Gating Spring Research Articles

Nonlinear compliance of NompC gating spring and its implication in mechanotransduction.

Cytoskeleton-tethered mechanosensitive channels (MSCs) utilize compliant proteins or protein domains called gating springs to convert mechanical stimuli into electric signals, enabling sound and touch sensation and proprioception. The mechanical properties of these gating springs, however, remain elusive. Here, we explored the mechanical properties of the homotetrameric NompC complex containing long ankyrin-repeat domains (ARDs). We developed a toehold-mediated strand displacement approach to tether single membrane proteins, allowing us to exert force on them and precisely measure their absolute extension using optical tweezers. Our findings revealed that each ARD has a low stiffness of ~0.7 pN/nm and begins to unfold stepwise at ~7 pN, leading to nonlinear compliance. Our calculations indicate that this nonlinear compliance may help regulate NompC's sensitivity, dynamic range, and kinetics to detect mechanical stimuli. Overall, our research highlights the importance of a compliant and unfolding-refolding gating spring in facilitating a graded response of MSC ion transduction across a wide spectrum of mechanical stimuli.

LHFPL5 is a key element in force transmission from the tip link to the hair cell mechanotransducer channel.

During auditory transduction, sound-evoked vibrations of the hair cell stereociliary bundles open mechanotransducer (MET) ion channels via tip links extending from one stereocilium to its neighbor. How tension in the tip link is delivered to the channel is not fully understood. The MET channel comprises a pore-forming subunit, transmembrane channel-like protein (TMC1 or TMC2), aided by several accessory proteins, including LHFPL5 (lipoma HMGIC fusion partner-like 5). We investigated the role of LHFPL5 in transduction by comparing MET channel activation in outer hair cells of Lhfpl5-/- knockout mice with those in Lhfpl5+/- heterozygotes. The 10 to 90 percent working range of transduction in Tmc1+/+; Lhfpl5+/- was 52 nm, from which the single-channel gating force, Z, was evaluated as 0.34 pN. However, in Tmc1+/+; Lhfpl5-/- mice, the working range increased to 123 nm and Z more than halved to 0.13 pN, indicating reduced sensitivity. Tip link tension is thought to activate the channel via a gating spring, whose stiffness is inferred from the stiffness change on tip link destruction. The gating stiffness was ~40 percent of the total bundle stiffness in wild type but was virtually abolished in Lhfpl5-/-, implicating LHFPL5 as a principal component of the gating spring. The mutation Tmc1 p.D569N reduced the LHFPL5 immunolabeling in the stereocilia and like Lhfpl5-/- doubled the MET working range, but other deafness mutations had no effect on the dynamic range. We conclude that tip-link tension is transmitted to the channel primarily via LHFPL5; residual activation without LHFPL5 may occur by direct interaction between PCDH15 and TMC1.

Nonlinear compliance of NOMPC gating spring and its implication in mechanotransduction.

NOMPC is a type of mechanosensitive ion channel (MSCs) that are tethered to cytoskeleton through gating springs and gated by forces applied to the cell, thereby mediating processes including hearing, touch sensation, and proprioception. The mechanical properties of the gating spring play crucial roles in force transmission and channel opening and adaptation, but have been poorly characterized so far. Here, we measured the stiffness and unfolding of ankyrin-repeat domains (ARDs), the gating spring in the tetrameric NOMPC complex, by pulling single or multiple ARDs using high-resolution optical tweezers. We found that each ARD has a low stiffness of ∼0.7 pN/nm and starts to unfold stepwise at ∼7 pN. Four ARDs show cooperativity in their mechanical properties through interactions at their lower contact sites. This find is further supported by cryo-EM structures of NOMPC containing full-length and truncated ARDs. Overall, our work demonstrates a compliant and unfolding-refolding gating spring essential for the function of NOMPC and likely other MSCs.

CAMP and voltage modulate rat auditory mechanotransduction by decreasing the stiffness of gating springs

Hair cells of the auditory and vestibular systems transform mechanical input into electrical potentials through the mechanoelectrical transduction process (MET). Deflection of the mechanosensory hair bundle increases tension in the gating springs that open MET channels. Regulation of MET channel sensitivity contributes to the auditory system's precision, wide dynamic range and, potentially, protection from overexcitation. Modulating the stiffness of the gating spring modulates the sensitivity of the MET process. Here, we investigated the role of cyclic adenosine monophosphate (cAMP) in rat outer hair cell MET and found that cAMP up-regulation lowers the sensitivity of the channel in a manner consistent with decreasing gating spring stiffness. Direct measurements of the mechanical properties of the hair bundle confirmed a decrease in gating spring stiffness with cAMP up-regulation. In parallel, we found that prolonged depolarization mirrored the effects of cAMP. Finally, a limited number of experiments implicate that cAMP activates the exchange protein directly activated by cAMP to mediate the changes in MET sensitivity. These results reveal that cAMP signaling modulates gating spring stiffness to affect auditory sensitivity.

Force From Filaments: The Role of the Cytoskeleton and Extracellular Matrix in the Gating of Mechanosensitive Channels.

The senses of proprioception, touch, hearing, and blood pressure on mechanosensitive ion channels that transduce mechanical stimuli with high sensitivity and speed. This conversion process is usually called mechanotransduction. From nematode MEC-4/10 to mammalian PIEZO1/2, mechanosensitive ion channels have evolved into several protein families that use variant gating models to convert different forms of mechanical force into electrical signals. In addition to the model of channel gating by stretching from lipid bilayers, another potent model is the opening of channels by force tethering: a membrane-bound channel is elastically tethered directly or indirectly between the cytoskeleton and the extracellular molecules, and the tethering molecules convey force to change the channel structure into an activation form. In general, the mechanical stimulation forces the extracellular structure to move relative to the cytoskeleton, deforming the most compliant component in the system that serves as a gating spring. Here we review recent studies focusing on the ion channel mechanically activated by a tethering force, the mechanotransduction-involved cytoskeletal protein, and the extracellular matrix. The mechanosensitive channel PIEZO2, DEG/ENaC family proteins such as acid-sensing ion channels, and transient receptor potential family members such as NompC are discussed. State-of-the-art techniques, such as polydimethylsiloxane indentation, the pillar array, and micropipette-guided ultrasound stimulation, which are beneficial tools for exploring the tether model, are also discussed.

The functional role of connectors in outer-hair-cell hair bundles

Our hearing relies on outer hair cells, which amplify sound-induced vibrations in our ears. Each hair cell has a hair bundle composed of stereocilia, “hair-like” structures protruding from the cell's apical surface. Two classes of links link stereocilia, gating springs and connectors, also known as top or shaft connectors, side, lateral, or ankle links. Gating springs link neighboring stereocilia of different heights, while connectors link all neighboring stereocilia. Sound-induced gating-spring oscillations open and close mechanoelectrical transduction channels attached to the gating springs, causing oscillations in the hair cell's sensory current.

Molecular Crux of Hair Cell Mechanotransduction Machinery

Although 62 years have elapsed since the first report of hereditary deafness in a mouse strain, the molecular mechanism of hair cell mechanotransduction remains elusive. Three recent studies present crucial insights into the molecular crux of hair cell mechanotransduction machinery.

Acoustic waves to brain signals: Identifying the gating spring

Acoustic waves to brain signals: Identifying the gating spring

Elasticity of individual protocadherin 15 molecules implicates tip links as the gating springs for hearing

Hair cells, the sensory receptors of the inner ear, respond to mechanical forces originating from sounds and accelerations. An essential feature of each hair cell is an array of filamentous tip links, consisting of the proteins protocadherin 15 (PCDH15) and cadherin 23 (CDH23), whose tension is thought to directly gate the cell's transduction channels. These links are considered far too stiff to represent the gating springs that convert hair bundle displacement into forces capable of opening the channels, and no mechanism has been suggested through which tip-link stiffness could be varied to accommodate hair cells of distinct frequency sensitivity in different receptor organs and animals. Consequently, the gating spring's identity and mechanism of operation remain central questions in sensory neuroscience. Using a high-precision optical trap, we show that an individual monomer of PCDH15 acts as an entropic spring that is much softer than its enthalpic stiffness alone would suggest. This low stiffness implies that the protein is a significant part of the gating spring that controls a hair cell's transduction channels. The tip link's entropic nature then allows for stiffness control through modulation of its tension. We find that a PCDH15 molecule is unstable under tension and exhibits a rich variety of reversible unfolding events that are augmented when the Ca2+ concentration is reduced to physiological levels. Therefore, tip link tension and Ca2+ concentration are likely parameters through which nature tunes a gating spring's mechanical properties.

Thermal Excitation of the Mechanotransduction Apparatus of Hair Cells

Although a hair bundle is normally deflected by mechanical stimuli, we found that irradiation of a hair cell from the bullfrog's sacculus with ultraviolet light causes rapid motion of the hair bundle toward its tall edge. This movement is associated with opening of mechanotransduction channels and disappears when tip links are disrupted. We localized the absorptive element responsible for the motion to the region directly below the hair bundle and measured an action spectrum similar to the absorption spectra of mitochondrial constituents. Temperature measurements revealed heating around the site of absorption; direct heating of the hair bundle confirmed that the response to light is mediated through heat. Although mechanical offsets of the hair bundle revealed that heat softens gating springs, it also acts directly to open transduction channels. This study identifies an unconventional method of hair-cell stimulation and clarifies the previously unexplained sensitivity of auditory organs to thermal stimulation.

Underestimated Sensitivity of Mammalian Cochlear Hair Cells Due to Splay between Stereociliary Columns

Current-displacement (I-X) and the force-displacement (F-X) relationships characterize hair-cell mechano-transduction in the inner ear. A common technique for measuring these relationships is to deliver mechanical stimulations to individual hair bundles with microprobes and measure whole cell transduction currents through patch pipette electrodes at the basolateral membrane. The sensitivity of hair-cell mechano-transduction is determined by two fundamental biophysical properties of the mechano-transduction channel, the stiffness of the putative gating spring and the gating swing, which are derived from the I-X and F-X relationships. Although the hair-cell stereocilia in vivo deflect <100 nm even at high sound pressure levels, often it takes >500 nm of stereocilia displacement to saturate hair-cell mechano-transduction in experiments with individual hair cells in vitro. Despite such discrepancy between in vivo and in vitro data, key biophysical properties of hair-cell mechano-transduction to define the transduction sensitivity have been estimated from in vitro experiments. Using three-dimensional finite-element methods, we modeled an inner hair-cell and an outer hair-cell stereocilia bundle and simulated the effect of probe stimulation. Unlike the natural situation where the tectorial membrane stimulates hair-cell stereocilia evenly, probes deflect stereocilia unevenly. Because of uneven stimulation, 1) the operating range (the 10–90% width of the I-X relationship) increases by a factor of 2–8 depending on probe shapes, 2) the I-X relationship changes from a symmetric to an asymmetric function, and 3) the bundle stiffness is underestimated. Our results indicate that the generally accepted assumption of parallel stimulation leads to an overestimation of the gating swing and underestimation of the gating spring stiffness by an order of magnitude.

The Rise and Fall of TRP-N, an Ancient Family of Mechanogated Ion Channels, in Metazoa.

Mechanoreception, the sensing of mechanical forces, is an ancient means of orientation and communication and tightly linked to the evolution of motile animals. In flies, the transient-receptor-potential N protein (TRP-N) was found to be a cilia-associated mechanoreceptor. TRP-N belongs to a large and diverse family of ion channels. Its unusually long N-terminal repeat of 28 ankyrin domains presumably acts as the gating spring by which mechanical energy induces channel gating. We analyzed the evolutionary origins and possible diversification of TRP-N. Using a custom-made set of highly discriminative sequence profiles we scanned a representative set of metazoan genomes and subsequently corrected several gene models. We find that, contrary to other ion channel families, TRP-N is remarkably conserved in its domain arrangements and copy number (1) in all Bilateria except for amniotes, even in the wake of several whole-genome duplications. TRP-N is absent in Porifera but present in Ctenophora and Placozoa. Exceptional multiplications of TRP-N occurred in Cnidaria, independently along the Hydra and the Nematostella lineage. Molecular signals of subfunctionalization can be attributed to different mechanisms of activation of the gating spring. In Hydra this is further supported by in situ hybridization and immune staining, suggesting that at least three paralogs adapted to nematocyte discharge, which is key for predation and defense. We propose that these new candidate proteins help explain the sensory complexity of Cnidaria which has been previously observed but so far has lacked a molecular underpinning. Also, the ancient appearance of TRP-N supports a common origin of important components of the nervous systems in Ctenophores, Cnidaria, and Bilateria.

Utrophin suppresses low frequency oscillations and coupled gating of mechanosensitive ion channels in dystrophic skeletal muscle

An absence of utrophin in muscle from mdx mice prolongs the open time of single mechanosensitive channels. On a time scale much longer than the duration of individual channel activations, genetic depletion of utrophin produces low frequency oscillations of channel open probability. Oscillatory channel opening occurred in the dystrophin/utrophin mutants, but was absent in wild-type and mdx fibers. By contrast, small conductance channels showed random gating behavior when present in the same patch. Applying a negative pressure to a patch on a DKO fiber produced a burst of mode II activity, but channels subsequently closed and remained silent for tens of seconds during the maintained pressure stimulus. In addition, simultaneous opening of multiple MS channels could be frequently observed in recordings from patches on DKO fibers, but only rarely in wild-type and mdx muscle. A model which accounts for the single-channel data is proposed in which utrophin acts as gating spring which maintains the mechanical stability a caveolar-like compartment. The state of this compartment is suggested to be dynamic; its continuity with the extracellular surface varying over seconds to minutes. Loss of the mechanical stability of this compartment contributes to pathogenic Ca2+ entry through MS channels in Duchenne dystrophy.

Mechanism of a Hair Cell Bioinspired Sensor with Ultrasensitivity to Weak and Low Frequency Vibration Signals

Although significant progresses have been made in sensor technology, it is still a challenging task to develop ultrasensitive sensors to monitor very weak and low frequency vibration signal for early warning of natural disasters and efficient structural health monitoring of infrastructures. It has been reported from previous experiments that some fishes have acute sensitivities to extremely low frequency linear acceleration due to the otolith organs of the inner ear. In this paper, based on the experimental results and qualitative mechanism of the infrasound sensitivity of some fishes conducted by other researchers, a bioinspired gating spring model with negative stiffness is established to simulate the mechanical-electricity transduction of the hair cell in fish ear. Then, numerical analyses of the mechanical model subject to static and dynamic loading are conducted, respectively. It is shown that the gating model has adaptive amplification capability to weak and low frequency excitation compared with the corresponding linear model. This mechanism can be used for the design of bioinspired ultrasensitive sensors for monitoring weak and low frequency vibration signal.

Mechanosensation: Tethered Ion Channels

Two new studies show that the Drosophila transient receptor potential (TRP) family member NOMPC forms both a mechanically gated ion channel and a fine filament that, by tethering the protein to microtubules, might act as a gating spring.

The elusive hair cell gating spring, a potential role for the lipid membrane

Deflection of auditory hair cell hair bundle results in a nonlinear (i.e., non Hookean) force-displacement relationships whose molecular mechanism remains elusive. A gating spring model posits that mechanosensitive channels are in series with a spring such that channel opening puts the activation gate in series with the spring, thus reducing spring extension until further stimulation is provided. Here we present a theoretical analysis of whether the lipid membrane might be the source of nonlinearity. A hair bundle kinematic model is coupled with a lipid membrane model that includes a diffusible compartment into which the tip-link embeds and a minimally diffusive reservoir pool. Using physiological parameters, this model was capable of reproducing nonlinear force-displacement plots, including a negative stiffness component but required a standing tip-link tension. In addition, this model suggests the mechanotransducer channel is most sensitive to curvature forces that are located within 2 nm of the tip-link. [Work supported in part by Grant Nos. R01-DC07910 and R01-DC03896 from the NIDCD of NIH and by The Timoshenko fund from Mechanical Engineering Department at Stanford University].

A NOMPC-Dependent Membrane-Microtubule Connector Is a Candidate for the Gating Spring in Fly Mechanoreceptors

Mechanoreceptors contain compliant elements, termed "gating springs," that transfer macroscopic stimuli impinging on the cells into microscopic stimuli that open the mechanosensitive channels. Evidence for gating springs comes from mechanical experiments; they have not been identified molecularly or ultrastructurally. We show that the filamentous structures that connect the plasma membrane to the microtubules are compliant structural elements in the mechanoreceptive organelle of fly campaniform receptors. These filaments colocalize with the ankyrin-repeat domain of the transient receptor potential (TRP) channel NOMPC. In addition, they resemble the purified ankyrin-repeat domain in size and shape. Most importantly, these filaments are nearly absent in nompC mutants and can be rescued by the nompC gene. Finally, mechanical modeling suggests that the filaments provide most of the compliance in the distal tip of the cell, thought to be the site of mechanotransduction. Our results provide strong evidence that the ankyrin-repeat domains of NOMPC structurally contribute to the membrane-microtubule connecting filaments. These filaments, as the most compliant element in the distal tip, are therefore good candidates for the gating springs.

Mechanism of Bio-Inspired Ultrasensitive Low Frequency Sensor with Mechanics Analysis

It is essential to develop ultrasensitive low frequency sensors for efficient structural health monitoring and early warning of natural disasters. Many fishes have been reported to have acute sensitivity to low frequency. Based on the mechanism of the infrasound sensitivity of fish, mechanism of bio-inspired ultrasensitive low frequency sensor is explored by a mechanical model with gating spring hypothesis for simulating the mechanical-electricity transduction of the hair cell in fish ear. Numerical analyses of the mechanical model subject to static and dynamic loading are conducted respectively by OpenSees. Under static loading, displacement response of gating model is more sensitive to weak loading due to the opening of gating spring. Under dynamic loading, the gating model is more acute sensitive to low frequency and weak loading due to the adaptive amplification of gating spring. This mechanical function can be used as the theoretical basis for the design of ultrasensitive bio-inspired low frequency sensors.

A mechanical model of stereocilia that demonstrates a shift in the high-sensitivity region due to the interplay of a negative stiffness and an adaptation mechanism

Stereocilia are the basic sensory units of nature's inertial sensors and are highly sensitive over broad dynamic ranges, which is a major challenge in the design of conventional engineering sensors. The high sensitivity that is maintained by stereocilia was hypothesized to exist due to a combination of adaptation and negative stiffness mechanisms, which shift the region of highest sensitivity toward the active operation range of the stereocilia bundle. To examine the adaptation hypothesis in terms of its potential applicability to future applications regarding the design of inertial sensors, we developed a mechanical mimicry of the interplay between negative stiffness and the adaptation of the stereocilia that produces spontaneous oscillation of the hair bundle. The mechanical model consists of an inverted pendulum and a fixed T-bar that mimic the interaction of two adjacent stereocilia. To focus on the interaction of one gating spring and the corresponding adaptation motor without the effect of coupling from the other gating springs attached to the neighboring stereocilia, we fixed one bar that contains the adaptation motor. To emulate the negative resistance of the tip-link due to the transient stiffness softening by the gating ion channel, a magnet pair was attached to the top of the inverted pendulum and the fixed T-bar. Readjustment of the tip-link tension by the ‘slipping down and climbing up’ motion of the adaptation molecular motors was demonstrated by the side-to-side movement of the magnet by a step motor. The negative stiffness region was observed near the equilibrium position and shifted with the activation of the adaptation motor. The temporal demonstration of the stiffness shift was measured as a spontaneous oscillation. The results showed that the interplay between the negative stiffness and the adaptation mechanism was mechanically produced by the combination of a repulsive force and its continuous readjustment and is better understood through a parameter study of a biomimetic mechanical system.

Direct gating and mechanical integrity of Drosophila auditory transducers require TRPN1

The elusive transduction channels for hearing are directly gated mechanically by the pull of gating springs. We found that the transient receptor potential (TRP) channel TRPN1 (NOMPC) is essential for this direct gating of Drosophila auditory transduction channels and that the channel-spring complex was disrupted if TRPN1 was lost. Our results identify TRPN1 as a mechanical constituent of the fly's auditory transduction complex that may act as the channel and/or gating spring.