binding. The activation phase was similarly recorded in HEK293 cells expressing P2X(2) even by inside-out patch clamp after intensive perfusion, excluding a possibility that the gating is due to block/unblock by endogenous blocker(s) of oocytes. We investigated its structural basis by substituting a glycine residue (G344) in the second transmembrane (TM) helix, which may provide a kink that could mediate "gating." We found that, instead of a gradual increase, the inward current through the G344A mutant increased instantaneously upon hyperpolarization, whereas a G344P mutant retained an activation phase that was slower than the wild type (WT). Using glycine-scanning mutagenesis in the background of G344A, we could recover the activation phase by introducing a glycine residue into the middle of second TM. These results demonstrate that the flexibility of G344 contributes to the voltage-dependent gating. Finally, we assumed a three-state model consisting of a fast ATP-binding step and a following gating step and estimated the rate constants for the latter in P2X(2)-WT. We then executed simulation analyses using the calculated rate constants and successfully reproduced the results observed experimentally, voltage-dependent activation that is accelerated by increases in [ATP].