纤维化疾病中的机械敏感性离子通道Piezo1

Piezo1是哺乳动物中新发现的一种机械敏感(mechanosensitive,MS)离子通道,在不同组织和器官中发挥着重要功能,包括骨骼、泌尿道、眼球和动脉等。然而,异常的Piezo1机械传导会造成多种疾病的发生并促进病程的发展。纤维化疾病几乎可以发生在任何一个组织和器官中,其主要特征是胶原蛋白和其他细胞外基质(extracellular matrix,ECM)成分的过度交联与累积,最终导致组织器官刚度增加,生理功能受到影响。目前,越来越多的研究表明,Piezo1在纤维化疾病的发生和发展中扮演着重要的调控作用,与其基质力学状态变化有着密切联系。本文叙述了Piezo1的结构和激活机理,并且系统地总结了Piezo1在心、肾、胰和肝等多种器官纤维化疾病中的研究进展,以期为纤维化疾病的治疗提供新的视角和策略。     

The "dashpot" mechanism of stretch-dependent gating in MscS

The crystal structure of the small conductance mechanosensitive channel (MscS) has been an invaluable tool in the search for the gating mechanism, however many functional aspects of the channel remain unsettled. Here we characterized the gating of MscS in Escherichia coli spheroplasts in a triple mutant (mscL-, mscS-, mscK-) background. We used a pressure clamp apparatus along with software developed in-lab to generate dose-response curves directly from two-channel recordings of current and pressure. In contrast to previous publications, we found that MscS exhibits essentially voltage-independent activation by tension, but at the same time strong voltage-dependent inactivation under depolarizing conditions. The MscS activation curves obtained under saturating ramps of pressure, at different voltages, gave estimates for the energy, area, and gating charge for the closed-to-open transition as 24 kT, 18 nm2, and +0.8, respectively. The character of activation and inactivation was similar in both K+ and Na+ buffers. Perhaps the most salient and intriguing property of MscS gating was a strong dependence on the rate of pressure application. Patches subjected to various pressure ramps from 2.7 to 240 mmHg/s revealed a midpoint of activation almost independent of rate. However, the resultant channel activity was dramatically lower when pressure was applied slowly, especially at depolarizing pipette voltages. It appears that MscS prefers to respond in full to abrupt stimuli but manages to ignore those applied slowly, as if the gate were connected to the tension-transmitting element via a velocity-sensitive "dashpot." With slower ramps, channels inactivate during the passage through a narrow region of pressures below the activation midpoint. This property of "dumping" a slowly applied force may be important in environmental situations where rehydration of cells occurs gradually and release of osmolytes is not desirable. MscS often enters the inactivated state through subconducting states favored by depolarizing voltage. The inactivation rate increases exponentially with depolarization. Based on these results we propose a kinetic scheme and gating mechanism to account for the observed phenomenology in the framework of available structural information.     

MSL1 is a mechanosensitive ion channel that dissipates mitochondrial membrane potential and maintains redox homeostasis in mitochondria during abiotic stress

Mitochondria must maintain tight control over the electrochemical gradient across their inner membrane to allow ATP synthesis while maintaining a redox‐balanced electron transport chain and avoiding excessive reactive oxygen species production. However, there is a scarcity of knowledge about the ion transporters in the inner mitochondrial membrane that contribute to control of membrane potential. We show that loss of MSL1, a member of a family of mechanosensitive ion channels related to the bacterial channel MscS, leads to increased membrane potential of Arabidopsis mitochondria under specific bioenergetic states. We demonstrate that MSL1 localises to the inner mitochondrial membrane. When expressed in Escherichia coli, MSL1 forms a stretch‐activated ion channel with a slight preference for anions and provides protection against hypo‐osmotic shock. In contrast, loss of MSL1 in Arabidopsis did not prevent swelling of isolated mitochondria in hypo‐osmotic conditions. Instead, our data suggest that ion transport by MSL1 leads to dissipation of mitochondrial membrane potential when it becomes too high. The importance of MSL1 function was demonstrated by the observation of a higher oxidation state of the mitochondrial glutathione pool in msl1‐1 mutants under moderate heat‐ and heavy‐metal‐stress. Furthermore, we show that MSL1 function is not directly implicated in mitochondrial membrane potential pulsing, but is complementary and appears to be important under similar conditions.     

Gadolinium Ions Block Mechanosensitive Channels by Altering the Packing and Lateral Pressure of Anionic Lipids

Effects of polyvalent ions on the lateral packing of phospholipids have been known for decades, but the physiological consequences have not been systematically studied. Gd³⁺ is a relatively nonspecific agent that blocks mechano-gated channels with a variable affinity. In this study, we show that the large mechanosensitive channel MscL of Escherichia coli is effectively blocked by Gd³⁺ only when reconstituted with negatively charged phospholipids (e.g., PS). Taking this lead, we studied effects of Gd³⁺ on monolayers and unilamellar vesicles made of natural brain PS, DMPS, and its mixtures with DMPC. In monolayer experiments, we found that μM Gd³⁺ present in the subphase leads to ∼8% lateral compaction of brain PS (at 35 mN/m). Gd³⁺ more strongly shrinks and rigidifies DMPS films causing a spontaneous liquid expanded-to-compact transition to the limiting 40 Ų/mol. Pressure-area isotherms of uncharged DMPC were unaffected by Gd³⁺, and neutralization of DMPS surface by low pH did not produce strong compaction. Upshifts of surface potential isotherms of DMPS monolayers reflected changes in the diffuse double layer due to neutralization of headgroup charges by Gd³⁺, whereas the increased packing density produced up to a 200 mV change in the interfacial dipole potential. The slopes of surface potential versus reciprocal area predicted that Gd³⁺ induced a modest (∼18%) increase in the magnitude of the individual lipid dipoles in DMPS. Isothermal titration calorimetry indicated that binding of Gd³⁺ to DMPS liposomes in the gel state is endothermic, whereas binding to liquid crystalline liposomes produces heat consistent with the isothermal liquid-to-gel phase transition induced by the ion. Both titration curves suggested a K_b of ∼10⁶ M⁻¹. We conclude that anionic phospholipids serve as high-affinity receptors for Gd³⁺ ions, and the ion-induced compaction generates a lateral pressure increase estimated as tens of mN/m. This pressure can “squeeze” the channel and shift the equilibrium toward the closed state.     

Identification of skeletal muscle autoantigens by expression library screening using sera from autoimmune rippling muscle disease (ARMD) patients

Novel forms of contractile regulation observed in skeletal muscle are evident in neuromuscular diseases like rippling muscle disease (RMD). Previous studies of an autoimmune form of RMD (ARMD) identified a very high molecular weight skeletal muscle protein antigen recognized by ARMD patient antisera. This study utilized ARMD and myasthenia gravis (MG) patient antisera, to screen a human skeletal muscle cDNA library that subsequently identified proteins that could play a role in ARMD. Based on nucleotide sequence analysis, three distinct ARMD antigens were identified: titin Isoform N2A, ATP synthase 6, and PPP1R3 (protein phosphatase 1 regulatory subunit 3). The region of titin identified by ARMD antisera is distinct from the main immunogenic region (MIR) recognized by classical MG antibodies. Sera from classical MG patient identifies an expressed sequence corresponding to the titin MIR. Although the mechanism of antibody penetration is not known, previous studies have shown that rippling muscle antibodies affect the contractile machinery of myofibers resulting in mechanical sensitivity. Titin's role as a modulator of muscle contractility makes it a potential target in understanding muscle mechanosensitive regulation.     

The dynamics of protein-protein interactions between domains of MscL at the cytoplasmic-lipid interface

The bacterial mechanosensitive channel of large conductance, MscL, is one of the best characterized mechanosensitive channels serving as a paradigm for how proteins can sense and transduce mechanical forces. The physiological role of MscL is that of an emergency release valve that opens a large pore upon a sudden drop in the osmolarity of the environment. A crystal structure of a closed state of MscL shows it as a homopentamer, with each subunit consisting of two transmembrane domains (TM). There is consensus that the TM helices move in an iris like manner tilting in the plane of the membrane while gating. An N-terminal amphipathic helix that lies along the cytoplasmic membrane (S1), and the portion of TM2 near the cytoplasmic interface (TM2_ci), are relatively close in the crystal structure, yet predicted to be dynamic upon gating. Here we determine how these two regions interact in the channel complex, and study how these interactions change as the channel opens. We have screened 143 double-cysteine mutants of E. coli MscL for their efficiency in disulfide bridging and generated a map of protein-protein interactions between these two regions. Interesting candidates have been further studied by patch clamp and show differences in channel activity under different redox potentials; the results suggest a model for the dynamics of these two domains during MscL gating.     

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics. However, the structural basis of the dynamic process from the closed to open states in MscL is not fully understood. In this study, we performed molecular dynamics (MD) simulations on the initial process of MscL opening in response to a tension increase in the lipid bilayer. To identify the tension-sensing site(s) in the channel protein, we calculated interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids. We found that Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL. Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78, and the force was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices in the inner leaflet of the bilayer. The radial dragging force induced radial sliding of the crossing portions, leading to a gate expansion. Calculated energy for this expansion is comparable to an experimentally estimated energy difference between the closed and the first subconductance state, suggesting that our model simulates the initial step toward the full opening of MscL. The model also successfully mimicked the behaviors of a gain of function mutant (G22N) and a loss of function mutant (F78N), strongly supporting that our MD model did simulate some essential biophysical aspects of the mechano-gating in MscL.     

The dynamics of protein-protein interactions between domains of MscL at the cytoplasmic-lipid interface

The bacterial mechanosensitive channel of large conductance, MscL, is one of the best characterized mechanosensitive channels serving as a paradigm for how proteins can sense and transduce mechanical forces. The physiological role of MscL is that of an emergency release valve that opens a large pore upon a sudden drop in the osmolarity of the environment. A crystal structure of a closed state of MscL shows it as a homopentamer, with each subunit consisting of two transmembrane domains (TM). There is consensus that the TM helices move in an iris like manner tilting in the plane of the membrane while gating. An N-terminal amphipathic helix that lies along the cytoplasmic membrane (S1), and the portion of TM2 near the cytoplasmic interface (TM2_ci), are relatively close in the crystal structure, yet predicted to be dynamic upon gating. Here we determine how these two regions interact in the channel complex, and study how these interactions change as the channel opens. We have screened 143 double-cysteine mutants of E. coli MscL for their efficiency in disulfide bridging and generated a map of protein-protein interactions between these two regions. Interesting candidates have been further studied by patch clamp and show differences in channel activity under different redox potentials; the results suggest a model for the dynamics of these two domains during MscL gating.     

Inactivation Kinetics and Mechanical Gating of Piezo1 Ion Channels Depend on Subdomains within the Cap

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