FUNDAMENTE TEORETICE SI METODE IN CERCETAREA BIOMEDICALA FUNDAMENTALA

Mai 2011

BAZELE MOLECULARE ALE PROCESELOR FIZIOLOGICE DE LA NIVELUL BIOMEMBRANELOR

Mai 2011

Esena vieii (fenomene biotice) const n capacitatea structurilor vii de a oxida gradat diferite substane, utiliznd mare parte din energia chimic astfel eliberat pentru desfurarea anumitor reacii chimice ce consum energie (endergonice). Ansamblul acestor procese de nivel molecular se numete metabolism i st la baza vieii, asigurnd condiiile energetice pentru fenomenele antientropice de pstrare i dezvoltare a structurilor proprii.

 Metabolismul cuprinde fenomene catabolice (litice) anabolice (sintetice) Catabolism nseamn deci scindarea substanelor complexe, cu eliberare de energie chimic de legtur, care este n parte stocat sub forma legturilor fosfat macroergice din diferii compui, n special adenozin-trifosfat (ATP). Lanurile de reacii catabolice realizeaz de fapt un transfer treptat de electroni ctre atomii de oxigen, rezultnd n final CO2 i H2O, precum i ali produi finali de catabolism, ce nu mai pot fi oxidai la nivel celular. Anabolism nseamn sintez de substane proprii, cu consum de energie, furnizat de catabolism prin ATP.

 Tesutul este o grupare de celule nvecinate ce aparin aceluiai tip tisular, adic au aceeai specializare morfofuncional. Tipurile tisulare principale sunt: epitelial, muscular, nervos, conjunctiv. Organele sunt grupri tisulare structurate n scop de integrare a funciilor, care pot astfel deveni mai complexe, cu elemente calitativ superioare ce nu se manifest ca atare la simplul nivel celular sau tisular. Tot astfel organele formeaz sisteme funcionale de organe, numite uneori aparate. Organismele sunt organizate n nivele superioare, pe plan ecologic (populaie, biocenoz / ecosistem, biosfer) i sistematic (populaie, specie, gen, familie, clas, ncrengtur, regn).

Pornind de la aspectele generale i particulare ale funciilor celulare, fiziologia descrie integrarea acestora la nivel de organ, sistem i organism n cadrul marilor funcii
de nutritiie (digestie, respiraie, excreie, circulaie), de reglare / comand / control (neuro-endocrine), de relaie (senzoriale, somatomotorii, psihocomp.).

 Intruct n organismul uman marea majoritate a celulelor nu pot ntreine schimburi directe cu mediul extern al acestuia, pentru viaa i activitatea lor, a fost necesar ca acestea s se diferenieze i s se organizeze n organe, aparate i sisteme care s poat s pun celulele n contact indirect, prin intermediul mediului intern, cu mediul nconjurtor. In acest scop s-au difereniat: aparatul digestiv care asigur aportul de substane nutritive i biologic active din mediul extern, sistemul hemodinamic care asigur distribuia sngelui purttor de nutrimente i principii biologic active tuturor esuturilor, aparatul respirator care asigur oxigenul necesar metabolismului i ndeprtarea bioxidului de carbon

La nivel celular se manifest toate proprietile generale, fundamentale, ale materiei vii: metabolismul, excitabilitatea, micarea, reproducerea. Ele sunt asigurate morfofuncional la nivel subcelular n mod difereniat, prin specializrile organitelor celulare. Unele aspecte ale vieii sunt prezente izolat, oarecum incomplet, i la nivel de organite izolate i chiar ansambluri supramoleculare reconstituite artificial. Pe de alt parte, n cadrul organismelor pluricelulare ele sunt integrate, rezultnd manifestri complexe la nivel de esut, organ, aparat, organism.

Celula este alctuit din membran, citoplasm i nucleu. Componenta principal a membranei este plasmalema, un bistrat fosfolipidic n care se gsesc proteine periferice i integrale, la care se adaug glicokalixul (cu compoziie predominant glicoproteic) la exterior i citoscheletul submembranar i reticulul superficial la interior. Citoplasma este compartimentat printr-un sistem de membrane fosfolipidice intracelulare. Organitele delimitate de membrane sunt de tip vezicular / canalicular i cuprind: reticulul endoplasmic, mitocondriile, lizozomii, aparatul Golgi, vezicule de endo- i exocitoz. Alte organite (nedelimitate de membrane) pot fi granulare, ca ribozomii i diverse incluziuni citoplasmatice, sau fibrilare, grupate ntr-un ansamblu morfo-funcional numit citoschelet.

In afar de organitele menionate, comune tuturor celulelor, exist varieti ale acestora, caracteristice anumitor tipuri celulare. - structurarea filamentelor de miozin i actin n miofibrile cu organizare sarcomeric n fibrele musculare striate. - n axonii neuronilor se constituie neurofilamente, n care tubulina particip la transportul axonal de substane - evaginri membranare filiforme ale plasmalemei numite cili, ce pot fi mobili prin prezena unei diferenieri citoscheletice specifice. - n timpul mitozei microtubulii formeaz fusul de diviziune, ce asigur migrarea ctre polii celulei a materialului genetic (cromatide n cazul mitozei sau cromozomi n cazul meiozei).

A. DEFINITII SI CLASIFICARI ALE BIOMEMBRANELOR B. PLASMALEMA SI STRUCTURILE ASOCIATE B1. Mozaicul fluid si diferentierile sale functionale: domenii membranare lipid rafts, caveole, poli de exocitoza B2. Structuri asociate cu plasmalema: glicokalix; citoschelet si reticul endoplasmic submembranar B4. Jonctiuni celulare B3. Transferul de substante prin plasmalema: definitii, clasificari si exemplificari (exceptand canalele ionice) B3.1. Canale ionice: operare, definitii si clasificri B3.2. Canalul de sodiu voltaj-dependent rapid B3.3. Canale de potasiu B3.4. Canale de clor B4. Potentiale membranare: asimetria ionica si ecuatia Goldman B4.1. Polarizari si depolarizari locale si globale B4.2. Potentiale de actiune

B5. Receptori membranari definitii, clasificari; sisteme de semnalizare B5.1. Receptori colinergici B5.2. Receptori adrenergici B5.3. Receptori pentru alte amine B5.4. Receptori purinergici B5.5. Receptori pentru aminoacizi B5.6. Receptori pentru peptide C. MEMBRANA RETICULARA: ARHTECTURA, DIFERENTIERI C1. Transferul de substante prin membrana reticulara: exemplificari C2. Sinteza proteica si procesarea post-translationala C3. Membrana lizozomala C4. Traficul fosfolipidelor membranare: sinteza D. MEMBRANE MITOCONDRIALE; FOSFORILAREA OXIDATIVA E. MEMBRANA NUCLEARA F. SINTEZA: PROCES, MEMBRANA, SUBSTRAT MOLECULAR

Plasmalema este un bistrat fosfolipidic ce conine i alte lipide, precum i proteine intrinseci (integrale, ce strbat ambele straturi) sau extrinseci (periferice, aparinnd unui singur strat). Plasmalema delimiteaz celula, asigurnd schimburi controlate de substan i informaie cu mediul extracelular. Ea prezint structuri specializate pentru diverse aspecte funcionale: transferul substanelor hidrofile (pori, canale, transportori), recunoaterea semnalelor biochimice (receptori), legtura cu celulele nvecinate (jonciuni strnse i comunicante), micarea celulei / lichidului extracelular (cili i flageli).

 a. deine rolul de barier de difuziune, adic de frontier fizic ntre mediul intra- i extracelular participnd astfel la meninerea diferenelor de concentraie a substanelor ntre aceste dou teritorii; b. asigur transportul anumitor substane. Aceste transporturi contribuind de asemeni la meninerea compoziiei mediului intracelular necesar derulrii tuturor reaciilor biochimice intracelulare; c. are rol fundamental n transmiterea informaiei ntre mediul intrai extracelular, dar n aceiai msur de la o celul la alta. n acest ultim domeniu, al transmiterii informaiei, membranele celulelor aa zis excitabile (neuronale, muchilor scheletici, netezi i cardiaci etc) joac un rol fundamental n cuplarea excitaiei proprii cu rspunsul fiziologic al celulei (eliberare de neuromediatori, contracie muscular etc). ectoplasm hialoplasm, mai vscoas i rigid dect granuloplasma suprafa celular = plasmalema propriu-zis acoperit de zona de microvecintate

LOG RAFTS

PLUTE DE BUSTENI

KON-TIKI

famous balsa raft floating on the Pacific

KON-TIKI ends up in museum

famous LIPID RAFTS

float forever in the membranes

LIPID RAFTS subset of membrane lateral heterogeneities due to lipid-lipid immiscibility

PLASMALEMMAL PROTEINS

= many in the liquid disordered regions = some preferentially in the ordered

raft domains

LIPID RAFTS

= highly dynamic, submicroscopic assemblies = rich in sphingolipids and cholesterol = float within the liquid phospholipid bilayer = coalesce upon clustering of their components

LIPID RAFTS

= platforms for protein attachment - when membranes are moved - during signal transduction

HISTORY

outer leaflet packing inner leaflet

Israelachvili 1973

lipid domains in biological membranes Karnovsky 1979

perturbation by free fatty acids regulation of receptor mobility

detergent-resistant membrane fractions

Brown 1992

resistance of lipid rafts to extraction by Triton X-100 at 4C

HISTORY In plasmalema exista microdomenii lipidice rezistente la detergenti, care au fluiditate mai redusa si organizare mai stabila decat marea fosfolipidica, si care pot fi implicate in dinamica proteinelor membranare
Rusu V, 1985, prelegere de biofizica pentru studentii la medicina

HISTORY
Simons K, Ikonen E (1997) Nature 387(6633):569-72 Functional rafts in cell membranes

1992-1997 1997-2005

~0015 papers ~2400 papers

~003/year ~300/year

Meder D,[...] Simons K (2006) Proc Natl Acad Sci U S A 103(2):329-34

LIPID RAFTS - NATURE / STRUCTURE

- outer leaflet (inner ?) - saturated hydrocarbon chains liquid-ordered = tightly packed - size and functions are debated - DRM = aggregates of rafts, not native state - too small to be optically resolved, untill recently
Gaus 2003, Parton 2003

- highly dynamic; partitioning kinetics

raft-associated proteins
GPI-anchored cholesterol-binding (caveolins) Src-TyrK (Lck, Fyn, Lyn; 2 acyl anchor) heterotrimeric GTP-binding hedgehog phospholipid-binding (annexins) palmitoylated & myristoylated (flotillins)
Chatterjee 2001 Kurzchalia 1999 Simons 2000 Mofett 2000 Karpen 2001 Babiychuk 2002 Rajendran 2003

GPI ANCHORS

LIPID RAFTS STRUCTURE

LIPID RAFTS ORGANISATION

FUNCTION
(non)caveolae-mediated endocytosis sorting in polarised epithelial cells virus budding immune receptor signalling

= membrane traffic + control = plasmalemmal signal transduction + receptor targeting / cycling domain receptor densities signal control

ENDOCYTOSIS MECHANISMS

LIPID RAFTS IN PLASMALEMMAL SIGNAL TRANSDUCTION

LIPID RAFTS

WHERE TO ?

 Excitabilitatea este proprietatea materiei vii de a rspunde, mai mult sau mai puin specific, la aciunea unui stimul adecvat. Stimulul (excitaia) este o variaie energetic din mediu i poate induce rspunsul dac este adecvat ca: form energetic, amplitudine, durat de aciune, bruschee (rat de transfer energetic). O form particular este aa numita excitabilitate electric, capacitatea unei membrane celulare de a genera i conduce semnale electrice speciale numite poteniale de aciune. Cuplarea dintre excitaie i rspuns este un fenomen deosebit de complex, care de obicei cuprinde o etap de transducie membranar i mecanisme de semnalizare intracelular.

Membrane Transport Proteins Transport of small molecules

Movement of molecules and maintenance of special environments within cells accomplished by special transmembrane proteins 15-30% of a cells genes encode membrane proteins 75% of cells metabolic energy in transport

Protein-free Lipid Bilayers

Eventually, most molecules will diffuse through down concentration gradient Rate differs dependent upon type of molecule Membrane virtually impermeable to charged ions

Membrane Transport Proteins

Biological membranes will also allow many molecules to permeate However, simple diffusion rate for many very slow or virtually nonexistent Thus, ions, sugars, amino acids, nucleotides, etc. assisted in movement by special proteins

Two types of membrane transport processes: Passive Transport Some carrier proteins All channel proteins Active Transport Some carrier proteins

Passive Transport
A gradient determines the direction of movement (downhill) All channel proteins and many carrier proteins allow solutes to passively diffuse (facilitated diffusion) across membranes

Active Transport
Sometimes it is necessary to transport a molecule against its concentration or electrochemical gradient (uphill) Process mediated by special carrier proteins, sometimes referred to as pumps This process requires energy and activity must be coupled to some source of metabolic energy

Transportul transmembranar se realizeaz ca macrotransfer / microtransfer. Dup sens, macrotransferul poate fi endocitoz / exocitoz, Dup cum vezicula de endocitoz conine sau nu material solid, aceasta se numete fagocitoz sau pinocitoz, iar dup dimensiuni poate fi vorba de macropinocitoz sau micropinocitoz. Microtransferul poate fi pasiv sau activ, dup cum se realizeaz n sensul sau mpotriva gradientului electrochimic transmembranar.

Transportul pasiv este un proces de difuziune prin membran. In general rata de difuzie printr-o membran este proporional cu temperatura absolut, gradientul de concentraie i coeficientul de permeabilitate, determinat la rndul su de coeficientul de partiie ntre membran i mediu. Substanele lipofile difuzeaz uor prin bistratul fosfolipidic, iar cele hidrofile utilizeaz diverse ci hidrofile. In sensul scderii ratei de transfer i al creterii selectivitii, precum i pe baza diferenelor structurale, cile hidrofile pot fi clasificate n pori, canale i transportori. Porii i canalele sunt structuri proteice care delimiteaz ci apoase transmembranare. Canalele prezint bariere de permeabilitate i de selectivitate, unele din ele operate chimic sau electric. Difuziunea transmembranar pe ci prefereniale specifice se numete facilitat, n opoziie cu difuziunea simpl.

 Difuziunea apei prin membran = osmoz Presiunea osmotic este egal cu presiunea care aplicat n compartimentul cu osmolaritate mai mare poate mpiedica osmoza. Osmolaritatea unei soluii este numrul total de particule aparinnd substanelor dizolvate raportat la numrul lui Avogadro i la volumul soluiei.

 In cazul transportorilor, transferul de substan presupune legarea acesteia de proteina transportoare pe o fa a membranei, o anume modificare conformaional a acesteia (flip-flop, ping-pong, situsuri succesive de legare), i eliberarea substanei transportate pe cealalt fa a membranei. Dup numrul de specii moleculare transferate pentru un ciclu transportor, exist uniport i cotransport. Acesta din urm se numete sinport dac transferul este n acelai sens i antiport dac sensul este opus.

 Transportul activ se realizeaz evident numai de ctre transportori proteici i poate fi primar sau secundar, dup cum proteina transportoare prezint sau nu activitate ATP-azic proprie. Transportul activ secundar este ntotdeauna cotransport, una din substane fiind transportat activ pe baza gradientului pentru o alta. Consumul de energie este indirect, realizndu-se la nivelul unui transportor activ primar ce menine gradientul menionat, necesar pentru funcionarea celui secundar.

Channel Proteins
Form hydrophilic pores across the membrane Not just simple aqueous pores Narrow . selectively permeable Transport through is always passive and rapid Allow inorganic ions to diffuse = ion channels Are gated; NOT continuously open

Channel Proteins
Form hydrophilic pores across the plasma membrane Narrow and must be selectively permeable Open and close as needed; transport through is rapid

Channel Proteins
Always passive transport; cannot be coupled to energy source Primarily function to allow inorganic ions to diffuse down electrochemical gradient When they transport inorganic ions, they are called ion channels

Ion Channels
Not just simple aqueous pores Display ion selectivity Are gated; NOT continuously open

Ion channels: up to 100 X 106 ions/second always passive transport Regulation of opening: gating

Kinds of stimuli that gate channels

Carrier proteins: activity somewhat resembles enzymatic activity

Carrier proteins: activity somewhat resembles enzymatic activity

protein contains specific binding sites for solute

carrier protein activity somewhat resembles enzymatic activity

reversible conformational changes involved

passive transport

Active Transport
Sometimes it is necessary to transport a molecule against its concentration or electrochemical gradient (uphill) Process mediated by special carrier proteins, sometimes referred to as pumps This process requires energy and activity must be coupled to some source of metabolic energy

Three ways of driving active transport

Three ways of driving active transport

Three ways of driving active transport

mainly found only in bacteria

Three types of carrier-mediated transport

coupled carriers can take advantage of energy stored in electrochemical gradient of one solute to drive transport of another

Active Transport

In the plasma membrane of animals, sodium is often the ion used to drive active transport Concentration is lower inside the cell than outside

Binding of ligands is cooperative Na+ binding enhances glucose binding Na + binding likely to happen in state A

Cellular pH

Proteins (and other molecules) are affected by pH Macromolecules generally have an optimal pH at which they function

Cellular pH
Cells must be able to control pH Accomplished for the cytosol two ways: sodium-proton exchanger sodium-driven chloride-bicarbonate exchanger

Cellular pH
Cells must be able to control pH Accomplished for the cytosol two ways: sodium-proton exchanger sodium-driven chloride-bicarbonate exchanger

Na+

CYTOSOL

H+

sodium-proton exchanger

Na+ and HCO3(HCl out)

(NaHCO3 in)

CYTOSOL

H+ and Cl-

sodium-driven chloride-bicarbonate exchanger

How do we get this essential sodium gradient?

Sodium low inside cell, potassium high Differences maintained by a special Na+-K+ pump Operates as an antiporter Uses ATP for energy

Plasma membrane Na+-K+ pump is an ATPase two ions transported against gradients

Ionophores: channel former mobile ion carrier

Produced by microorganisms as biological weapons

Used by biologists to increase permeability of membranes

Gramicidin A: -antibiotic Calcium transporter: -turn on cell signaling

Electrochemical Gradient
Charged molecules move along a gradient that is a combination of: concentration gradient electrical gradient Membrane potential: electrical potential difference across membrane

Ion Channels and Electrical Properties of Cells

A membrane potential arises when there is a difference in electrical charge on the two sides of a membrane These differences can result from active ion pumping or from passive diffusion

Ion Channels and Electrical Properties of Cells

Sodium concentrations are low within a cell In contrast, there are many negatively charged fixed anions There must be other cations to balance the negative charges

Potassium Channels
Balance of internal negative charges accomplished largely by potassium Actively pumped into the cell by Na+-K+ pump Potassium can also move freely in and out through K+ leak channels

Potassium Channels and Electrical Properties of Cells

Potassium leak channels always open Potassium, through these two types of transport, reaches a balance between concentration gradient and electrochemical gradient

Potassium Channels and Electrical Properties of Cells

When equilibrium is reached, with no net ion flow, there is a resting membrane potential A slight change or discrepancy in the arrangement of charges across a membrane can quickly cause a change in membrane potential

Membrane potential in animal cells depends on K+ leak channels and the K+ gradient across membrane

Na+/K+ pump and channels that allow K+ to leak in enable K+ to approach equilibrium e.g., if no membrane potential, but K+ abundant in cells. K+ leak out due to concentration gradient. K+ movement will cease because a negative charge inside cell will result. When concentration and electrical gradients are balanced, resting membrane potential is reached

Calcium Gradient Essential

MUST be maintained at very low intracellular concentrations (interferes with phosphatebased energy system) Steep gradient maintained by active pumps that use the power of ATP to pump Ca2+ out of the cell Calcium evolved to be a crucial signaling molecule Increases in response to certain extracellular signals to induce a very rapid response

Calcium Gradient in Muscle

Muscle cells contain sarcoplasmic reticulum Special organelle that acts as internal store of calcium Upon reception of signal to contract muscle, store of calcium rapidly released, causes cellular response

Calcium Gradient in Muscle

Once contraction no longer needed, Ca2+ actively pumped out of cytosol back into sarcoplasmic reticulum This ends the muscle contraction, but requires input of energy, such as from ATP

Calcium Gradient in Muscle

Relates to rigor mortis that sets in after death As cells die and signaling pathways fall apart, calcium is released from sarcoplasmic reticulum -- muscles contract Cells are dead and cannot generate or maintain the ATP needed to actively transport Ca2+ back out -- muscles remain contracted

CALCIUM INFLUX AND FLUXES AND SMOOTH MUSCLE CONTRACTION

studies on contribution of L-type channels studies on contribution of L-type reticular store, and store-operated channels

and store-operated channels

D. N. Serban D. N. Serban Iasi - - Romania Iasi Romania

Cytosolic calcium signals participate in any celullar activity, from the coupling of excitation to responses (contractile / secretory) and control of metabolism, gene expression (differentiation / adaptation) and division (proliferation) down to functional alterations and celullar death, incidental (necrosis) or programmed (apoptosis).

RETICULAR CALCIUM RELEASE RyR & InR RETICULAR UPTAKE SERCA

EXTRACELULLAR CALCIUM INFLUX CaL, ROC, SOC PLASMALEMMAL EXTRUSION PMCA & Na/Ca

CYTOSOLIC CALCIUM CALCIUM-CALMODULIN-MLCK

MYOSIN LIGHT CHAIN PHOSPHORYLATION FORCE DEVELOPMENT STABLE BRIDGES ACTIN-DEPENDENT MECHANISMS

FORCE MAINTENANCE CONTRACTILE STATUS FIBRE LENGTH - CIRCUMFERINTA HEMODYNAMIC RESISTANCE SECTORIAL PRESSURE PARIETAL TENSION VASCULAR COMPLIENCE BLOOD FLOW

SOC

Berridge 1995

store-operated channels = plasmalemmal Ca+conducting channels activated by depletion of Ca+ stores mediate capacitive influx
Putney 1986, Casteels 1981

many issues are still debated - molecular architecture - activation mechanism

HOW MANY SOCS highly calcium-selective current ICRAC in nonexcitable cells is generated by Ca+- SOC
Lewis 2001

ICRAC similar; pCa2+/pNa+ = 1 to 40; cat-SOC

Skryma 2000, Albert 2003, Abeele 2004

activated by the same mechanisms sensitive to the same inhibitors

Albert 2002, Liu 2003, Hunton 2004

 Ca+- SOC (pCa2+/pNa+ ~ 1000) in rat basophilic leukemia cells (RBL) Cat-SOC (pCa2+/pNa+ = 1 ) in smooth muscle cells (SMC)
Trepakova 2001, Smani 2003

Ca+- SOC n RBL Cat - SOC (pCa2+/pNa+ = 40) in human submandibular gland cells Cat - SOC (pCa2+/pNa+ = 4) in human parotid gland cells
Ambudkar 2003

cat SOC
Na+ influx depolarization voltage-gated Ca2+ channels SOC & CaL co-activation in gallbladder
Poteser, 2003 Morales 2004

capacitive influx contraction // depolarization Na+ influx Ca2+ influx via Na/Ca in pulmonary artery reverse Na/Ca contributes to store depletion mediated increase in cytosolic calcium
Zhang 2005 Arnon 2000, Lee 2002

CIF-iPLA2-SOC
Ca2+- SOC (RBL, Jurkat) and cat-SOC (SMC) triggered by CIF (Ca2+ influx factor, partially purified) CIF produced upon Ca2+ store depletion displaces inhibitory CaM from iPLA2 lysophospholipids which activate SOC store refilling CIF CaM rebinds to iPLA2 inactive SOCs Ca2+ influx
Csutora 1999, Trepakova 2000

CIF may be sphingosine 1-phosphate in human neutrophils and HL60

Itagaki 2003

IP3R-SOC
conformational coupling model
Irvine 1990, Petersen 1996

direct coupling of SOCs with IP3 receptors (IP3R) store depletion SOC in two different ways CIF vs conformational coupling
Prevarskaya 2004

two distinct classes of Ca2+-conducting channels: SOC activated by depletion via CIF-iPLA2 no IP3 is needed IP3ROC activated by IP3R by direct coupling no store depletion is needed >50% fast capacitive <50% slow non-capacitive
Bolotina 2004

bovine trachea depletion

Bazan-Perkins 2003

 possible compartmentalization of the storeoperated pathway in areas away from receptors and other Ca2+ influxes
Ambudkar 2004

variety of Ca2+ channels Ca2+-SOC, cat-SOC, IP3ROC, voltage-gated influx to trigger / maintain different physiological responses in various cells