We show that in the potassium channel KcsA, proton-dependent activation is followed by an inactivation process similar to C-type inactivation, and this process is suppressed by an E71A mutation in the pore helix. EPR spectroscopy demonstrates that the inner gate opens maximally at low pH regardless of the magnitude of the single-channel-open probability, implying that stationary gating originates mostly from rearrangements at the selectivity filter. Two E71A crystal structures obtained at 2.5 A reveal large structural excursions of the selectivity filter during ion conduction and provide a glimpse of the range of conformations available to this region of the channel during gating. These data establish a mechanistic basis for the role of the selectivity filter during channel activation and inactivation.

The coupled interplay between activation and inactivation gating is a functional hallmark of K+ channels1,2. This coupling has been experimentally demonstrated from ion interaction effects3,4, cysteine accessibility1 and is associated with a well-defined boundary of energetically coupled residues2. The structure of KcsA in its fully open conformation, as well as four other partial openings, richly illustrates the structural basis of activation-inactivation gating5. Here, we have identified the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that as a consequence of the hinge bending and rotation of TM2, the aromatic ring of Phe103 tilts towards residues Thr74 and Thr75 in the pore helix as well as Ile100 in the neighboring subunit. This allows the network of hydrogen bonds among residues W67, E71, and D80 to destabilize the selectivity filter6,7, facilitating entry to its non-conductive conformation. Mutations at position 103, affect gating kinetics in a size-dependent way: small side chain substitutions F103A and F103C severely impair inactivation kinetics, while larger side chains (F103W) have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open state interaction-energies between Phe103 and surrounding residues. Similar interactions were probed in the Shaker K-channel where inactivation was impaired in the mutant I470A. We propose that side chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K channels.

Snakes possess a unique sensory system for detecting infrared radiation, enabling them to generate a ‘thermal image’ of predators or prey. Infrared signals are initially received by the pit organ, a highly specialized facial structure that is innervated by nerve fibers of the somatosensory system. How this organ detects and transduces infrared signals into nerve impulses is not known. Here we use an unbiased transcriptional profiling approach to identify TRPA1 channels as infrared receptors on sensory nerve fibers that innervate the pit organ. TRPA1 orthologues from pit bearing snakes (vipers, pythons, and boas) are the most heat sensitive vertebrate ion channels thus far identified, consistent with their role as primary transducers of infrared stimuli. Thus, snakes detect infrared signals through a mechanism involving radiant heating of the pit organ, rather than photochemical transduction. These findings illustrate the broad evolutionary tuning of TRP channels as thermosensors in the vertebrate nervous system.

Summary The capsaicin receptor, TRPV1, is regulated by phosphatidylinositol-4,5-bisphosphate (PIP2), although the precise nature of this effect (i.e., positive or negative) remains controversial. Here, we reconstitute purified TRPV1 into artificial liposomes, where it is gated robustly by capsaicin, protons, spider toxins and, notably, heat, demonstrating intrinsic sensitivity of the channel to both chemical and thermal stimuli. TRPV1 is fully functional in the absence of phosphoinositides, arguing against their proposed obligatory role in channel activation. Rather, introduction of various phosphoinositides, including PIP2, PI4P and PI, inhibits TRPV1, supporting a model whereby phosphoinositide turnover contributes to thermal hyperalgesia by disinhibiting the channel. Using an orthogonal chemical strategy, we show that association of the TRPV1 C-terminus with the bilayer modulates channel gating, consistent with phylogenetic data implicating this domain as a key regulatory site for tuning stimulus sensitivity. Beyond TRPV1, these findings are relevant to understanding how membrane lipids modulate other “receptor-operated” TRP channels.

Mechanosensitive ion channels rely on membrane composition to transduce physical stimuli into electrical signals. The Piezo1 channel mediates mechanoelectrical transduction and regulates crucial physiological processes, including vascular architecture and remodeling, cell migration, and erythrocyte volume. The identity of the membrane components that modulate Piezo1 function remain largely unknown. Using lipid profiling analyses, we here identify dietary fatty acids that tune Piezo1 mechanical response. We find that margaric acid, a saturated fatty acid present in dairy products and fish, inhibits Piezo1 activation and polyunsaturated fatty acids (PUFAs), present in fish oils, modulate channel inactivation. Force measurements reveal that margaric acid increases membrane bending stiffness, whereas PUFAs decrease it. We use fatty acid supplementation to abrogate the phenotype of gain-of-function Piezo1 mutations causing human dehydrated hereditary stomatocytosis. Beyond Piezo1, our findings demonstrate that cell-intrinsic lipid profile and changes in the fatty acid metabolism can dictate the cell’s response to mechanical cues.

Transient receptor potential (TRP) channels are polymodal signal detectors that respond to a wide array of physical and chemical stimuli, making them important components of sensory systems in both vertebrate and invertebrate organisms. Mammalian TRPA1 channels are activated by chemically reactive irritants, whereas snake and Drosophila TRPA1 orthologs are preferentially activated by heat. By comparing human and rattlesnake TRPA1 channels, we have identified two portable heat-sensitive modules within the ankyrin repeat-rich aminoterminal cytoplasmic domain of the snake ortholog. Chimeric channel studies further demonstrate that sensitivity to chemical stimuli and modulation by intracellular calcium also localize to the N-terminal ankyrin repeat-rich domain, identifying this region as an integrator of diverse physiological signals that regulate sensory neuron excitability. These findings provide a framework for understanding how restricted changes in TRPA1 sequence account for evolution of physiologically diverse channels, also identifying portable modules that specify thermosensitivity.chemosensation | pain | thermosensation | calcium modulation | somatosensation P rimary afferent (somatosensory) neurons detect a range of physical and chemical stimuli, including temperature, pressure, and noxious irritants (1). The transient receptor potential (TRP) channel family has been shown to play a predominant role in these processes, particularly in regard to thermosensitivity and chemosensitivity (2-5). TRPA1, otherwise known as the "wasabi receptor," plays a key role in somatosensation in evolutionarily diverse phyla, including vertebrate and invertebrate species. Mammalian TRPA1 is expressed by primary afferent sensory neurons of the pain pathway, where it functions as a sensor of environmental and endogenous chemical irritants, such as allyl isothiocyanate (AITC), acrolein, and 4-hydroxynonena, and contributes to cellular mechanisms underlying inflammatory pain (6-9).TRPA1 channels show species-specific functional variation to suit their physiological roles. For example, snakes are unique among vertebrates in that their TRPA1 channels are heat-sensitive, which some species (rattlesnakes, boas, and pythons) have exploited to detect infrared radiation (10). Similarly, insect TRPA1 channels are heat-sensitive and contribute to thermal avoidance behaviors (11)(12)(13)(14). In these cases, however, thermosensitivity comes at the expense of chemosensitivity, such that AITC and other chemical irritants still activate these channels but with reduced potency compared with mammalian orthologs (10). Despite clear physiological differences between snake and mammalian TRPA1 channels, they share significant amino acid identity (56%), providing a unique opportunity to exploit sequence comparisons and domain swaps to pinpoint structural elements associated with stimulus detection and/or gating. Delineating elements that contribute to TRPA1 function will provide insights into the evolutionary process whereby structural changes lead t...

The prokaryotic K+ channel KcsA is activated by intracellular protons and its gating is modulated by transmembrane voltage. Typically, KcsA functions have been studied under steady-state conditions, using macroscopic Rb+-flux experiments and single-channel current measurements. These studies have provided limited insights into the gating kinetics of KcsA due to its low open probability, uncertainties in the number of channels in the patch, and a very strong intrinsic kinetic variability. In this work, we have carried out a detailed analysis of KcsA gating under nonstationary conditions by examining the influence of pH and voltage on the activation, deactivation, and slow-inactivation gating events. We find that activation and deactivation gating of KcsA are predominantly modulated by pH without a significant effect of voltage. Activation gating showed sigmoidal pH dependence with a pKa of ∼4.2 and a Hill coefficient of ∼2. In the sustained presence of proton, KcsA undergoes a time-dependent decay of conductance. This inactivation process is pH independent but is modulated by voltage and the nature of permeant ion. Recovery from inactivation occurs via deactivation and also appears to be voltage dependent. We further find that inactivation in KcsA is not entirely a property of the open-conducting channel but can also occur from partially “activated” closed states. The time course of onset and recovery of the inactivation process from these pre-open closed states appears to be different from the open-state inactivation, suggesting the presence of multiple inactivated states with diverse kinetic pathways. This information has been analyzed together with a detailed study of KcsA single-channel behavior (in the accompanying paper) in the framework of a kinetic model. Taken together our data constitutes the first quantitative description of KcsA gating.

The mechanosensitive channel of small conductance (MscS) is a key determinant in the prokaryotic response to osmotic challenges. Here, we have determined the structural rearrangements associated with MscS activation in membranes using patch-clamp, EPR spectroscopy, and computational analyses. MscS was trapped in its open conformation after modifying the transbilayer pressure profile through the asymmetric incorporation of lysophospholipids. The transition from the closed to the open state is accompanied by the downward tilting of the TM1-TM2 hairpin, and by the expansion, tilt, and rotation of the TM3 helices. These movements expand the permeation pathway, leading to an increase in water accessibility around TM3. Our open MscS model is compatible with single channel conductance measurements and supports the notion that helix tilting is associated with efficient pore widening in mechanosensitive channels.Mechanosensation is involved in many physiological roles, including osmotic balance, touch, and hearing (1,2). At the molecular level, mechanosensitivity relies on the activity of ion channels that transduce a variety of mechanical stimuli to open a conductive pore. Mechanosensitive (MS) channels are grouped by function rather than sequence similarity (3,4). In prokaryotic systems, MS channels respond directly to bilayer deformations, with a transduction mechanism defined at the protein-lipid interface (5,6). Although this is also true for some eukaryotic MS channels (7), many also respond to mechanical deformations through their association with the cytoskeletal network (8).While the molecular identification of eukaryotic MS channels remains challenging (2,9,10), the biophysical and structural properties of prokaryotic MS channels have proved far more tractable at the molecular level. The crystal structures for the MS channels of large (MscL) and small (MscS) conductance (11-13), have provided a molecular framework to interpret functional and biophysical data and have helped establish the basic mechanistic principles by which these two distinct channels sense the physical state of the bilayer (14-17). Nevertheless, given the critical role that lipid-protein interactions play in prokaryotic Correspondence should be addressed to E.P at eperozo@uchicago.edu. Supporting Online Material Material and Methods Fig. S1 Functional, spectroscopic, and computational studies have shown that in the pentameric MscL activation gating proceeds as a result of a large tilt of both transmembrane (TM) segments (14,17,18). Concerted helical rotation and tilting generates a large aqueous pore, much as in the iris of a camera lens. However, an equivalent gating mechanism is not as obvious in the case of MscS. With three TM segments arranged as a homoheptamer (12), the structural design of MscS is very different to that of MscL. Furthermore, while the MscL crystal structure appears to be a good representation of the closed conformation in its native environment (19,20), the functional state represented by the MscS crystal struc...