PERSPECTIVE Feeling the hidden mechanical forces in bilayer is an original sense Andriy Anishkina, Stephen H. Loukinb, Jinfeng Tengb, and Ching Kungb,c,1 aDepartment of and Center for Computational Proteomics at the Huck Institute of Life Sciences, Pennsylvania State University, University Park, PA 16802; and bLaboratory of Molecular Biology and cDepartment of Genetics, University of Wisconsin–Madison, Madison, WI 53706

Edited by Roderick MacKinnon, The Rockefeller University and Howard Hughes Medical Institute, New York, NY, and approved April 15, 2014 (received for review January 21, 2014)

Life’s origin entails enclosing a compartment to hoard material, energy, and information.The envelope necessarily comprises amphipaths, such as prebiotic fatty acids, to partition the two aqueous domains. The self-assembled comes with a set of properties including its strong anisotropic internal forces that are chemically or physically malleable. Added bilayer stretch can alter force vectors on embedded to effect conformational change. The force-from-lipid principle was demonstrated 25 y ago when stretches opened purified Escherichia coli MscL channels reconstituted into artificial bilayers. This reductionistic exercise has rigorously been recapitulated recently with two + vertebrate mechanosensitive K channels (TREK1 and TRAAK). Membrane stretches have also been known to activate various voltage-, ligand-, + or Ca2 -gated channels. Careful analyses showed that Kv, the canonical voltage-gated channel, is in fact exquisitely sensitive even to very small tension. In an unexpected context, the canonical transient-receptor-potential channels in the Drosophila eye, long presumed to open by ligand

binding, is apparently opened by membrane force due to PIP2 hydrolysis-induced changes in bilayer strain. Being the intimate medium, govern membrane proteins by physics as well as chemistry.This principle should not be a surprise because it parallels water’s paramount role in the structure and function of soluble proteins. Today, overt or covert mechanical forces govern cell biological processes and produce sensations. At the genesis, a bilayer’s response to osmotic force is likely among the first senses to deal with the capricious primordial sea.

mechanosensitivity | force sensing | channel gating | bilayer mechanics | touch

To compete successfully, organisms must α-helixcantiltit.Aforcecanalsostrainthe multiple short- and long-distance forces evolve effective reactions to earth’s physical bond and displace the partners, like stretch- among atoms and feeding them into New- stimuli: heat, force, voltage, and chemical ing or bending the helix. The bond breaks tonian laws of motion is the basis of mo- ligands. Emil Fischer’s lock-and-key binding when work (force times displacement) lecular dynamics simulation (MDS), which between molecules is well known and exceeds the bond energy. For example, it in recent decades has successfully analyzed − explains such overt senses as vision, smell, takes ∼4pN(4× 10 12 N) to break a hy- and explained the many workings of mac- and most tastes. We also understand voltage drogen bond, ∼1,600 pN to break a C–C romolecules and their assembly (4, 5). Some sensing. For example, a charge-bearing helix covalent bond (Fig. S1). Ligand– or forces relevant to biology and used in MDS traversing the electric field across the lipid protein–protein binding also mechanically are listed in Fig. S1. Some findings might bilayer can mechanically drive conforma- strains the bonds between protein do- surprise us. For example, most of the time tional changes of embedded ion channels mains to effect long-range conformational in catalysis is spent on the diffusion to and or (1). Heat and force are universal, changes. The last 100 y of mechanochem- from the catalytic site, mechanically posi- governing all matters and reactions. A chal- istry have shown how mechanical forces tioning the substrates and products; whereas lenge to current biology is to understand govern even inorganic chemical reactions the very stage of chemical transformation, of how Nature chooses and uses her molecular (2). Many steps in biochemical reactions or which we tend to fixate, happens almost thermometers and force gauges. We review other molecular processes can therefore be instantaneously. the basis of force sensing, emphasizing the simulated by explicitly stating the New- roles of the lipid bilayer mechanics. tonian forces involved and following the The Mechanics of the Lipid Bilayer motions of the components—an approach A surface tension appears at any liquid–liquid Biological Mechanisms Are Ultimately bringing invaluable insights into molecular or liquid–gas interface. Recall that at a Mechanics underpinnings of life. The 2013 Chemistry hexane–water interface, the surface tension Textbooks describe the energy minimum Nobel was awarded for the development appears because water molecules there are when atoms are brought to an equilibrium of molecular dynamics and quantum me- deprived of half of their hydrogen-bonding distance (bond length), at which, there is zero chanical approaches to simulate complex partners they enjoy in the bulk. Thermal force between them because their mutual chemical systems. How the paired atoms attraction balances repulsion. Thermal oscil- move upon a perturbation can be described Author contributions: A.A., S.H.L., J.T., and C.K. wrote the paper.

lations or external forces change the bond by a set of laws and numerical parame- The authors declare no conflict of interest. “ ” length and generate compressive or expan- ters called the force field (3). In macro- This article is a PNAS Direct Submission. sive forces in the bond, much like with molecules, long-distance Lennard-Jones 1To whom correspondence should be addressed. E-mail: ckung@ a spring. External force acting on an atom and electrostatic forces come into play wisc.edu.

can simply move the whole bonded assem- during conformational change. Reposition- This article contains supporting information online at www.pnas.org/ bly. For example, pulling one end of an ing atoms by iteratively calculating the lookup/suppl/doi:10.1073/pnas.1313364111/-/DCSupplemental.

7898–7905 | PNAS | June 3, 2014 | vol. 111 | no. 22 www.pnas.org/cgi/doi/10.1073/pnas.1313364111 Downloaded by guest on October 1, 2021 dispersion of the surface also makes mole- to take a better-matched conformation. positive curvature. Asymmetric insertions PERSPECTIVE cules less densely packed in the few inter- Reducing the acyl chain length from PC20 to of exogenous amphipaths activate these facial layers, thus decreasing the repulsion PC18 converts the reconstituted gramicidin channels (14), consistent with the bilayer- between the neighbors (6). Qualitatively A from a stretch-activated to a stretch- coupling hypothesis (26). The lipid force similar interfacial forces develop in the inactivated channel (12). Besides thinning, a profile and how stretch relates to MscL lipid bilayer. However, there are critical stretch can also change the magnitude and gating has been examined by MDS (10). In distinctions. Molecules at a liquid interface direction of the force vectors on the protein. line with the role of the bilayer’s focused are not restrained from freely joining the Chemically changing bilayer composition by lateral tension (Fig. 1B), random and scan- bulk. Water in a sea of hexane will shrink inserting impurities or through lipid me- ning mutagenesis showed that replacing hy- into a sphere. Adding more water increases tabolism can have the same effect (Fig. 1B). drophobic with hydrophilic residues in M1 the sphere’s volume but reduces the relative Although these principles are general, we or M2 at the outer rim of MscL removes number of water at the surface, with no chose to discuss ion channels among mem- mechanosensitivity in vitro and in vivo (27). change in surface tension. In contrast, the braneproteinsbecausetheiractivitiescan The miniprotein antibiotic gramicidin A polar lipid head is stuck with its nonpolar be examined easily and quantitatively, even from Bacillus brevis readily forms cation tail making the bilayer a “surface without at the level of single proteins and down channels in bilayers by pair-wise stacking. It bulk.” Typical membrane lipids tend to to a microsecond time resolution (13) by has been used to examine the role of vari- compact into a bilayer sheet instead of electric means. ous chemical and physical properties of a droplet. Adding lipids enlarges the sheet the bilayer in quantitative details (28). Over but does not change the exposed-to-hidden The Force-from-Lipid Principle the years, the force-from-lipid principle has surface ratio per molecule. Water molecules The force-from-lipid principle was estab- gradually been used to understand eukary- strongly bond with the polar atoms of the lished some 25 y ago from the study of the otic processes as well. More recently, this head groups or the glycerol, although bar- mechanosensitive channels from Escherichia principle has been rigorously extended to red from the nonpolar hydrocarbon tails. coli (14, 15). Their discovery, atomic struc- animal ion channels as summarized below. tures, and biological roles have been peri- Thus, a sharp lateral tension develops at the + level of the lipid neck. As a self-assembled odically reviewed (see refs. 16 and 17 and Mechanosensitive Two-Pore-Domain K citations therein). Briefly, the key observa- Channels entity at equilibrium, this tension is bal- + anced by the large repulsion from interior tion is that purified MscL or MscS can be Microbes have more varieties of K channels tail movement and the smaller head–head reconstituted into artificial bilayers and than animals (29). The simplest are homo- “ ” “ repulsion in a relaxed membrane. The rel- retain their mechanosensitivity, excluding tetramers of S1-P-S2 subunits ( S for seg- ative contribution of the two repulsions the need of cytoskeleton, external tether, or ment” in “α-helical segment”; “P” for “pore” depends on the head-to-tail width ratio, accessory channel subunits as force trans- in “pore domain”, the filter structure), such as which corresponds to the spontaneous cur- mitters (18, 19). Because bilayers of phos- KcsA from the Gram-positive Streptomyces, vature of the lipid leaflet, with a larger head pholipids of different head groups (20) or the first ion channel of known crystal struc- favoring positive curvature. This layered fatty-acid lengths (21) support the mecha- ture (30). A variant of this motif are two- + internal force distribution has been calcu- nosensitivity of MscL, a lock-and-key li- pore-domain K channel (K2p)dimers lated from thermodynamics (7, 8) and ex- gand-gated mechanism need not apply. of S1-P1-S2-S3-P2-S4 subunits. Human K2p amined by MDS (9, 10) (Fig. 1B, Upper Left). Judging by the slope of the Boltzmann family comprises 15 fairly disparate mem- Thus, unlike soluble proteins, which are distribution of the open probability against bers, several of which can be stretched open bombarded by particles from all directions bilayer tension, MscL protein expands by in clamped patches. The structures of two ∼ 2 Δ – (Fig. 1A, Left), embedded proteins as a part 20 nm . This expansion in area ( A) K2p (31 33) are now known. The mechano- of the membrane ensemble are subjected to requires 220 pN to stretch its 22-nm pe- sensitive TRAAK (TWIK-related arachidonic − + anisotropic push and pull (Fig. 1A, Right), rimeter, doing 2 × 10 23 J (50 kT) work. acid-stimulated K ) (34) has been solved in especially sharply focused suction. A stretch Half of the channels are open at 10–12 open states at 3.8 Å (32) (Fig. 2 A and B)and will deform the water sphere in hexane pN/nm, not far from the lytic tension of then at 2.75 Å resolution revealing a domain imagined above, changing the surface-to- lipid bilayer (22, 23). The ΔAestimatedfrom swap (33). TRAAK has features not found volume ratio but not the surface tension. activities approximates the structural esti- in KcsA, two of which may be related to However, a stretch force can only thin the mate from crystallography (24) and elec- mechanosensitivity: Of the four inner helices, bilayer (until it breaks) and thereby increase tron paramagnetic resonance spectroscopy the two S2 are long, each having a kink that the lateral tension. This general description (25). MscL is a homopentamer of subunits makes the lower portion lying almost flat. should not obscure local variations. The with two transmembrane helices, M1 and This portion is amphipathic with hydro- contour and hydrophobicity of the protein M2. To open, these helices apparently lean philic basic residues facing the membrane– surface need to match that of the lipids. down and move outward making a much cytoplasm interface (Fig. 2A, purple) and The bilayer structure can therefore be thinner structure, as if to meet a thinned hydrophobic residues facing the membrane distorted, e.g., being curved, compressed, or bilayer (25). Bilayers with shorter chain interior (green), placing the two peptides at otherwise deformed at the protein-lipid in- length favor opening, but tension is still the membrane–cytoplasm interface of the terface. The distortion spreads over a few required. However, an asymmetric addi- inner leaflet, rich in acidic lipids in vivo. layers of annular lipids and even enables a tion of lysolipids to either one of the Interestingly, the inner transmembrane half mechanical cross-talk between the proteins monolayers opens the channel in a relaxed of K2p channels (31) is even fenestrated. In or protein domains through the lipid me- membrane (21), suggesting involvement of TRAAK (32, 33), a sizable portion of chan- dium (11). Added external stretch can thin additional physical parameters, such as nel’s lower half is not enclosed by protein, the bilayer generating a hydrophobic mis- a mismatch of tension in the two leaflets leaving a 5-Å-wide gap presumably filled match at the interface, driving the protein and/or bending of annular lipids favored by with lipids in vivo, extending from the

Anishkin et al. PNAS | June 3, 2014 | vol. 111 | no. 22 | 7899 Downloaded by guest on October 1, 2021 bottom of the filter to the lower edge of the lysophospholipids, polyunsaturated fatty these channels displayed spontaneous ac- channel (Fig. 2B, yellow). acids (PUFAs), volatile general anesthetics tivities that could not be further increased by K2p control the resting membrane poten- (36, 37) and morphine (38). pipet suctions. Positive pipet pressure pulses, tialbypassingleakcurrent(35).Theyare however, could proportionally reduce their Reconstitution of Vertebrate regulated by disparate stimuli: pH, tempera- + activity to total closure. These authors rea- ture, lipidic ligands, and membrane stretch. Mechanosensitive K Channels, K2p soned that open probability has been max- Among the diverse K2p, the mechanosensitive Although there are antecedents (39, 40), re- imized by inherent tension in the patches + TREK-1 (TWIK-related K -1) and TRAAK cent works generate new excitement because (41). Such a tension is likely generated have been extensively studied by Patel, of the deep knowledge on the structure and during the gigaOhm-seal formation, bond- Honore, and coworkers (36, 37). Whole- function of K2p summarized above. Berrier ing the bilayer lipids to the pipet glass sur- cell TREK-1 current is strongly suppressed et al. (41) have recently reconstituted face (42–45). by hyperosmolarity. In patches from ex- an enriched fraction of mouse TREK-1 In a recent rigorous study, Brohawn et al. pressing cells, pressure in either direction into liposomes and examined patches ex- (46) purified zebrafish TREK-1 and human activates TREK-1, although suction is more cised from them. They observed the ∼80-pS TRAAK to homogeneity, as evidenced by + effective. It is also activated by negatively K -specific outward-rectifying chlorproma- monodispersion of column-elution profiles charged phospholipids such as PIP2,by zine-sensitive conductance. Surprisingly, (Fig. 2C), and also reconstituted them into liposomes made of mixed-length phophati- dylcholine extracted from soybean azolectin. In excised patches with multiple channels, A they found the channels to respond in- stantaneouslytoappliedforce(Fig.2D). These channels are also activated by arach- idonic acid (the PUFA embedded in the name TRAAK). These K2p respond propor- tionally to either negative or positive pres- sures much like the bacterial MscL in a similar setting (21). This is also true when TRAAKs are expressed in cultured cells, wheretheyshouldbeinthesameorientation (Fig. 2 E and F). This indifference to pressure direction leaves no doubt that it is the added bilayer tension that gates the channels. Be- yond demonstrating the force-from-lipid biophysical principle for animal channels, B these authors also expressed TRAAK in cul- tured mouse Neuro2A cells. Prodding them with a probe evokes a K2p-dependent out- ward current that counteracts the native in- ward current through Piezo1. Details aside, both sets of reconstitution experiments clearly showed that vertebrate K2p,muchlike MscL and MscS, are themselves sensitive to stretch force from the lipid bilayer, requiring no additional subunits, cytoskeleton, or ex- tracellular matrix tethers. Precisely how lipid forces operate TREK-1 + or TRAAK is not yet clear. Like many K channels, including the budding-yeast K2p (47), TREK-1 apparently uses a dynamic fil- ter, where several gain-of-function mutations are found (48). Its quaternary ammonium (QA)-binding site seems accessible from the inside before channel activation, suggesting a static open inner gate (49), although QAs Fig. 1. Anisotropic forces of the lipid bilayer and their changes that can reshape embedded proteins. (A)A could reach the site through the fenestration diagram comparing a protein in the cytoplasm, near-isotropically compressed by the bombardment of neigh- boring particles (Left) and one embedded in a bilayer, subjected to large lateral tension at the two polar– now shown in the atomic structure (32, 33) nonpolar interfaces opposed by compression forces at the other levels of the bilayer (Right). (B)Thelateral (Fig. 2B). TREK-1 is activated by negatively tension at the interface is balanced by the pressure in the head groups and hydrophobic tails (Upper Left)and charged phospholipids such as PIP2,and this equilibrium can match a certain protein conformation, say, the closed state of a channel protein (Upper inhibited by polycations or agents that cause Right). External stretch force (Lower, broad red arrows) and/or amphipaths with positive (red triangle) or negative (green triangles) spontaneous curvature asymmetrically added to one leaflet can thin and bend the PIP2 hydrolysis (36, 37). These observations bilayer at the protein–lipid interface, changing the force vectors (small arrows) and therefore prefer a better can be explained by the interaction of the matched protein conformation, say, the open state. negatively charged inner leaflet with a

7900 | www.pnas.org/cgi/doi/10.1073/pnas.1313364111 Anishkin et al. Downloaded by guest on October 1, 2021 proximal peptide segment, rich in basic membrane potential, the voltage sensors, shows drastically different behavior in whole PERSPECTIVE amino acids, which immediately follows the acting through the S4-S5 linkers, mechanically cells, in patches, or upon reconstitution into end of the inner helix S4. The bilayer constrict the lower gate comprising the four planar bilayers. MacKinnon and coworkers crenators (trinitrophenol, lysolecithin) acti- S6. Depolarization moves the sensors and found the differences best explained by vate, whereas cup formers (chlorpromazine, linkers, releasing the constriction (51). These changes in tension on the membrane, held tetracaine) inhibit TREK-1 (36, 37), consis- actionslikelyalsomakeuseoftheintimately low when it is locally constrained by sub- tent with the bilayer-coupling hypothesis (14, attached lipids (52). Here the channel opens cortical cytoskeleton, but increases when the 26). TREK-1 is estimated to expand by ∼2to not by removing a plug, but by a lateral membrane is detached and adheres to the 4nm2 in the membrane plane when opens expansion of the cytoplasmic side of the surface of the glass electrode (43, 44). For (45). It remains reasonable that the inner channel with a ΔA. If Kv is built only for example, the tendency to open, as indicated helices (S2 and/or S4) converge to close and interactions between its protein domains, by the Boltzmann slope and midpoint volt- + diverge to open like other K channels as was one might expect the domains to be densely age, is much higher for channels in an on-cell supposed by earlier homology models (50), packed to minimize the contact with the patch than those in the rest of the same even if the inner gate cooperates with the membrane—a potential source of noise. cell after the patch is broken (Fig. 3 A–D). filter gate. The two S2 inner helices, having However, the association between the pore Quantitative analyses of kinetic schemes in- amphipathicity (Fig. 2 A and B) that matches and the peripheral domains is limited to the dicate that the tension-sensitive step is the the inner leaflet naturally rich in acidic inner S4-S5 link and a contact between S1 and finalopeningofthegateafterthefourvoltage phospholipids and being at the level where S6 toward the outer side (52, 53). As a result, sensors have moved, i.e., at the point when Δ peak lateral tension is expected, would pre- all domains, including the gate-bearing core A takes place. Thus, tension mainly acts on dict that they are subjected to that tension are in extensive contact with lipids. This coupling the sensors to the gate, presumably and its changes during membrane stretch. unusual design implies roles of lipids in Kv through lipids, and not on the sensors them- selves. The tension in the patch generated + function. Indeed, the action of the voltage The Voltage-Dependent K Channel Is as sensor with its positive arginines (the gating by lipid adhesion to glass is only about 0.5– Sensitive to Bilayer Force as It Is to charges) engages the negative phosphate 4 mN/m (42). Free-energy changes, as es- Voltage oxygens at the lipid neck (54, 55). However, timated from the changes of the rate of + The celebrated voltage-dependent K chan- why should the pore domain also be sur- opening step with or without added ten- Δ – 2 nel, Kv, is a tetramer of S1-S2-S3-S4-S5-P-S6 rounded by lipids? sion, indicate a A of some 3 4nm, subunits in essence (1). Like the S1-P-S2 of The free-energy change associated with the consistent with structural information (44). KcsA, the four S5-P-S6 helical pairs come bilayer deformation is estimated to be com- Because this tension-dependent behavior is together to form the pore domain housing parable to that of the voltage-dependent part observed in excised patches and varies with the filter and the gate. The four S1-S4 form of the total gating energy (56). Although not lipid compositions upon Kv reconstitution the four peripheral domains, each bearing commonly known, Kv (e.g., the Kv1.2 paddle (43), the tension must come from the lip- a voltage sensor. Driven by the resting chimera of known crystal structure) (52) ids. The effect of this small tension is not trivial. “Kv channel is as much a mechano- sensitive channel as it is a voltage-dependent channel” (44, p. 10356); open probability ABC rises from 0 to 0.65 between −60 and +50 mV, but rises from 0 to 1.0 upon suction at midrange voltages. Kv, being a model for out Nav, Cav, and TRPs, indicates that the lipid- tension effect is much more general than we commonly thought. The generality of channel 900 mechanosensitivity has been advocated by Morris and coworkers (ref. 57 and refer- in ences therein). D EFPhysics or Physiology? Any entity that deforms in the direction 2000 negative of force is, by physical definition, mechano- 1500 pressure )

A sensistive. So does the mechanosensitivity of (p t 1000

ren Kv simply reflect its necessary expansion in r

Cu 500 positive pressure the membrane plane under stress, or does 0 it have any biological significance? As de- 0 20 40 60 Absolute value of applied pressure (mmHg) scribed above, heterologous TRAAK can pass outward current that counteracts in piezo1 Fig. 2. The atomic structures and the reconstituted activities of TRAAK. (A and B) TRAAK structures from two inward current in cells responding to external ’ perspectives showing the two S2 amphipathic peptides [hydrophobic (green) and charged (purple) residues] and one force (46). Given evolution sopportunism,it of the two prominent fenestrations (yellow) (from ref. 32). (C) Monodispersion of column eluate indicates the purity of seems unlikely that this exquisite sensitivity the TRAAK protein produced. (D) Such protein is reconstituted into liposomes. TRAAK channels in a patch excised from is not exploited. Hypoosmotic swelling opens such a proteoliposome are activated (upper traces) in proportion to the suction applied to patch (bottom traces). (E) TRAAKs in a patch excised from an expressing CHO cell, in which they should have the same orientation, respond Kv in cultured cells (44), presumably by (upper traces) to either suction (black) or pressure (red). (F) Activation by pressures in either direction are quantitatively stretching small membrane domains confined similar, indicating that it is the membrane stretch that activates (modified from ref. 46). by cytoskeletons (Fig. 3D). Thus, osmotic

Anishkin et al. PNAS | June 3, 2014 | vol. 111 | no. 22 | 7901 Downloaded by guest on October 1, 2021 changes, muscle contractions, or lipid-com- physical change in the membrane. Aston- this lipid with negative spontaneous curva- position changes (below) may activate or ishingly, light induces a near-micrometer ture in the inner monolayer would favor modulate Kv, Cav, etc., at least in principle. shrinkage of the ommatidia (units in the higher curvature, thinner microvilli, and “ ” Mechanosensitivity is most palpable in compound eye) that is directly visible under lateral-stack shrinkage. Elsewhere, isolated the sense of touch. Interestingly, a recent a light microscope and measured with an microvilli, cilia, filopodia, or dendritic spines report shows that, when certain dorsal-root- atomic force microscope (Fig. 4 C and D). are common, where such physical move- ganglion neurons are prodded, channels with This contraction begins before the channel + −/− ment cannot easily be detected. Nonetheless Kv1.1 subunits pass K outward, acting as current. Deleting the PLC (norpA ) a brake (58) to oppose the depolarization by takes away the contraction; deleting the they house many signal-transduction path- the inward current through other touch- channels does not. The authors reason that ways that deploy receptors, G proteins, PLC, sensitive channels yet to be defined. It is beheading PIP2 to make the smaller DAG and TRPs. Such pathways are commonly reasoned that these Kv1.1 subunit-containing increases tension in the inner membrane explored according to the current dominant channels therefore set the threshold of the leaflet to pull open channels (Fig. 4A). To paradigm, with the ultimate explanation touch-induced action potential or tune the test, the well-established mechanosensitive being protein phosphorylations due to PLC’s firing adaptation. Mice expressing a domi- channel, gramicidin A (28) was added to the activation of protein kinase C (PKC) (71). nant negative allele of Kv1.1 suffer severe photoreceptor cell and found to pass current One wonders how much the PLC-induced proportional to light intensity. Note that go- mechanical allodynia, interpreting touch changes in bilayer tension discussed here in as hurt (58) (Fig. 3E). ing from PIP2 to DAG is not an isometric fact participate in these pathways (72). Evolution also exploits Kv’smechano- shrinkage. That PIP2 rod becoming the DAG sensitivity negatively. The Chilean rose ta- cone (Fig. 4B) should increase bilayer asym- rantula paralyzes its preys with venom that metry and curvature, and would change includes the toxin VSTx1, which enters the the bilayer force profile (Fig. 1). Although − membrane to bind the Kv2.1 sensor at 10 8 we do not know the exact changes in mem- M specificity (59). Strangely, VSTx1 has no brane thickness, curvature, or forces in the effect on Kv in slack membranes, and causes channel’s vicinity, diverse cationic amphi- the channel in the tense bilayer to behave as paths that are expected to enter and crowd in a slack membrane (43), as if the toxin the inner leaflet (thus reducing lateral tension perturbs the local lipid arrangement to block there) did inhibit light-induced current. The case of membrane forces controlling TRP/ voltage sensor-gate coupling and/or the final + gate opening. TRPL therefore seems convincing. Still, H (65) and PUFAs (products of innaE) (66) The Photomechanics of Fly Vision are also involved, so photomechanics may In the 1970s, the Pak laboratory and others not be the whole story. (60) began a systematic genetic dissection of The Drosophila work coined the term the Drosophila visual transduction. Blind flies “TRP,” which is honored as the founder of were sorted by complementation grouping the TRPC subfamily (below), with “C” being and by the wave of discharges from “canonical”. Canonical or not, is this a special the compound eye during a light flash case? Rods and cones do not work this way: (electroretinography). Mutants display dif- The rhodopsin-Gt-phosphodiesterase cascade ferent corruptions of the wild-type wave- leads to a cGMP drop that closes the cGMP- forms. For examples: “norpA” for “no gated channel, shutting off dark current. receptor Potential, complementation group However, the vertebrate retina also contains A” later proves to encode phospholipase C; non–image-forming but intrinsically photo- “innaE” for “inactivation, no afterpotential, sensitive ganglion cells for circadian entrain- group E” encodes a diacylglycerol lipase, ment. Here the molecular pathway directly etc., implicating the involvement of lipid parallels that of the insect, beginning with metabolism. melanopsin and ending in the activation of “trp” stands for “transient receptor poten- TRPC6/7 (67). The conservation is striking, Fig. 3. The mechanosensitivity of Kv. (A–C)TheKv1.2 tial” (61), which was the first blind fly gene with melanopsin being far more similar to paddle chimera expressed in an Sf-9 cell responds to cloned (62). trp and its homolog trpl (trp- the insect rhodopsin than vertebrate rho- voltage steps ranging from −100to40mV.The like) were later shown to encode pore-form- responses in on-cell mode (A) are drastically different dopsin (68). Similar pathways may be used from those in the whole-cell mode (B). The normalized ing channel subunits (63). Much research for other TRP channels in melanocyte (69) conductances are graphically compared in C.Asinter- implicates the cascade of rhodopsin-Gq- and keratinocyte (70) of the skin. preted in D, the large differences originate from the ∼ high pipet tension in the on-cell patch due to lipid–glass phospholipase C (PLC, from norpA) That the 5-Å PIP2-to-DAG head shrink hydrolyses PIP (phosphatidylinositol can become a near-micrometer contraction adhesion, but the lack of it in the whole-cell membrane, 2 where the cortical cytoskeleton prevents the local 4,5-bisphosphate) into DAG (diacylglycerol), has to do with the unusual anatomy of the membranes from being stretched (from ref. 44). (E)The although how these lipids activate the chan- insect photoreceptor cell, which sums the role of Kv1.1’s mechanosensitivity in countering im- nels was unclear. Thinking being chemical in minute shrinkage of the thousands of stacked pact-induced excitation in vivo is made evident when acopyofadominant-negativealleleofKv1.1 is our era, one tends to assume that these lipids microvilli it bears (Fig. 4E). Potentially, there + − expressed in the mouse (mceph / ) causing it to be bind a specific site to control the channels. It is a direct mechanical link between the ac- overly sensitive to gentle poking, as indicated by the low is therefore a great surprise when Hardie and cumulation of DAG and the size of a stack of limb-withdrawal threshold of prod strength (from ref. Franze (64) showed that there is a global microvilli. For example, increased content of 58). ns, not significant; **P < 0.01.

7902 | www.pnas.org/cgi/doi/10.1073/pnas.1313364111 Anishkin et al. Downloaded by guest on October 1, 2021 TRP Superfamily membrane of the budding yeast presents a Given the divergence among yeast, toad, PERSPECTIVE TRPs form a loosely connected family: The 300-pS inward rectifier, favoring the flow of and rat, it is difficult to imagine how het- + mammalian subtype TRP-A, -C, -M, -ML, cations including vacuolar Ca2 into the cy- erologous cytoskeleton or other tether can -N, -P, and -V are only similar in the S5-P-S6 toplasm, observed upon hyperosmotic shock. reconnect to transmit force. core sequence. Although known since the It opens to membrane stretch in excised Accessories 1970s, it is the 1997 discovery of the “pepper patches. Forward and reverse genetics point channel” (TRPV1) being a molecular ther- to the importance of aromatic residues, which Besides the definitive reconstitution experi- mometer (73) that caught imagination and tend to locate at the polar–nonpolar interfaces ments, there are other evidences that general physical properties instead of specific chem- popularized TRP research, resulting in co- of the bilayer. The S5-P-S6 core appears to be piousandcomplexfindingsthatdefyany the force sensor because insertion of amor- ical binding govern mechanosensitivity. By comprehensive review today. Besides the phous peptides covalently disrupting the various criteria, reported mechanosensitive channels include MscL, MscS, Mec-4/10, subfamily diversities, TRP channels are connection between the core from the S1-S4 polymodal, meaning that each can receive peripheral domains has little effect on chan- TRP,Piezo,K2p,Kv,KCa,Nav,NMDA and integrate multiple inputs, including nel mechansoensitivity (78). receptor, CFTR, AChR, etc. (see ref. 83 for 2+ The atomic structure of TRPV1 has re- references). These proteins have no simi- cytoplasmic ligands, pH, Ca , membrane “ cently been solved by cryoelectron micros- larities and no recognizable force-sensing lipids and other amphipaths, , ” osmolarity, and mechanical force. TRPC1, copy showing a Kv-like basic structure with domain comparable to voltage sensor or C6, M3, M4, M7, P2, V1, V2, and V4 are several differences (79, 80). It has also been ligand-binding sites. We emphasize the judged as mechanosensitive, by widely dif- purified and reconstituted into lipid bilayers, nonspecific physical interaction between em- ferent criteria. The risk seems not that their showing that this minimal assembly is suffi- bedded protein and the bilayer here partly mechanosensitivity is in doubt but that cient for temperature sensing (81). There because it is often ignored. There are, of other TRPs are not so assigned, given that has been no report on TRP channel re- course, examples of specific binding of cer- the mechanosensitivity can so easily be constitution experiments to directly test the tain lipids (e.g., PIP2), anesthetics, or drugs, masked, as in Kv and the Drosophila TRP. principle of lipid-force sensing. However, that act on membrane proteins. The cortical cytoskeleton protects the For example, TRPV4 has long been thought indirect evidence is accumulating, arguing – to be activated by a lipid ligand (74) but can against force transmission from partner membrane from being stretch (Fig. 3 A D)in in fact be stretched open (75, 76). proteins. For example, rat TRPV4 has been animal cells in general.Treatmentwithcyto- chalasins, actin-filament dissociating agents, Besides mammals and insects, TRPs are expressed in budding yeast and remains ’ found in protists, algae, diatoms, fungi, etc., responsive to osmotic stimulation (82) and ease the stretch activation of myocyte snative although not in prokaryotes (16). The ex- in Xenopus oocytes displaying mechano- unitary conductances, the first such activation tensively studied TRPY1 (77) in the vacuolar sensitive unitary conductances (75, 76). ever described (84). Similar observations have also been made on mechanosensitive chan- nels of known molecular identity. For exam- ple, treatment with latrunculin, another such A B agent, greatly enhances the suction-induced in current through TREK-1–expressed in COS cells (85). Mutating β-spectrin, which links the membrane to the cytoskeleton, reduces the force needed to pull membrane tether from cultured touch-receptor neurons of the worm with an atomic force microscope (86). out E Theelaborateanatomyofthevertebrate cochlear or the insect Johnston’sorgan C D illustrates how additional structures are built to enhance mechanosensations. At the level of molecules, channels are accessorized to harness or amplify the signals. Some chan- nels clearly choose their own lipids, e.g., the negatively charged PIP2 as deduced by its effects. The binding can even be tight enough for resolution in crystal structure, such as Kir (87). Tightly bound lipids can be considered subunits of such proteins. Many channels seem to preferentially reside in raft-like Fig. 4. The photomechanical transduction pathway in the Drosophila compound eye. (A) A diagram showing the microdomains. Mechanosensitive channels pathway, where the G protein-activated PLC converts PIP to DAG, concentrated in the inner leaflet (upper half of the 2 are found to be in ordered and thicker bilayer in this diagram). (B) Lipid structures emphasize that the beheading of the rod-shaped PIP2 converts it into the cone-shaped DAG, yielding change in both the volume of lipid and spontaneous curvature in the inner leaflet domains with cholesterol and sphingolipids (modified from ref. 64). (C) The arrangement of the cantilever of an atomic force microscope on a retina. (D) Brief (83). Accessory proteins such as cadherins or flashes (bump on the center line) leads to shrinkages as measured by the atomic force microscope, proportional to integrins also form tethers to transmit force light intensity (lower curves). The shrinkage precedes the phototransduction current (upper curves) (from ref. 64). E shows the structure of an ommatidium, the unit of the compound eye, which comprises thousands of microvilli inward. There is little doubt that the trans- where the phototransduction takes place. It is the sum of the small shrinkage in each microvillus that is measured by mitted force gate mechanosensitive channels, the atomic force microscope (modified from ref. 100). as in the case of the hair-cell transduction

Anishkin et al. PNAS | June 3, 2014 | vol. 111 | no. 22 | 7903 Downloaded by guest on October 1, 2021 channel. However, it is unclear whether the or vesicles through entropy-driven hydro- hearing, the Bayliss effect, electromechani- channels bind the tether directly or indirectly phobic interactions. This spontaneity is the cal feedback of the heart, the monitoring of through other proteins to receive the force. key, requiring no additional material or in- blood pressure. Nor do we know how organ- The channel can even receive the force from formation. The self-assembled bilayer comes isms sense gravitation, wind, waves, drought, a lipid microdomain, which is pulled by the with its physical properties and abilities. and flood. The growing literature on the tether (83). The hair-cell tip link produces Even in a naked lipid bilayer, lateral forces importance of lipids and amphipaths leads tenting that appears to tense up the mem- profile respond to deformation (10), heat us to advocate a paradigm that combines brane (88). For cells less specialized than the (96), pH (97), ions (98), chemical mod- the chemistry and the mechanics of the hair cells, a common conception is that the ifications by light (99), etc. It could work bilayer beyond lock-and-key thinking. We channel is pulled by the cytoskeleton. As as an elementary sensor in response to review here the recent extension of the described above, the cortical cytoskeleton changes, including osmotic changes, in force-from-lipid principle from bacterial to prevents rather than activates mechano- the violent primordial soup. The responses animal ion channels. This extension showed sensitive channels (44) (Fig. 3 A–D). Al- may appear crude now, but allow for evo- that membrane proteins are universally gov- though tugging on the stress fibers can lutionary improvements by the embedment erned by the mechanical properties of sur- indeed open certain channels (89), it is of impurities, such as peptides. We are rounding lipids throughout evolution. This again unclear whether we are pulling the now at awe by the sophistication of modern review on the extension of force-from-lipid channel or the surrounding lipids. membranes, which house the key energy principle is meant to remind us that certain If a channel with a small free-energy dif- converters: photosynthesis and oxidative physical principles are so basic that they ference between its closed and open state has phosphorylation. However, at the most basic underlie all molecules, whether they reside to expand a sizable gate to allow ion passage, level, we are still struggling to understand in bacteria, however lowly, or in humans, it must be very sensitive to membrane ten- how physical forces govern various cell bi- however lofty. sion, as in the case of Kv. Additional struc- ological, genetic, and developmental pro- tures may well be needed to prevent ac- cesses. The 21st century remains surprisingly ACKNOWLEDGMENTS. Work in our laboratories is sup- ported by the Huck Institute of Life Sciences (A.A.) and cidental opening. The peripheral domains ignorant of mechanosensitive processes. National Institutes of Health Grant GM096088 and the Vilas in Kv and its related tetrameric channels We know not the molecular bases of touch, Trust of the University of Wisconsin–Madison (to C.K.). may well be such structures. 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