Have we found the tip link,transduction channel,and gating spring of the hair cell?

Recent reports have offered candidates for key components of the apparatus used for mechanotransduction in hair cells. TRPA1 and cadherin 23 have been proposed to be the transduction channel and component of the tip link, respectively; moreover, ankyrin repeats in TRPA1 have been proposed to be the gating spring. Although these are excellent candidates for the three components, definitive experiments supporting each identification have yet to be performed.     

The ankyrin repeat domain of the TRPA protein painless is important for thermal nociception but not mechanical nociception

The Drosophila TRPA channel Painless is required for the function of polymodal nociceptors which detect noxious heat and noxious mechanical stimuli. These functions of Painless are reminiscent of mammalian TRPA channels that have also been implicated in thermal and mechanical nociception. A popular hypothesis to explain the mechanosensory functions of certain TRP channels proposes that a string of ankyrin repeats at the amino termini of these channels acts as an intracellular spring that senses force. Here, we describe the identification of two previously unknown Painless protein isoforms which have fewer ankyrin repeats than the canonical Painless protein. We show that one of these Painless isoforms, that essentially lacks ankyrin repeats, is sufficient to rescue mechanical nociception phenotypes of painless mutant animals but does not rescue thermal nociception phenotypes. In contrast, canonical Painless, which contains Ankyrin repeats, is sufficient to largely rescue thermal nociception but is not capable of rescuing mechanical nociception. Thus, we propose that in the case of Painless, ankryin repeats are important for thermal nociception but not for mechanical nociception.     

Full Reconstruction of a Vectorial Protein Folding Pathway by Atomic Force Microscopy and Molecular Dynamics Simulations

During co-translational folding, the nascent polypeptide chain is extruded sequentially from the ribosome exit tunnel and, under severe conformational constraints, is dictated by its one-dimensional geometry. How do such vectorial constraints impact the folding pathway? Here, we combine single-molecule atomic force spectroscopy and steered molecular dynamics simulations to examine protein folding in the presence of one-dimensional constraints that are similar to those imposed on the nascent polypeptide chain. The simulations exquisitely reproduced the experimental unfolding and refolding force extension relationships and led to the full reconstruction of the vectorial folding pathway of a large polypeptide, the 253-residue consensus ankyrin repeat protein, NI6C. We show that fully stretched and then relaxed NI6C starts folding by the formation of local secondary structures, followed by the nucleation of three N-terminal repeats. This rate-limiting step is then followed by the vectorial and sequential folding of the remaining repeats. However, after partial unfolding, when allowed to refold, the C-terminal repeats successively regain structures without any nucleation step by using the intact N-terminal repeats as a template. These results suggest a pathway for the co-translational folding of repeat proteins and have implications for mechanotransduction.     

Structure of the N-terminal ankyrin repeat domain of the TRPV2 ion channel

The TRPV ion channels mediate responses to many sensory stimuli including heat, low pH, neuropeptides, and chemical ligands. All TRPV subfamily members contain an intracellular N-terminal ankyrin repeat domain (ARD), a prevalent protein interaction motif. The 1.6-A crystal structure of the TRPV2-ARD, with six ankyrin repeats, reveals several atypical structural features. Repeats one through three display unusually long and flexible fingers with a large number of exposed aromatic residues, whereas repeats five and six have unusually long outer helices. Furthermore, a large counterclockwise twist observed in the stacking of repeats four and five breaks the regularity of the domain, altering the shape of surfaces available for interactions with proteins or other cellular ligands. Both solution studies and crystal packing interactions indicate that the TRPV2-ARD does not form homo-oligomers, suggesting that the ARD of TRPV ion channels may be used for interactions with regulatory factors rather than in promoting tetrameric assembly of the ion channels.     

Crystal structure of the human TRPV2 channel ankyrin repeat domain

TRPV channels are important polymodal integrators of noxious stimuli mediating thermosensation and nociception. An ankyrin repeat domain (ARD), which is a common protein-protein recognition domain, is conserved in the N-terminal intracellular domain of all TRPV channels and predicted to contain three to four ankyrin repeats. Here we report the first structure from the TRPV channel subfamily, a 1.7 A resolution crystal structure of the human TRPV2 ARD. Our crystal structure reveals a six ankyrin repeat stack with multiple insertions in each repeat generating several unique features compared with a canonical ARD. The surface typically used for ligand recognition, the ankyrin groove, contains extended loops with an exposed hydrophobic patch and a prominent kink resulting from a large rotational shift of the last two repeats. The TRPV2 ARD provides the first structural insight into a domain that coordinates nociceptive sensory transduction and is likely to be a prototype for other TRPV channel ARDs.     

A finite element framework for studying the mechanical response of macromolecules: application to the gating of the mechanosensitive channel MscL

The gating pathways of mechanosensitive channels of large conductance (MscL) in two bacteria (Mycobacterium tuberculosis and Escherichia coli) are studied using the finite element method. The phenomenological model treats transmembrane helices as elastic rods and the lipid membrane as an elastic sheet of finite thickness; the model is inspired by the crystal structure of MscL. The interactions between various continuum components are derived from molecular-mechanics energy calculations using the CHARMM all-atom force field. Both bacterial MscLs open fully upon in-plane tension in the membrane and the variation of pore diameter with membrane tension is found to be essentially linear. The estimated gating tension is close to the experimental value. The structural variations along the gating pathway are consistent with previous analyses based on structural models with experimental constraints and biased atomistic molecular-dynamics simulations. Upon membrane bending, neither MscL opens substantially, although there is notable and nonmonotonic variation in the pore radius. This emphasizes that the gating behavior of MscL depends critically on the form of the mechanical perturbation and reinforces the idea that the crucial gating parameter is lateral tension in the membrane rather than the curvature of the membrane. Compared to popular all-atom-based techniques such as targeted or steered molecular-dynamics simulations, the finite element method-based continuum-mechanics framework offers a unique alternative to bridge detailed intermolecular interactions and biological processes occurring at large spatial scales and long timescales. It is envisioned that such a hierarchical multiscale framework will find great value in the study of a variety of biological processes involving complex mechanical deformations such as muscle contraction and mechanotransduction.     

Stereocilia membrane deformation: implications for the gating spring and mechanotransduction channel

In hair cells, although mechanotransduction channels have been localized to tips of shorter stereocilia of the mechanically sensitive hair bundle, little is known about how force is transmitted to the channel. Here, we use a biophysical model of the membrane-channel complex to analyze the nature of the gating spring compliance and channel arrangement. We use a triangulated surface model and Monte Carlo simulation to compute the deformation of the membrane under the action of tip link force. We show that depending on the gating spring stiffness, the compliant component of the gating spring arises from either the membrane alone or a combination of the membrane and a tether that connects the channel to the actin cytoskeleton. If a bundle is characterized by relatively soft gating springs, such as those of the bullfrog sacculus, the need for membrane reinforcement by channel tethering then depends on membrane parameters. With stiffer gating springs, such as those from rat outer hair cells, the channel must be tethered for all biophysically realistic parameters of the membrane. We compute the membrane forces (resultants), which depend on membrane tension, bending modulus, and curvature, and show that they can determine the fate of the channel.     

Regulation of the transient receptor potential channel TRPA1 by its N-terminal ankyrin repeat domain

The transient receptor potential channel A1 (TRPA1) is unique among ion channels of higher vertebrates in that it harbors a large ankyrin repeat domain. The TRPA1 channel is expressed in the inner ear and in nociceptive neurons. It is involved in hearing as well as in the perception of pungent and irritant chemicals. The ankyrin repeat domain has special mechanical properties, which allows it to function as a soft spring that can be extended over a large range while maintaining structural integrity. A calcium-binding site has been experimentally identified within the ankyrin repeats. We built a model of the N-terminal 17 ankyrin repeat structure, including the calcium-binding EF-hand. In our simulations we find the calcium-bound state to be rigid as compared to the calcium-free state. While the end-to-end distance can change by almost 50% in the apo form, these fluctuations are strongly reduced by calcium binding. This increase in stiffness that constraints the end-to-end distance in the holo form is predicted to affect the force acting on the gate of the TRPA1 channel, thereby changing its open probability. Simulations of the transmembrane domain of TRPA1 show that residue N855, which has been associated with familial episodic pain syndrome, forms a strong link between the S4-S5 connecting helix and S1, thereby creating a direct force link between the N-terminus and the gate. The N855S mutation weakens this interaction, thereby reducing the communication between the N-terminus and the transmembrane part of TRPA1.     

Elastic versus brittle mechanical responses predicted for dimeric cadherin complexes

Cadherins are a superfamily of adhesion proteins involved in a variety of biological processes that include the formation of intercellular contacts, the maintenance of tissue integrity, and the development of neuronal circuits. These transmembrane proteins are characterized by ectodomains composed of a variable number of extracellular cadherin (EC) repeats that are similar but not identical in sequence and fold. E-cadherin, along with desmoglein and desmocollin proteins, are three classical-type cadherins that have slightly curved ectodomains and engage in homophilic and heterophilic interactions through an exchange of conserved tryptophan residues in their N-terminal EC1 repeat. In contrast, clustered protocadherins are straighter than classical cadherins and interact through an antiparallel homophilic binding interface that involves overlapped EC1 to EC4 repeats. Here we present molecular dynamics simulations that model the adhesive domains of these cadherins using available crystal structures, with systems encompassing up to 2.8 million atoms. Simulations of complete classical cadherin ectodomain dimers predict a two-phased elastic response to force in which these complexes first softly unbend and then stiffen to unbind without unfolding. Simulated α, β, and γ clustered protocadherin homodimers lack a two-phased elastic response, are brittle and stiffer than classical cadherins and exhibit complex unbinding pathways that in some cases involve transient intermediates. We propose that these distinct mechanical responses are important for function, with classical cadherin ectodomains acting as molecular shock absorbers and with stiffer clustered protocadherin ectodomains facilitating overlap that favors binding specificity over mechanical resilience. Overall, our simulations provide insights into the molecular mechanics of single cadherin dimers relevant in the formation of cellular junctions essential for tissue function.     

A primer on ankyrin repeat function in TRP channels and beyond

Transient receptor potential (TRP) channels are rapidly gaining attention as important receptors and transducers of diverse sensory and environmental cues. Recent progress in the field has provided new insights into the structure and function of the ankyrin repeat motifs present in the N-terminal cytosolic domain of many TRP channels. The topics addressed in this Highlight include the structural features of canonical ankyrin repeats, new clues into the functions these repeats perform in cells, and how this information can be applied to develop further experiments on TRP channels and other proteins containing ankyrin repeats.     

Vinculin, cadherin mechanotransduction and homeostasis of cell–cell junctions

Cell adhesion junctions characteristically arise from the cooperative integration of adhesion receptors, cell signalling pathways and the cytoskeleton. This is exemplified by cell–cell interactions mediated by classical cadherin adhesion receptors. These junctions are sites where cadherin adhesion systems functionally couple to the dynamic actin cytoskeleton, a process that entails physical interactions with many actin regulators and regulation by cell signalling pathways. Such integration implies a potential role for molecules that may stand at the interface between adhesion, signalling and the cytoskeleton. One such candidate is the cortical scaffolding protein, vinculin, which is a component of both cell–cell and cell–matrix adhesions. While its contribution to integrin-based adhesions has been extensively studied, less is known about how vinculin contributes to cell–cell adhesions. A major recent advance has come with the realisation that cadherin adhesions are active mechanical structures, where cadherin serves as part of a mechanotransduction pathway by which junctions sense and elicit cellular responses to mechanical stimuli. Vinculin has emerged as an important element in cadherin mechanotransduction, a perspective that illuminates its role in cell–cell interactions. We now review its role as a cortical scaffold and its role in cadherin mechanotransduction.     

Compliance of the hair bundle associated with gating of mechanoelectrical transduction channels in the bullfrog's saccular hair cell

Mechanical stimuli are thought to open the transduction channels of a hair cell by tensing elastic components, the gating springs, that pull directly on the channels. To test this model, we measured the stiffness of hair bundles during mechanical stimulation. A bundle's compliance increased by about 40% at the position where half of the channels opened. This we attribute to conformational changes of transduction channels as they open and close. The magnitude and displacement dependence of the gating compliance provide quantitative information about the molecular basis of mechanoelectrical transduction: the force required to open each channel, the number of transduction channels per hair cell, the stiffness of a gating spring, and the swing of a channel's gate as it opens.     

Structural analyses of the ankyrin repeat domain of TRPV6 and related TRPV ion channels

Transient receptor potential (TRP) proteins are cation channels composed of a transmembrane domain flanked by large N- and C-terminal cytoplasmic domains. All members of the vanilloid family of TRP channels (TRPV) possess an N-terminal ankyrin repeat domain (ARD). The ARD of mammalian TRPV6, an important regulator of calcium uptake and homeostasis, is essential for channel assembly and regulation. The 1.7 A crystal structure of the TRPV6-ARD reveals conserved structural elements unique to the ARDs of TRPV proteins. First, a large twist between the fourth and fifth repeats is induced by residues conserved in all TRPV ARDs. Second, the third finger loop is the most variable region in sequence, length and conformation. In TRPV6, a number of putative regulatory phosphorylation sites map to the base of this third finger. Size exclusion chromatography and crystal packing indicate that the TRPV6-ARD does not assemble as a tetramer and is monomeric in solution. Adenosine triphosphate-agarose and calmodulin-agarose pull-down assays show that the TRPV6-ARD does not interact with either ligand, indicating a different functional role for the TRPV6-ARD than in the paralogous thermosensitive TRPV1 channel. Similar biochemical findings are also presented for the highly homologous mammalian TRPV5-ARD. The implications of the structural and biochemical data on the role of the ankyrin repeats in different TRPV channels are discussed.     

Mechano-electrical Transduction: New Insights into Old Ideas

The gating-spring theory of hair cell mechanotransduction channel activation was first postulated over twenty years ago. The basic tenets of this hypothesis have been reaffirmed in hair cells from both auditory and vestibular systems and across species. In fact, the basic findings have been reproduced in every hair cell type tested. A great deal of information regarding the structural, mechanical, molecular and biophysical properties of the sensory hair bundle and the mechanotransducer channel has accumulated over the past twenty years. The goal of this review is to investigate new data, using the gating spring hypothesis as the framework for discussion. Mechanisms of channel gating are presented in reference to the need for a molecular gating spring or for tethering to the intra- or extracellular compartments. Dynamics of the sensory hair bundle and the presence of motor proteins are discussed in reference to passive contributions of the hair bundle to gating compliance. And finally, the molecular identity of the channel is discussed in reference to known intrinsic properties of the native transducer channel.     

Structural biology of thermoTRPV channels

Essential for physiology, transient receptor potential (TRP) channels constitute a large and diverse family of cation channels functioning as cellular sensors responding to a vast array of physical and chemical stimuli. Detailed understanding of the inner workings of TRP channels has been hampered by a lack of atomic structures, though structural biology of TRP channels has been an enthusiastic endeavor since their molecular identification two decades ago. These multi-domain integral membrane proteins, exhibiting complex polymodal gating behavior, have been a challenge for traditional X-ray crystallography, which requires formation of well-ordered protein crystals. X-ray structures remain limited to a few TRP channel proteins to date. Fortunately, recent breakthroughs in single-particle cryo-electron microscopy (cryo-EM) have enabled rapid growth of the number of TRP channel structures, providing tremendous insights into channel gating and regulation mechanisms and serving as foundations for further mechanistic investigations. This brief review focuses on recent exciting developments in structural biology of a subset of TRP channels, the calcium-permeable, non-selective and thermosensitive vanilloid subfamily of TRP channels (TRPV1-4), and the permeation and gating mechanisms revealed by structures.     

Nanomechanics of the cadherin ectodomain: "canalization" by Ca2+ binding results in a new mechanical element

Cadherins form a large family of calcium-dependent cell-cell adhesion receptors involved in development, morphogenesis, synaptogenesis, differentiation, and carcinogenesis through signal mechanotransduction using an adaptor complex that connects them to the cytoskeleton. However, the molecular mechanisms underlying mechanotransduction through cadherins remain unknown, although their extracellular region (ectodomain) is thought to be critical in this process. By single molecule force spectroscopy, molecular dynamics simulations, and protein engineering, here we have directly examined the nanomechanics of the C-cadherin ectodomain and found it to be strongly dependent on the calcium concentration. In the presence of calcium, the ectodomain extends through a defined ("canalized") pathway that involves two mechanical resistance elements: a mechanical clamp from the cadherin domains and a novel mechanostable component from the interdomain calcium-binding regions ("calcium rivet") that is abolished by magnesium replacement and in a mutant intended to impede calcium coordination. By contrast, in the absence of calcium, the mechanical response of the ectodomain becomes largely "decanalized" and destabilized. The cadherin ectodomain may therefore behave as a calcium-switched "mechanical antenna" with very different mechanical responses depending on calcium concentration (which would affect its mechanical integrity and force transmission capability). The versatile mechanical design of the cadherin ectodomain and its dependence on extracellular calcium facilitate a variety of mechanical responses that, we hypothesize, could influence the various adhesive properties mediated by cadherins in tissue morphogenesis, synaptic plasticity, and disease. Our work represents the first step toward the mechanical characterization of the cadherin system, opening the door to understanding the mechanical bases of its mechanotransduction.     

Examination of a novel head-stalk protein family in Giardia lamblia characterised by the pairing of ankyrin repeats and coiled-coil domains

The intestinal pathogen Giardia lamblia possesses several unusual organelle features, including two equivalent nuclei, no mitochondria or peroxisomes, and a developmentally regulated rough endoplasmic reticulum and Golgi. Giardia also possesses a number of complex and unique cytoskeleton structures that dictate cell shape, motility and attachment. Our investigations of cytoskeletal proteins have revealed the presence of a new protein family. Proteins in this family contain both ankyrin repeats and coiled-coil domains; although these are common protein motifs, their pairing is unique, thus establishing a new class of head-stalk proteins. Examination of the G. lamblia genome shows evidence for at least 18 genes coding for proteins with a series of ankyrin repeats followed by a lengthy coiled-coil domain and at least an additional 14 genes coding for proteins with a prominent coiled-coil domain flanked by two series of ankyrin repeats. We have examined one of these proteins, Giardia Axoneme Associated Protein (GASP-180), in detail. GASP-180 is a 180 kDa protein containing five ankyrin repeats in a 200 amino acid N-terminal domain separated by a short spacer from an approximately 1375 amino acid coiled-coil domain. Using anti-peptide antibodies raised against a unique 20 amino acid sequence found at the C-terminus, we have determined that GASP-180 is present in cytoskeleton extractions of the parasite and localises to the proximal base of the anterior flagellar axonemes. The combination of the localisation and the structural and functional motifs of GASP-180 make it a strong candidate to participate in control of flagellar activity.