Lesson 3: 1. Phylogenesis of the eukaryotic cell. 2. Metabolism, the role of membranes (ion channels). 3. Molecular receptors. SECTION 1. CELL AS AN INTEGRAL, DYNAMIC SYSTEM
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Lesson 3:1. Phylogenesis of the eukaryotic cell.2. Metabolism, the role of membranes (ion channels).3. Molecular receptors.
SECTION 1. CELL AS AN INTEGRAL, DYNAMIC SYSTEM
Филогенез эукариотическойклетки
CELL
Procariot Eucariot
Kingdom Bacteria
Kingdom Archea
Bacilla
Cocci
Only unicellularand bacterial communities(films and mats)
Uni- and multicellular
kingdom plant Kingdom fungi Kingdom
animals
The realm of viruses – perhaps it is not a living form, but a way of sharing genetic information in asexual reproduction
Vibrio
Spirochete
3V. A. Kozlov, full Bilogical Science doctor, Ph.D. Medical Science, professor
Last Eukaryote Common Ancestor
THREE LIFE DOMAINSBacteries Eucariotes
(fungi,plants,animals)
Archaea
Lost ancestral branches
In 1905 K.S. Merezhkovsky suggested that the eukaryotic cell was formed as a result of symbiogenesis – the fusion of bacterial cells and cellular ancestors of mitochondria in animals or chloroplasts and mitochondria in fungi and plants. It is possible that the cell nucleus was formed from a DNA virus that was once absorbed by such a symbiotic cell.
Theory of symbiogenesisan
aero
bes
aero
bes
Bacteria
Archeae
Mitochondrion
Chloroplasts
Last Eukaryote Common AncestorVirus DNA
Protista
Plants - chloroplasts and mitochondria,mainly tissue,autotrophsAnimals are only mitochondria,tissues and organs,heterotrophs
?
1905 «Uber Natur und Ursprung der Chromatophoren im Pflanzen reiche», K.S. Merezhkovsky
Margulis, Lynn, 1970, Origin of Eukaryotic Cells, Yale University Press, ISBN 0-300-01353-1
Intracellular parasitism↓
Commensalism↓
Symbiosis↓
Eukaryotic cell
6
7V. A. Kozlov, full Bilogical Science doctor, Ph.D. Medical Science, professor
R. Gupta suggested that the eukaryotic cell arose as a result of a symbiosis of thegram-negative bacteria belonging to the proteobacteria and the archaea. Thissymbiosis was created in an oxygen environment rich in antibiotics, secreted byother microorganisms. Partners, have entered into a symbiosis, provided that:Archaea - resistant to antibiotics,proteobacteria - tolerant to oxygen.The intracellular Archean symbionte was subsequently surrounded by a bacterialcell membrane that protected it from the effects of oxygen, which gave rise to theendoplasmic reticulum and nuclear membrane (R.S. Gupta, 2005).Gupta R.S. Molecularsequences and the early history of life // Microbial Phylogeny and Evolution / Ed. J. Sapp. Oxford Univ. Press, 2005. Р. 160–183.
It is possible that the DNA-kov virus became the basis of the cell nucleus, in which the genomes of the proteobacteria and the archaea gradually completely migrated.At the final stage, the symbiosis included mitochondria and chloroplasts.
Radhey S. Gupta (Professor) Department of Biochemistry and Biomedical Sciences (HSC- 4H2) 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5
The last eukaryotic common ancestor had a much more complex genome than the modern multicellular genome.Multicellularity was realized through the loss of unnecessary regions of the genome.In the process of combining the genomes, the ring DNA was opened and transformed into linear DNA, perhaps this caused the formation of so-called intron sections of DNA, which are virtually absent in prokaryotes and which in the early stages of symbiogenesis were needed to "match" the genomes of different organisms to each other.
Types of cell connection
Connection of three cells by nexus
nexus
CELLULARCONTACTS
Cell - Matrix
1) hemidesmosomes
2) focal contacts
keratin filaments
cadgerin
actin filaments
adhesion contact
finger joint
integrin
keratin filaments
adhesivebelt
gap junction
Cell - Cell1) contacts of a simple typea) adhesiveb) interdigitation (finger joints)2) contacts of coupling type
a) desmosomes and adhesive bands3) contacts of the locking typea) tight connection4) communication contacts
a) nexus
Intercellular contacts are necessary forcell connectionsinformation exchangea) intercellularb) with the external environment
For this, the cell createsion channels
Ion channels
ion channelsselective
non selective
Potential-controlled are channels whose activity is
determined by the transmembrane electric field
The receptor-driven receptors are channels whose
activity is determined by interaction with the mediator
Na+-channels
K+-channels
Ca+-channels Ligand-driven Mechanically-
controlled
General representation of the ion channels structure and function
Channel-forming protein molecules of all ion channels have some common structural features and are usually represented by large transmembrane proteins with molecular masses above 250 kD.
Consist of several subunits.
Usually the most important properties of the channels are determined by their
α-subunit.
Other subunits, which are part of the structure of ion channels, play an auxiliary role, modulating the properties of channels.
It takes part in the formation of the ion selectiveburrow, the sensory mechanism of thetransmembrane potential difference (channelgate), and has binding sites for exogenous andendogenous ligands.
Three-dimensional spatial structurechanneling molecule of the protein is located
in the cytoplasmicmembrane
the mouth of the canal, facing the outer and inner sides of the membrane,
pore filled with water
gate channel
are formed by a portion of the peptide chain that can easily change its conformation and determine the open or closed state of the channel.
on its dimensions and charge dependselectivity and permeability of the
ion channel.The permeability of the channel for a given ion is determined by 1) the dimensions,2) the magnitude of the charge and3) a hydrated shell.
is forms
Described> 100 varieties of ion channels, different approaches are used for their classification.One of them is based on taking into account differences in the structure of channels and in the mechanisms of functioning.Ionic channels can be divided into several types:passive ion channels, or quiescent channels;channels of slotted contacts;channels, the state of which
the influence on their gate mechanismmechanical factors (mechanically sensitive channels),potential differences on the membrane (potential-dependent channels)or ligands that bind to the channeling protein on the outer or inner side of the membrane, or the channel pore (ligand-dependent channels).
1) closed2) opend
3) inactivated
Passive Channels
can be open (active) in resting cells, i. e. in the absence of any impactsare not strictly selective,through them, the cell membrane can "leak" for several ions, for example К+ и CI–; К+ и Na+; Ca2+. Therefore, the second name of these channels is the leak channels.
channels of cytoplasmic membranes ofnerve fibers and their endings, cells oftransversely striated, smooth muscles,myocardium and other tissues
Form and maintain the resting potential of thecytoplasmic cell membrane
Channels of slotted (dense) cell contacts
Channels of gap junctions are formed in the area of contact of two adjacent cells, very close (3.5 nm) adjacent to each other.
In the membrane of each contacting cell, six subunits of connexin proteins form a hexagonal structure - connexon, in the center of which a pore or ion channel is formed (the next slide).
At the contact point, a mirror structure of two connexons is formed in the membranes of both cells, and the ion channel between them becomes common.
Through such ion channels from cell to cell can travel1) various mineral ions, including Ca2+,2) low-molecular organic substances.
The channels of the slotted cell contacts provide information transfer between the cells of the myocardium, smooth muscles, the retina of the eye, and the nervous system.
Membranestwo neighboringcells
ChannelSlitcontact
3,5 nm
Connexin –protein
Connexon is a functional complex of connexins
Mirror structure of two connexons
Ion channelsclosed and opened
are in response to
mechano-dependent
potential-dependent
ligand-dependentinteraction of the low-
molecular mediator with the receptor site of the
channel
mechanical deformation
change in membrane resting potential
Potential-dependent channels• The state of these channels is controlled by the forces of the electric field created
by the magnitude of the potential difference (∆ϕ) on the membrane.• They can be in the inactive (closed), active (open) and inactivated states, which
are controlled by the state of the activation and inactivation gate, which depends on ∆ϕ on the membrane.
• In a resting cell, the potential dependent channel is usually in a closed state, from which it can be opened or activated.
• The probability of its independent discovery is low, in a state of rest in the membrane, a small number of these channels are open.
• Depolarization of the membrane (decrease of ∆ϕ) activates the channel, increasing the probability of its opening. To open the channel, it is necessary to achieve a critical level of depolarization.
• The function of activation gates is performed by an electrically charged amino acid (lysine or arginine located in every third position on a given site of the molecule) closing the entrance to the mouth of the canal.
• Lysine and arginine, like polar amino acids, are the sensor ∆ϕ on the membrane; when the critical membrane depolarization level is reached, the charged part of the sensor molecule shifts towards the lipid microenvironment of the channeling molecule and the gate opens the entrance to the channel mouth (see the next slide).
The potential-dependent channel can be in three consecutive states:
closed;open;inactivated
The status of the channel dependsof the value of the membrane potentialGo to 1-2-3 is carried out abruptly,Go to 3-1 for a "long" period of timePotential-dependent channel
Time, ms1
2
3
Pore
Domen
Intracellular liquid (out)
Transmembrane integral protein channel forming
Selective filter
α-subunitβ2-subunit
β1-subunit
voltage sensor
Cytoskeleton protein
Activation gate
Inactivation gate
The intracellular medium (in)
The opening speed of the activation gate can bevery high
and low.For this indicator, the potential-dependent ion channels are divided intoFast (for example, fast potential-dependent Na+ channels) and slow (for example, slow potential-dependent Ca2+ channels).The speed of the activation of the channel depends on the mobility of the activation gates.The fast channels open instantly (μs) and remain open for an average of 1 ms. Their activation is accompanied by a rapid avalanche increase in the permeability of the channel for certain ions – the throughput of the open channel is striking: the ion current occurs at a speed of up to 100,000,000 ions / s.Slow channels open with fast, but the speed of the activation gate in them is much smaller, so the movement of ions through them begins later.
Activation gate – a sequence of amino acids in the form of a dense ball (coil) on the filament, located at the outlet of the mouth of the canal. When the sign of the charge changes on the membrane from (+) to (–), the ball closes the outlet from the mouth, the channel becomes impermeable (closed) to the ion, but the channel can be activated by a threshold or large stimulus.Inactivation gates are also a sequence of amino acids. Inactivation is accompanied by a cessation of movement of ions through the channel and can proceed as fast as activation, or slowly – for seconds or even minutes. All this time the channel can not be opened at any excitation power, until the closed state passes.
Channelclosed
Repolarization
Depolarization or an ligand
addition
Activation gate
Inactivation gate
the channel is inactivated
the channel is activatedfurther depolarization
There are substances that block the work of potential-dependent ion channels. Thus, another state of the ion channel is possible-blocked. Such substances are called ion channel blockers.One of the first blockers was described the substance tetrodotoxin (formed in the body of fugu fish), a blocker of potential-dependent sodium channels. In the experiment, when it was introduced into the animals, loss of sensation, muscle relaxation, immobility, respiratory arrest, and death were noted.The clinic uses lidocaine, novocaine, procaine – substances, when administered to the body in small doses, a blockade of potential sodium channels of nerve fibers develops and the transmission to the central nervous system of signals from pain receptors is blocked, these are local anesthetics.
All the ion channel blockers operate in the same way - they are integrated as a plug in the channel lumen. Differences between blockers are due to their binding site to the channel molecules.
Like a cork, tetrodotoxin enters the outer vestibule of the sodium channel. In this case, each of its active group interacts with its amino acid residue
The batrachotoxin molecule sits in the channel and does not allow it to close, passing sodium ions. The arrow shows the ion current
batrachotoxin
tetrodotoxin
The pore-forming part of the ion channel can be1) a single polypeptide, organized in the form of several identical transmembrane domains,2) several protein subunits, which can be either the same (homo-oligomer) or unequal (hetero-oligomer) in its structure.
Each pore-forming subunit or domain is formed by several transmembrane α-helical segments with N- and C-terminal protein domains directed intracellularly or extracellularly.
One of the transmembrane segments of the subunit of the potential-activated channels contains a unique set of positive charges and functions as a potential sensor (Armstrong, Hille, 1998, Hille, 2001).
Virtually all channels in the composition of the channel-forming subunits have regulatory domains that bind to different regulatory molecules.
The channels have the property of selectively passing ions, which is realized at the narrowest point of the channel – the selective filter.
The structure of the Na+-ion channel of the cell membrane:a – two-dimensional structure of the α-subunit of the ion channel of the cell membrane;b – on the left – a sodium channel consisting of 4 α-subunits and two β-subunits (sideview); on the right – the form of the sodium channel from above.Figures I. II. III. IV domains of α-subunit
Potential-depended channel
Extracellularspace
view from above
Cytosol
a
b
Disruption of the ion channels often leads to diseases -canalopathies. The main cause of such violations -hereditary mutations, affecting the structure of the channel.
Examples of canalopathies:- cystic fibrosis- cardiac arrhythmias- Brugada syndrome (sodium channel of cardiomyocytes)- Syndrome Timothy (anomalies of calcium channels)- generalized epilepsy
Ion channel operation(different colors are allocated to different proteins that form domains)
the capacity of the open channel is striking:the ion current occurs at a rate of up to 100,000,000 ions / s
Ligand-dependent ion channels
Formed by protein macromolecules, simultaneously performing the function
ion channelsand selective receptors of ligands.
Therefore, they were followed by different synonymous names, for example: a synaptic receptor, a ligand-dependent channel.
Unlike potential-dependent ion channels, the opening of which is in response to a decrease in ∆ϕ, the ligand-dependent ion channels open (activated) by the interaction of the recognition site of the molecule receptor ligand protein with the ligand-substance to which the receptor recognition site has a high affinity.
Ligand-dependent channel
The ligand (transmitter) binding center
Cell membrane
2α, β, γ, δ protein subunits forming an ion recognition system
Hydrophobic surface
Hydrophilic cavity
pore
Intracellular liquid
Cytosol
gateM2-spiral
14 nanometers
12 nanometers
Receptor`s antagonist(blocker)
Transmembrane domain
The ligand-binding domain
Amino-terminal domain
Molecular (protein) structure of the ligand-dependent ion channel(different colors are allocated to different proteins that form domains)
Ligand-dependent ion channels are usually located in the postsynaptic membranes of nerve cells and their processes, as well as muscle fibers.
Typical examples of ligand-dependent ion channels are the channels of pre- and postsynaptic membranes, activated by acetylcholine, glutamate, aspartate, γ-aminobutyric acid, catecholamines, glycine and other synaptic neurotransmitters.
Usually the name of the channel (receptor) reflects the type of neurotransmitter, which in its natural conditions is its ligand. So, if it is the channels of the neuromuscular synapse, in which the neurotransmitter acetylcholine is used, the term "acetylcholine receptor" is used, if it is also sensitive to nicotine, then it is called a nicotine-sensitive or simply n-acetylcholine receptor (n-cholinoreceptor , N-ACH).
Ligand-dependent channels can change permeability for Na + and K + cations or for K+ and CI– anions. This selectivity of ligand binding and changes in ion permeability is genetically fixed in the spatial structure of the macromolecule.
If the interaction of the mediator and the receptor part of the macromolecule forming the ion channel directly changes the permeability of the channel, then within a few milliseconds this changes the permeability of the postsynaptic membrane for mineral ions and the magnitude of the postsynaptic potential, for example, acetylcholine and glutamate channels.Such channels are called fast and localized, for example, in the postsynaptic membrane of axo-dendritic excitatory synapses and axosomatic inhibitory synapses.
There are slow ligand-dependent ion channels.Their discovery is realized through a chain of successive events:the primary mediator (ligand, neurotransmitter) activates G-protein → it interacts with GTP → which triggers the synthesis of secondary mediators in the intracellular signaling → which phosphorylate the ion channel → this changes its permeability for mineral ions → and, as a consequence, the magnitude of the postsynaptic ∆ϕ.
This chain of events is carried out for hundreds of milliseconds.With such slow ligand-dependent ion channels, we will meet in studying the mechanisms of regulation of the heart, smooth muscles.
Connection of the ion channel to the receptorAcetylcholine excites the m2-receptor, it through the system of blast proteins leads to the opening of the K + -channel of the sinoatrial node, the entrance of K + into the cell, the hyperpolarization of its membrane and the decrease of its excitability.
A special type of ligand-dependent channels are the channels localized in membranes of the endoplasmic reticulum of the smooth muscle cell.Their ligand is a secondary mediator of intracellular signaling of inositol triphosphate signal - IF3.
Ionic channels characterized by some structural and functional properties inherent in both voltage-dependent and ligand-dependent ion channels are described. They are potential-NO-sensitive ion channels, the state of the gate mechanism of which is controlled by cyclic nucleotides (cAMP and cGMP).Cyclic nucleotides bind to the intracellular COOH terminal of the channeling protein molecule and activate the channel. These channels are less selective for cations and regulate permeability for other ions.Thus, Ca2+, through activated channels from the extracellular medium, block the permeability of the channels for Na+ .Such channels are found in the retina rods, their permeability for Ca2+ and Na+ is determined by the level of cGMP.
Ligand-dependent ion channels are widely represented in membrane structures that provide synaptic signaling from a number of sensory receptors in the CNS; transmission of signals in the synapses of the nervous system and from neurons to effector cells.
Receptors
RECEPTION is the process of perceiving and transforming (converting) the energy of an external stimulus into a nerve impulse or a complex sequence of intracellular processes, through membrane and / or cytoplasmic processes. The receptive function is performed by special formations – receptors.
SensoryMembrane Intracellular
Specialized cells:1) mechanoreceptors, react tomechanical compression orstretching of the receptor oradjacent tissues (tactile,auditory, gravitational,equilibration, joint-muscle);2) thermoreceptors (thermal andcold), perceive temperaturechanges;3) nociceptors (pain receptors);4) electromagnetic, perceive lighton the retina of the eye;5) chemoreceptors (taste, smell,[Na+], osmosis)
With its own enzymatic activity
Conjugated with enzymes
Conjugated with G-proteins
Receptors Molecular
Passes the ion in response to mechanical deformation of the cell membrane - mechanically dependent channels of tactile, gravitational, painful neuronal receptors and receptors of equilibrium of the otolith apparatus.
Mechano-dependent channel
stretching
cytoskeleton
stretching
Sensory receptors
mechanoreceptorsof the skin, muscles,of cardio-vascularsystem, internalorgans
mechanoreceptorsof the hearing andvestibular organs
Mechanoreceptors
Secondary sensory:perception of irritation occurs in one cell, and nerve impulses arise in
another, closely related to the first
Primary sensory:perception of irritation and the emergence of a nerve impulse occur in the same
cell
Corti's organ
The equilibrium body
Merkel disc Meissner's body Fatera-Pacini body
liquid internal spaceneuron cellsend
otolits macula
supporting cells Neuronssensitive neural pathways
Gravitation
free nerve endings
muscle spindle Golgi tendon organ
Peripheral
unencapsulated nerve endings:skin, subcutaneous tissues,dermal and subcutaneousvessels
the medial preoptic region ofthe hypothalamus (centralneurons-thermosensors), thereticular formation of themiddle and spinal cord
Termoreceptors
Central
Reaction to the temperature Difference between deep andsurface receptors
corpuscle Ruffini cone Krause
Nociceptors
Free nerve endings are demyelinic(slow nerve fibers)and myelin (fast nerve fibers)
Reaction to exceeding the threshold of irritation.1 type: mechano- and heat-receptors;2 type – chemoreceptors
Electromagnetic receptors
Under the action of a photon (the carrier of electromagnetism), rhodopsin is destroyed by lumirodopsin and metarodopsin, which hydrolyses with water to form 11-trans-retinal and opsin. Highlighted for 200 femtoseconds! in this process, the energy excites the action potential in the neurons of the retina.
3D retina organization
blind spotRetina
Ligth
Retina
stics cones pigment epithelial cells
sticks 125 million cone 7 million
cone
The central (medullary) chemoreceptors are located directly in the rostral parts of the ventral respiratory group, in the structures of the blue spot (locus coeruleus), the reticular nuclei of the brain stem seam, react to hydrogen ions in the intercellular fluid of the brain surrounding them
Peripheral (arterial) chemoreceptors are located in carotid bodies in the region of bifurcation of common carotid arteries and in aortic bodies in the region of the aortic arch, respond both to the change in the concentration of hydrogen ions
and the partial pressure of oxygen in the arterial blood.Olfactory (smells - dangerous, neutral, pleasant, pheromones).
Taste (bitter, sour, sweet, salty, minds).Sodium (located in a dense substance of nephrons).
Chemoreceptors
Osmoreceptors
They are located in the hypothalamus and liver.They react to changes in the osmotic pressure of the blood plasma. Between the osmoreceptors of the liver and the hypothalamus, as well as nephrons of the kidneys, there are direct reflex connections.
Molecular receptors
Structure of receptorsa) ionotropic and b) bound to the G-protein
receptormediatora b
mediator AdenilatciclaseChannelOut cellular
space
Intra cellular space
Channel pore
receptor G-protein
Out cellular space
Intra cellular space
Membrane receptors coupled with ion channelsThe mechanism of work:Activation of the receptorOpening of the ion channelChanging the electrical potential of the cell membrane
Change in the functional state of the cell
NMDA-receptor is a receptor-ionophore complex, which includes:1) site for specific binding of the mediator (L-glutamic acid);2) regulatory, or coactivating site for specific binding of glycine;3) allosteric modulator sites located on the membrane (polyamine) and in the ion channel (binding sites of phencyclidine, divalent cations and a potential-dependent Mg2+ binding site).
Molecular organization of the ionotropic receptor
Membrane receptors coupled with G-proteinsThe mechanism of work:Activation of the receptorActivation of G-proteinActivation of the enzyme catalyzing the formation of a
second mediatorFormation of second intermediaryActivation of protein kinasePhosphorylation of effector proteinChange in the functional state of the cell
The ligand binds to the receptor recognition site, this changes its conformation and triggers the activity of guanosine phosphatase, which forms GTP from GDP, the latter activates adenylate cyclase, which converts adenosine monophosphate into its cyclic form. cAMP activates a cascade of intracellular kinases, the latter of which triggers a cellular response.
ligand
Ligand-receptor complex Adenilatciclase
GDP GDP
cAMP
Molecular organization of receptors associated with G-protein
ADENILATCICLASE SYSTEM
Molecular organization of receptors associated with G-protein
PHOSPHOOSYSTEID SYSTEMLigand
Recepror
G-protein G-protein Phospholipase C
Proteincinase C Adenilatcyclase
cAMP formation
Isolation of Ca from intracellular depot
Phosphorylation of cell proteins
G-proteins of two types:GS – stimulating cellular responseGI – inhibitory cellular responseLigands (primary mediators) are hydrophilic amines, amine-like substances, peptides:acetylcholine, histamine, catecholamines (dopamine, norepinephrine, adrenaline), serotonin, pituitary hormonesSecondary intermediaries (messengers):cyclic nucleotides (cAMP, cGMP – guanylate cyclase system),diacylglycerol (DAG)inositol diphosphate (ITP2)inositol triphosphate (ITP3)
Mediator
Receptor
G-protein
Effector protein
Second manager
Finish ffector
Resalt
Noradrenalin
β-adrenergic
Adenylatecyclase
cAMP
Proteincinase A
Adenylatecyclase
cAMP
Proteincinase A
Glutamate Dopamine
Phospholipase C
DAG IP3
Proteincinase C Emission of Ca2+
Enhancement of proteinphosphorylation and activation
of calcium binding proteins
Proteinphosphorylation
eincrease
Proteinphosphorylation
edecrease
Molecular organization of receptorswith its own enzymatic activity
Ligands:factors of growth of nerves, thrombocytes, etc.,insulin
Growth factor
Recognition Center
a b c dChange in the
conformation, dimer formation
Tyrosine autophosphorylation
SH2-domain binding and phosphorylation
Transmembrane spiral
Tyrosincinasedomain Tyrosine
remains
Proteine SH2-domain recognizes specific sites
containing phosphotyrosinetyrosine kinase
Enzimeactivation
Transcription Factor Activation
Modificationof gene
transcription
Tissue answer
Intracellular (nuclear) receptors - transcription factors
Only hydrophobic molecules!
Ligands of nuclear receptors (fat-soluble substances):
diiodotyrosine, triiodothyrosine,
retinoids,
steroid hormones (cholecalciferols, glucocorticoids, mineralocorticoids, dihydrotestosterone, progestins, estrogens)
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