Gating-by-tilt of mechanosensitive membrane channels

We propose an alternative mechanism for the gating of biological membrane channels in response to membrane tension that involves a change in the slope of the membrane near the channel. Under biological membrane tensions we show that the energy difference between the closed (tilted) and open (untilted) states can far exceed kBT and is comparable to what is available under simple ilational gating. Recent experiments demonstrate that membrane leaflet asymmetries (spontaneous curvature) can strong effect the gating of some channels. Such a phenomenon would be more easy to explain under gating-by-tilt, given its novel intrinsic sensitivity to such asymmetry.Comment: 10 pages, 2 figure

The Regulation of Medicines in Croatia - a Contribution to Public Health

Regulatorni sustav za lijekove uključuje postojanje zakonodavnog okvira te agencije za lijekove kao regulatornog tijela. Do sada su provedene tri regulatorne reforme kako bi zakonski okvir bio što bolje usklađen s pravnom stečevinom EU-a, u kojoj lijekovi predstavljaju jedno od najbolje reguliranih i usklađenih područja. U svrhu reguliranja hrvatskog tržišta lijekova, 2003. godine osnovana je Agencija za lijekove i medicinske proizvode, koja provodi postupak davanja odobrenja za stavljanje lijeka u promet, nadzor nad neželjenim učincima lijeka, laboratorijsku provjeru kakvoće lijekova i cjepiva uzorkovanih iz prometa, izdavanje dozvola za proizvodnju i distribuciju lijekova, praćenje potrošnje lijekova te informiranje o lijekovima i promicanje njihove racionalne uporabe. Medicinski proizvodi su regulirani posebnim zakonom te Agencija na tom polju provodi upis u očevidnik i vigilanciju medicinskih proizvoda. Na ovaj su način proizvodi za zdravstvo koji se nalaze na hrvatskom tržištu odgovarajuće kakvoće, sigurnosti i djelotvornosti te su nadalje pod stalnim nadzorom nadležnog tijela koje ocjenjuje njihov omjer koristi i rizika. Pristupom Republike Hrvatske punopravnom članstvu u EU Agencija će se uključiti u europske postupke odobravanja lijekova, kao što su centralizirani postupak pri Europskoj agenciji za lijekove (EMA), ali prije svega postupak uzajamnog priznavanja te decentralizirani postupak, gdje je veća uloga agencija zemalja članica EU-a. U ovom članku dan je pregled najvažnijih regulatornih aktivnosti iz područja lijekova te spremnost Agencije na funkcioniranje u budućem integriranom europskom regulatornom prostoru.The regulatory system for medicinal products includes the existence of a legislative framework and a medicines agency as the regulatory body. The legislative framework for medicinal products has been amended several times so as to align it with the EU acquis communautaire, where medicinal products represent one of the best regulated and aligned areas. For the purpose of regulating the Croatian medicines market, the Agency for Medicinal Products and Medical Devices was established in 2003 to implement the procedure of granting marketing authorisation for medicinal products, to supervise the adverse reactions of medicinal products, to conduct laboratory tests of the quality of medicines and vaccines sampled from the market, to issue licences for the manufacture and distribution of medicinal products, to monitor medicine consumption, and to inform about medicines and promote their rational use. Medical devices are regulated under a special act, and the Agency conducts entries into the register in that field and carries out vigilance over medical devices. In this way, products intended for health care on the Croatian market are of the appropriate quality, safety and efficacy, and are under the constant supervision of the competent body that assesses their risk-to-benefit ratio. Upon accession of the Republic of Croatia to full membership in the European Union, the Agency will be included in the European authorisation procedures for medicines, such as the centralised procedure in the European Medicines Agency (EMA), and above all, the mutual recognition procedure and decentralised procedure in which the role of the agencies of EU Member States is greater. This article gives an overview of the most important regulatory activities in the field of medicinal products, and the readiness of the Agency to function in the future integrated European regulatory area

Disruption of membrane cholesterol organization impairs the activity of PIEZO1 channel clusters

The human mechanosensitive ion channel PIEZO1 is gated by membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently, little is known about PIEZO1 plasma membrane localization and organization. Using a PIEZO1-GFP fusion protein, we investigated whether cholesterol enrichment or depletion by methyl-β-cyclodextrin (MBCD) and disruption of membrane cholesterol organization by dynasore affects PIEZO1-GFP’s response to mechanical force. Electrophysiological recordings in the cell-attached configuration revealed that MBCD caused a rightward shift in the PIEZO1-GFP pressure–response curve, increased channel latency in response to mechanical stimuli, and markedly slowed channel inactivation. The same effects were seen in native PIEZO1 in N2A cells. STORM superresolution imaging revealed that, at the nanoscale, PIEZO1-GFP channels in the membrane associate as clusters sensitive to membrane manipulation. Both cluster distribution and diffusion rates were affected by treatment with MBCD (5 mM). Supplementation of polyunsaturated fatty acids appeared to sensitize the PIEZO1-GFP response to applied pressure. Together, our results indicate that PIEZO1 function is directly dependent on the membrane composition and lateral organization of membrane cholesterol domains, which coordinate the activity of clustered PIEZO1 channels

Heterologously-expressed and Liposome-reconstituted Human Transient Receptor Potential Melastatin 4 Channel (TRPM4) is a Functional Tetramer

Mutation, irregular expression and sustained activation of the Transient Receptor Potential Channel, type Melastatin 4 (TRPM4), have been linked to various cardiovascular diseases. However, much remains unknown about the structure of this important ion channel. Here, we have purified a heterologously expressed TRPM4-eGFP fusion protein and investigated the oligomeric state of TRPM4-eGFP in detergent micelles using crosslinking, native gel electrophoresis, multi-angle laser light scattering and electron microscopy. Our data indicate that TRPM4 is tetrameric, like other TRP channels studied to date. Furthermore, the functionality of liposome reconstituted TRPM4-eGFP was examined using electrophysiology. Single-channel recordings from TRPM4-eGFP proteoliposomes showed inhibition of the channel using Flufenamic acid, a well-established inhibitor of TRPM4, suggesting that the channels are functional upon reconstitution. Our characterisation of the oligomeric structure of TRPM4 and the ability to reconstitute functional channels in liposomes should facilitate future studies into the structure, function and pharmacology of this therapeutically relevant channel

Inhibitory Control Over Ca2+ Sparks via Mechanosensitive Channels Is Disrupted in Dystrophin Deficient Muscle but Restored by Mini-Dystrophin Expression

Background: In dystrophic skeletal muscle, osmotic stimuli somehow relieve inhibitory control of dihydropyridine receptors (DHPR) on spontaneous sarcoplasmic reticulum elementary Ca release events (ECRE) in high Ca external environments. Such 'uncontrolled' Ca sparks were suggested to act as dystrophic signals. They may be related to mechanosensitive pathways but the mechanisms are elusive. Also, it is not known whether truncated dystrophins can correct the dystrophic disinhibition. Methodology/Principal Findings: We recorded ECRE activity in single intact fibers from adult wt, mdx and mini-dystrophin expressing mice (MinD) under resting isotonic conditions and following hyper-/ hypo-osmolar external shock using confocal microscopy and imaging techniques. Isotonic ECRE frequencies were small in wt and MinD fibers, but were markedly increased in mdx fibers. Osmotic challenge dramatically increased ECRE activity in mdx fibers. Sustained osmotic challenge induced marked exponential ECRE activity adaptation that was three times faster in mdx compared to wt and MinD fibers. Rising external Ca concentrations amplified osmotic ECRE responses. The eliminated ECRE suppression in intact osmotically stressed mdx fibers was completely and reversibly resuscitated by streptomycine (200 μM), spider peptide GsMTx-4 (5 μM) and Gd (20 μM) that block unspecific, specific cationic and Ca selective mechanosensitive channels (MsC), respectively. ECRE morphology was not substantially altered by membrane stress. During hyperosmotic challenge, membrane potentials were polarised and a putative depolarisation through aberrant MsC negligible excluding direct activation of ECRE through tubular depolarisation. Conclusions/Significance: Dystrophin suppresses spontaneous ECRE activity by control of mechanosensitive pathways which are suggested to interact with the inhibitory DHPR loop to the ryanodine receptor. MsC-related disinhibition prevails in dystrophic muscle and can be resuscitated by transgenic mini-dystrophin expression. Our results have important implications for the pathophysiology of DMD where abnormal MsC in dystrophic muscle confer disruption of microdomain Ca homeostasis. MsC blockers should have considerable therapeutic potential if more muscle specific compounds can be found

TMEM87a/Elkin1, a component of a novel mechanoelectrical transduction pathway, modulates melanoma adhesion and migration

Mechanoelectrical transduction is a cellular signalling pathway where physical stimuli are converted into electro-chemical signals by mechanically activated ion channels. We describe here the presence of mechanically activated currents in melanoma cells that are dependent on TMEM87a, which we have renamed Elkin1. Heterologous expression of this protein in PIEZO1-deficient cells, that exhibit no baseline mechanosensitivity, is sufficient to reconstitute mechanically activated currents. Melanoma cells lacking functional Elkin1 exhibit defective mechanoelectrical transduction, decreased motility and increased dissociation from organotypic spheroids. By analysing cell adhesion properties, we demonstrate that Elkin1 deletion is associated with increased cell-substrate adhesion and decreased homotypic cell-cell adhesion strength. We therefore conclude that Elkin1 supports a PIEZO1-independent mechanoelectrical transduction pathway and modulates cellular adhesions and regulates melanoma cell migration and cell-cell interactions

Dobra proizvođačka praksa: uloga domaćih proizvođača i nadležnih tijela

In every country, a manufacturer of medicinal products for either human or veterinary use is required to operate in compliance with local legislation effect that they are committed to abide by the same standards. The candidate countries transpose the acquis into their national legislation, including the good manufacturing practice (GMP). Consequently, the local manufacturer is required to strictly comply with GMP and the manufacturing licence, including for medicinal products exclusively intended for export. A vital role is also played by national regulatory authorities, in Croatia by the Agency for Medicinal Products and Medical Devices which issues the manufacturing licence, GMP certifi cate, and the Certifi cate of a Pharmaceutical Product (CPP) and conducts laboratory control of products. GMP inspection is carried out by the Pharmaceutical Inspectorate with the Ministry of Health and Social Welfare. Both authorities are responsible only for human medicines. There are legislative issues not yet harmonised with the acquis, but as a country aspiring for the EU membership, Croatia is expected to demonstrate that its industry and competent authorities are able to conform to current requirements and thus fully adhere to the integrated European regulatory network. Hence the importance of strengthening the institutional capacity of the competent authorities, as insuffi cient resources may have a direct bearing on patients by limiting their access to affordable treatment.U svakoj zemlji proizvođač lijekova za humanu ili za veterinarsku uporabu obavezan je poslovati sukladno lokalnom zakonodavstvu, koje je u EU usklađeno za sve članice koje moraju poštivati jednake standarde. Zemlje kandidati za članstvo ugrađuju europsko zakonodavstvo u nacionalno i na taj način implementiraju dobru proizvođačku praksu (GMP). Sukladno tomu, proizvođač lijeka obvezan je osigurati da se svi proizvodni postupci za lijekove izvode u skladu s dobrom proizvođačkom praksom i proizvodnom dozvolom uključujući i lijekove koji su namijenjeni samo za izvoz. Ovdje je nezaobilazna i uloga nacionalnih regulatornih tijela posebno Agencije za lijekove i medicinske proizvode koja izdaje proizvodnu dozvolu, GMP certifi kate i certifi kate o farmaceutskom proizvodu - lijeku (CPP) te provodi laboratorijsku kontrolu proizvoda. GMP inspekciju provodi farmaceutski inspektorat koji je u sastavu Ministarstva zdravstva i socijalne skrbi. Oba su tijela nadležna samo za lijekove za humanu uporabu. Postoje još neka neusklađena pitanja što se tiče prihvaćanja Pravne stečevine na ovome polju, ali kako je Hrvatska zemlja kandidat za punopravno članstvo u EU, očekuje se da će moći demonstrirati da njezina industrija i nadležna tijela poštuju važeće zahtjeve EU i tako potpuno pristupaju europskoj regulatornoj mreži. Stoga je važno jačati institucionalni kapacitet nadležnih tijela, jer nedostatni potencijali mogu izravno utjecati na pacijente ograničavajući im pristup dostupnim terapijama

MscS-like mechanosensitive channels in plants and microbes

The challenge of osmotic stress is something all living organisms must face as a result of environmental dynamics. Over the past three decades, innovative research and cooperation across disciplines have irrefutably established that cells utilize mechanically gated ion channels to release osmolytes and prevent cell lysis during hypoosmotic stress. Early electrophysiological analysis of the inner membrane of Escherichia coli identified the presence of three distinct mechanosensitive activities. The subsequent discoveries of the genes responsible for two of these activities, the mechanosensitive channels of large (MscL) and small (MscS) conductance, led to the identification of two diverse families of mechanosensitive channels. The latter of these two families, the MscS family, consists of members from bacteria, archaea, fungi, and plants. Genetic and electrophysiological analysis of these family members has provided insight into how organisms use mechanosensitive channels for osmotic regulation in response to changing environmental and developmental circumstances. Furthermore, determining the crystal structure of E. coli MscS and several homologues in several conformational states has contributed to our understanding of the gating mechanisms of these channels. Here we summarize our current knowledge of MscS homologues from all three domains of life and address their structure, proposed physiological functions, electrophysiological behaviors, and topological diversity

Building programmable multicompartment artificial cells incorporating remotely activated protein channels using microfluidics and acoustic levitation

Abstract: Intracellular compartments are functional units that support the metabolism within living cells, through spatiotemporal regulation of chemical reactions and biological processes. Consequently, as a step forward in the bottom-up creation of artificial cells, building analogous intracellular architectures is essential for the expansion of cell-mimicking functionality. Herein, we report the development of a droplet laboratory platform to engineer complex emulsion-based, multicompartment artificial cells, using microfluidics and acoustic levitation. Such levitated models provide free-standing, dynamic, definable droplet networks for the compartmentalisation of chemical species. Equally, they can be remotely operated with pneumatic, heating, and magnetic elements for post-processing, including the incorporation of membrane proteins; alpha-hemolysin; and mechanosensitive channel of large-conductance. The assembly of droplet networks is three-dimensionally patterned with fluidic input configurations determining droplet contents and connectivity, whilst acoustic manipulation can be harnessed to reconfigure the droplet network in situ. The mechanosensitive channel can be repeatedly activated and deactivated in the levitated artificial cell by the application of acoustic and magnetic fields to modulate membrane tension on demand. This offers possibilities beyond one-time chemically mediated activation to provide repeated, non-contact, control of membrane protein function. Collectively, this expands our growing capability to program and operate increasingly sophisticated artificial cells as life-like materials