Molecular Cell Biology Notes PDF

MOLECULAR CELL BIOLOGY  CELL STRUCTURE & FUNCTION The Cell Basic structural building block of living organisms Has protoplasm (the living part of the cell that has all the organelles) There are about 37 trillion cells in our bodies (almost the same as bacteria ce...

MOLECULAR CELL BIOLOGY  CELL STRUCTURE & FUNCTION The Cell Basic structural building block of living organisms Has protoplasm (the living part of the cell that has all the organelles) There are about 37 trillion cells in our bodies (almost the same as bacteria cells) More than 200 different cell types Parts of the Cell Ribosomes: Large subunit (60s) and small subunit (40s) Has rRNA j Produced in nucleolus Translates mRNA to protein Golgi Apparatus: Polysaccharide synthesis Modifies, sorts, packages, concentrates, and stores secretory products (like proteins and lipids) & Closely linked to the Endoplasmic Reticulum (ER) Endoplasmic Reticulum (ER): Synthesises, packs and processes molecules Rough Endoplasmic Reticulum (RER) helps with protein * synthesis and Core glycosylation (putting sugar molecules on to proteins) Smooth Endoplasmic Reticulum (SER) participates in lipid metabolism, steroid hormone synthesis, gluconeogensis (making glucose), and detoxification If it has ribosomes it is rough Vesicles: - They are small cellular containers that have many functions (have hydrophilic head and hydrophobic tail) Examples: 1. Granules: secretory vesicles (move molecules outside of the cell by exocytosis) & 2. Lysosomes: break down excess or worn-out cell parts (hydrolytic enzymes) 3. Peroxysomes: generate hydrogen peroxide to destroy the JOIN THE DARKSIDE excess. Also breaks down fatty acids Mitochondria: Power-house of the cell Membrane bound (static) Have their own DNA and some proteins Grow and reproduce Move fast and change shape Outer membrane is permeable to molecules less than 10 kDa Inner membrane is impermeable to protons Shuttle systems/carrier proteins allow passage in the inner membrane Krebs cycle and oxidative phosphorylation happen in mitochondria Functions: energy production, produce heat, hormone signalling, steroid synthesis, etc. Nucleus: Where DNA replication occurs (transcription) Contains the genome (entire set of DNA instructions) Multinuclear cells have more than 1 nucleus Lamins are filaments (long chains of protein subunits) on the inside of the nuclear membrane They regulate size, shape, chromatin pattern (important for transcription) Nucleolus Ribosomes are made here Nuclear Pore: Monitor and regulates passage of substances Composed of several glycoproteins that build up nuclear pore complexes (this is what regulates the passage of substances) Proteins that go to nucleus have a nuclear localisation signal Cytoskeleton: It is a mesh of filaments called microtubules, microfilaments, and intermediate filaments (helical arrangements) Provides support to the cell and plasma membrane Allows the cell to move Involved in cell division (mitosis) JOIN THE DARKSIDE Microtubules: A tublin is on the negative Created from the Microtubule Organising Centre (MTOC) located in end and B tublin is on positive end the centrosome (centre of the cell) Subunits called tubulin heterodimers (alpha and beta tubulin balls) These tubulins line up (this line is called a protofilament). 13 protofilaments create 1 microtubule (has a positive and negative end). Function: in mitosis they form the mitotic spindle, deploy organelles, transport organelles There are 9 microtubule structure in one cilia (found on the surface of cells) & Types of cilia: 1. Primary: cell signalling 2. Motile: movement 3. Nodal: left-right development (embryo) Microfilaments (actin filaments): Thin and flexible Polar Made of actin monomers (intertwine to form a double helical filament) Forms: 1. Stress fiber (contractile bundle) 2. Cell cortex (gel-like network) 3. Lamellipodium (dendritic network) 4.Filopodium (tight parallel bundle) Functions: Endocytosis and exocytosis Maintenance of cell shape Cell movement Extension and contraction of filopodia Intermediate filaments: Long protein molecules with a central helical domain (rope structure) Highly dynamic (easy to bend hard to break) Type 1 and 11 (epithelial): keratins Type 111: vimentin, desmin, peripherin and glial fibrillary acidic protein (mainly others) Type 1V (axonal): neurofilaments Type V (nucleus): lamins (stabilise the nuclear membrane) Common in cells with high mechanical stress (skin cells) Stabilise cell Anchor to adhesion complexes JOIN THE DARKSIDE  Cell-cell junctions Junctions provide contact (adhesion) between cells or a cell and the extracellular matrix Cell Membrane: Made up of a phospholipid bilayer (hydrophilic head & hydrophobic tail) Relatively impermeable membrane Thermodynamically stable Has cholesterol Functions: 1. Compartmentalisation: provides organisation and separates organelles 2. Scaffold for biochemical activities: provide the cell with necessary molecules for biochemical activities 3. Selective permeability: controlled entry and exit of substances Membrane Trafficking: The process by which proteins and other molecules are distributed within the cell or into extracellular space Relatively fast process It is fundamental in building compartments, sending signals, storing, and separating harmful reactions Process: Example) 1. RER: newly synthesised proteins are moved into the cisternae. Chaperones help fold these proteins. Oligosaccharides are added. 2. Cis Golgi Network: mannose-6-phosphate is added to the proteins that later become lysosomal enzymes. Oligosaccharides get modified. 3. Medial Golgi Cistern: proteins begin to get sorted. Glycolysation of lipids occurs and Oligosaccharides get modified all the way. Glycoproteins and glycolipids begin to get sorted. 4. Trans Golgi Network: final modification of sugars. Specific vesicles go to their destinations and get stored. JOIN THE DARKSIDE  Molecular Cell Biology Cell Homeostasis Maintaining a constant stable internal environment Action Potential The cell membrane has integral (in the bilayer) and peripheral proteins (attached Rapid sequence of changes in voltage to the surface of the bilayer) that contribute to 50% of the weight across a membrane The membrane is fluid due to the different movements of the lipid bilayer (lateral 1. at rest there are more positively diffusion, flip-flop, flexion, rotation) charged ions outside than inside Integral proteins can diffuse and move around causing the membrane to be fluid (resting membrane potential) (membrane is asymmetrical due to this) 2. Ion channels allow the ions to The reason why the membrane is a platform of exchange is to maintain move in homeostasis 3. when the voltage reaches a It exchanges water, gasses, sugars, ions, proteins, signals, and toxic waste certain point (-55 or higher) the products sodium channel opens and sodium ions rush into the cell. Types of Transport Because of this the membrane Passive: no energy required, with the concentration gradient, small molecules, potential becomes more positive goes from high to low (depolarisation) Disease related to passive is Glut 1 deficiency syndrome 4. When it reaches a certain level the Facilitated diffusion (uniport) are carrier mediated and channel mediated while channel becomes inactivated simple diffusion is freely moving on its own meaning no more sodium can Active: energy required (ATP), against the concentration gradient, small enter molecules, low to high 5. The potsssium channels open Deficiency related to active is cystic fibrosis making potsssium ions move to Uniport and contransport are primary active transport and they are pump the outside making it eventually mediated while antiport and symport are secondary and are carrier mediated negatube in the cell Cystic fibrosis occurs because of a malfunctioning chloride channel where the (repolarisation) water does not follow the chloride ions out of the cell leading to thick mucus 6. Since the potsssium channels are LARGE molecules are transported by active transport as well by endocytosis slow at closing the cell is and exocytosis hyperpolarised for a little while This requires the cytoskeleton and specific receptors (more negative) Endocytosis: bringing extracellular substances inside by 7. The sodium potssium pump that has been working all this time engulfing them in vesicles brings the cell back to its RMP 1. Phagocytosis (cell eating, large molecules) 2. Pinocytosis (fluids, small molecules) 3. Receptor- mediated (triggered by ligand signal) Exocytosis: secretion and excretion of substances from the cell 1. Constitutive secretory pathway ( directly) 2. Regulated secretory pathway (specialised cells and specific cargo) 3. Lysosomal secretory pathway (involves fusion of lysosomes with plasma membrane) Membrane potential is the difference in electrical potential (amount of work) between the inside and outside of the cell Inside of cell is always negative (between -90 and +40) Ions: K, Na, Cl, Ca Membrane polarisation is the presence of positively charged ions on one side and negatively charged ions on the other Membrane depolarisation is when there is an inward flow of positive ions across the membrane causing the electrical potential inside to become more positive compared to the outside Hyperpolarised is when the cell has a more negative potential compared to the outside JOIN THE DARKSIDE  Cell Signalling Principles of Cell Signalling: 1. Reception 2. Transduction 3. Response Extracellular signal molecule (ligand)-> receptor protein-> transduction (Intracellular signalling proteins)-> response (effector proteins)-> metabolic enzyme, transcription regulatory protein, cytoskeletal protein Characteristics of Signalling: 1. Specificity 2. Amplification 3. Network 4. Feedback Types of Intracellular Signalling: 1. Juxtacrine (contact dependent) 2. Paracrine (short signalling) 3. Synaptic (neuronal) 4. Endocrine (uses circulatory system-> bloodstream) Hormone Synthesis: Proteins and peptides DNA mRNA protein route Lipid based- synthesis through cholesterol Cells that secrete steroid hormones have more SER Cells that secrete peptide hormones have more RER Types of Receptiom: 1. Cell surface- most ligands 2. Intracellular- steroid and thyroid hormones Types of Responses: 1. Slow- transduction to nucleus-> alters protein synthesis-> alters cytoplasmic machinery-> alters cell behaviour (minutes to hours) 2. Fast- transduction alters protein function-> alters cytoplasmic machinery-> alters cell behaviour (seconds to minutes) Nuclear Receptors Found in nucleus or cytoplasm Regulate gene transcription Domains: ligand binding (binds to specific ligands) DNA binding (conserved) activation function (can have more than 1 and is involved in recruiting gene regulators for transcription) Steroid Receptors Regulate transcription Exist as cytoplasmic complexes with Heat Shock Proteins (help folding and keeping proteins in check) HSP gets dissociated when ligand binds to receptor Binds to specific DNA elements T3 Signalling Acts on gene regulation directly On nuclear and mitochondrial genes Signalling happens when it binds to thyroid receptors and coactivators JOIN THE DARKSIDE Types of Cell Surface Receptors Ion- Channeled couple Ligand gated, voltage gated, mechanically gated Fast synaptic signalling Mediate most forms of Electrical signalling in NS GABAA G-coupled Ligand activated Passes membrane 7 times (slower but longer lasting) Largest family Linked to cAMP or phophoinositol signalling pathway Types: 1. Gs (stimulates cAMP). cAMP amplifies the signal as a second messenger 2. Gi (inhibits cAMP meaning slows its production down) 3. Gq (activates phospholipase C which is an enzyme that converts a molecule into second messengers which then release calcium and protein kinase) GABAB Note: serotonin and acetylcholine are both ligand gated and g coupled Adrenergenic receptors are a group of G coupled receptors Alpha receptor 1 is Gq Alpha receptor 2 is Gi Beta receptors are all Gs TSH signalling: 1. TSH binds to TSH receptor which activates Gs and Gq 2. Gs and Gq actuvate 2 different pathways (cAMP) and (PLC) Enzyme-linked Function as enzymes or closely linked Passes membrane once Types: Receptor tyrosine kinases- receptor is a kinase Cytoplasmic tyrosine kinase- the receptor is not the kinase but is linked to it Phosphorylation is the addition of a phosphate group to a molecule Molecular switches are molecules that can switch back and forth between 2 stable states 1. Signalling by phosphorylation results in an alteration in molecular structure and this leads to functional change 2. Signalling by GTP binding is when it alters between an active and inactive form Types of Intracellular signalling complexes 1. On scaffold proteins- receptor molecule has attachment piece where other intracellular signalling proteins attach and link together 2. On phospoinositide docking sites- phosphorylated lipids ( hyperphosphorylated phosphoinositide ) integrate into membrane and relay signals upon activation 3. On an activated receptor- toriginal receptor is the docking station itself and it activates intracellular signalling proteins Complex signalling is all of them together Desensitisation is the decrease in receptor response to chemical activation JOIN THE DARKSIDE  Genes & Transcription - Central Dogma of Molecular Biology The flow of information from DNA to RNA to proteins Replication -> Transcription -> Translation Part 1: The Basics of DNA Structure Storing genetic information happens through DNA DNA stands for deoxyribonucleic acid It’s made of subunits called nucleotides Nucleotides contain: De Sugar molecules (deoxyribose) La Phosphate molecule Nitrogen containing side group (known as a base) 3 Nucleoside is a nucleotide without the phosphate Bases: Adenine (A) Guanine (G) Cytosine (C) Thymine (T)- replaced by Uracil (U) in RNA The reason DNA has Thymine is because it has a high resistance to mutation. RNA is short lived so it doesn’t matter. Nucleotides are divided into: 1. Purines- A and G 2. Pyrimidines- C, T, U DNA Structure The 2 strands form a double helix (strands run in opposite directions) Nucleotides are joined together by sugar-phosphate linkages DNA is read from 5’ end (has phosphorylation group attached) to 3’ end (at the end it has a hydroxyl group attached) Note: polarity exists between strands Bonding Nucleotides within a strand are bonded by strong covalent bonds (phosphodiester bond) Nucleotides between strands are held together weakly by hydrogen bonds DNA Storage Exists in a highly condensed form in chromosomes (contain long lists of genes) Specialised proteins bind to and fold DNA, this creates coils and loops in an organised way DNA Binding Proteins 1. Histone proteins (packaging the chromosome) 2. Non histone chromosomal proteins Chromatin is a mixture of DNA and proteins that form chromosomes DNA + Histone= Nucleosome Each nucleosome contains: 2 H2A, 2 H2B, 2 H3, 2 H4 Histones can be modified JOIN THE DARKSIDE Part 2: DNA Replication & Genetic Inheritance Replication occurs in a semi-conservative manner The weak bonds allow DNA to be pulled apart without damage Replication relies on base complementarity This is facilitated by enzymes DNA Replication New DNA strand is called primer strand DNA polymerase (enzyme) facilitates synthesis of primer strand by creating a phosphodiester bond between the 3’ OH and phosphorus of incoming dNTP (doexynucelosidetriphosphate) Okazaki fragments: transient pieces that are joined together after synthesis Info: the leading strand runs from 5’ to 3’ continuously Types of DNA Polymerase 1. Alpha, delta, epsilon- nuclear DNA 2. Gamma- mitochondrial DNA 3. Beta- DNA repair and gap filling Terms: Phenotype: physical appearance of trait Gene: unit of inheritance behind trait Allele: different forms of gene Genotype: genetic makeup of individual Homozygous: same 2 alleles Heterozygous: different alleles Dominant allele: one or 2 copies ( capital) Recessive allele: has to be copies. (Less common) Part 3: Transcription Transcription: template DNA strand gets replicated producing RNA RNA is flexible compared to DNA due to having only one strand (can lead to multiple shapes) Classes of RNA: 1. Ribosomal 2. Messenger 3. Transfer RNA strand is same as DNA non template strand (U instead of T) Transcription steps: 1. Unwinding small portion of DNA double helix 2. TF11D binds to TATA box (DNA sequence) on promoter 3. TF11H uses energy from ATP to break DNA strand apart 4. TF11H phosphorylates RNA polymerase 11 to start RNA synthesis (elongation ) 5. Termination happens when polymerase encounters termination signal (AAUAAA) Processing Steps: 1. Addition of 5’ cap 2. Addition of poly A tail at 3’ end 3. Introns (non coding regions) are spliced out while exons (Coding regions) are brought together Spliceosomes do the splicing JOIN THE DARKSIDE Part 4: Translation and Post-Translational Modifications Happens outside of nucleus Happens on free ribosomes RER is the site of protein production mRNA encodes genetic info rRNA catalyses formation of peptide bonds tRNA translates nuclei c acids to proteins Peptide bonds are what hold one amino acid together Important DNA is read 5’ to 3’ Replicated 5’ to 3’ Transcribed 5’ to 3’ Translated 5’ to 3’ Ribosomes sites: 1. A site 2. P site 3. E site JOIN THE DARKSIDE  Protein Biochemistry Proteins are complex biopolymers whose structure is determined by the Ubiquitination sequence of amino acids 8.5 kDa protein Amino Acids Marks proteins for degradation Amino acids contain a carboxyl, amino, and side chain group Binds to certain amino acids Amino acids are asymmetric due to side chain Regulates major cellular processes (cell Methionine and Cysteine contain sulphur division, immune responses, embryonic Methionine is always the first amino acid in a chain development Cysteine can form disulfide bonds The proteasome degrades/recycles unwanted proteins Primary Structure Carboxyl group of 1 amino acid is attached to amino group of another Functions of Proteins: This determines how the peptide folds Structural proteins- Example: non polar amino acids are found tucked inside Extracellular Matrix Proteins Secondary Structure Muscle Depends on hydrogen bonding Cytoskeletal The folding pattern of the chain Immunity Proteins Types: Transport 1. Alpha helixes: H bonds between carboxyl and amino groups DNA binding 2. Beta sheets: flatter, thinner, more flexible, more stable (h bonds again) Pro and anti coagulant 3. U Turns: 3-4 amino acids, form a short loop which is strong Catalytic Tertiary Structure Signalling 3D Membrane transport Includes all secondary structures Involved in cell adhesion and recognition Quaternary Structure Several subunits Held by non covalent bonds Several tertiary structures Effects of abnormal primary structure Misfolding: sickle cell disease Trapping protein in ER: cystic fibrosis Premature stop codons: Duchene Muscular Dystrophy Insulin Synthesis 1. Pro insulin contains AA sequence of insulin 2. I’m Golgi disulphide bonds are established 3. Insulin is stored in vesicles 4. Amounts of insulin and C PEPTIDE are released Abnormal Secondary Structure Amyloidosis- group of diseases caused by improper protein folding (Beta sheets) Prions- infectious particles (alpha helix changes to beta sheet) Protein detection Staining Immunoblotting (separate proteins by size) Mass spectrometry (feed in a sample) Crystallography (usually used to find electronic structure of proteins by making crystals out of proteins) Edman sequencing (analysing 1 amino acid at a time) Proteins can be manipulated in many ways JOIN THE DARKSIDE  The Cell Cycle & Apoptosis Cellular Reproduction (also known as multiplication by division) 4 phases 1. Cell growth (G1) 2. DNA replication (S) 3. Preparation for division (G2) 4. Cell divides to form 2 cells (M) Terms Cell cycle= reproductive cycle Mitosis= division of parent cell Cytokinesis= physical process of cell division (cytoplasm gets divided) G1 Phase Rapid cell growth Duplication of organelles and cytosolic components High metabolic activity Centrioles begin duplication The cells that don’t replicate of to G zero phase and undergo apoptosis S Phase DNA replication and more growth G2 Phase Cell growth continues (final growth) Preparation for cell division Enzymes and proteins are synthesized Replication of centrioles completed Note: these 3 are apart of interphase DNA Packaging 8 Histone proteins DNA + Hist one => Nucleosome only present cell during division Histones are small and this allows for better binding They are rich in arginine and lysine JOIN THE DARKSIDE TERMS Chromatin: complex of DNA and Histones Nucleosome: short DNA wrapped around 8 Histone proteins Chromosome: condensed DNA Chromatid: 1 copy of a chromosome Centromere: holds 2 sister chromatids together Centrosome: organelle that organizes the microtubule Spindle: formed by the microtubules on opposite ends of the cell Kinetochore: protein complex that binds to a region on the chromosome M Phase Prophase Spindle fibers appear and chromosomes condense (microtubule starts organizing itself) Prometaphase Spindle fibers attach to chromosomes and chromosomes condense further Metaphase Chromosomes align in the middle Anaphase Centromeres divide and sister chromatids move to opposite ends of cell (form individual chromatids) Telophase Nuclear membrane reforms and chromosomes decondense Cytokinesis Cytoplasm divides (contractile ring made of actin and myosin) and 2 daughter cells are made Motor proteins involved in interpolar microtubules Dyenin Kinesin Meiosis is a special type of cell division where the result is only 1 from each pair (forms sperm and egg cells) JOIN THE DARKSIDE Checkpoints Regulate division Responds to info received from cells Triggers internal and external, events and ensures coordination and timing Binary biochemical switches Launch events smoothly Cyclin is a protein that travels through the cell cycle as a backup mechanism Cytogenetics The study of the microscopic structure of chromosomes by chromosome banding Chromosomes are most condensed in metaphase Apoptosis (shrinkage) Programmed cell death Fast process Does not lead to bursting of cells Avoids damage to neighbouring cells Steps 1. Membrane blending (roughening of membrane 2. Cell shrinkage 3. Nuclear fragmentation (breaks into small pieces) 4. Chromatin condensation 5. chromosomal DNA fragmentation 6. mRNA degradation 7. Separation of cell fragments unit apoptotic bodies 8. Organelle and membrane still intact (contents don’t spill out) Signal Transduction in Apoptosis Irreversible process Caspases (cysteine enzymes) Two pathways: Extrinsic- triggered by cell surface death receptors Steps: FASL ligand binds to FAS receptor (death receptor) which activates FADD which binds to the initiator caspase ( Pro caspase 8) to activate it. This then cleaves Pro caspase 3 to activate it (executioner caspase) and then cleave the substrate Intrinsic- triggered by chemicals and depends on mitochondria apoptotic stimulus to activate proaptotic B. 1-13- only protein ( Bad/Bim ). This activates anti-apoptotic protein which frees up other proteins and aggregates active pro-apoptotic effectors proteins (eg. Bax/Bak) which forms a pore on mitochondria realeasing intermembrane proteins (IMP). One of these is called cytochrome c which activated procaspase 9 that binds to apoptosome. This cleaves caspase 3 to cleave substrate. JOIN THE DARKSIDE  Enzyme Kinetics Enzymes are proteins that are folded to create active sites (catalytic sites) Enzyme Kinetics that allow the specific recognition of the substrates that they transform. They are very specific (but can have more than one substrate they bind to) They can be regulated according to the organisms needs Classes of Enzymes Catalysts increases the rate of a reaction and it does not change its structure Enzymes lower the activation rate (energy required to start a reaction). Models of Enzymes: 1. The induced fit model: a substrate binds to an active site and both the enzyme and substrate change shape to fit each other. It can also bind to substrates together or unbind them. The enzyme reverts back to its original shape afterwards. 2. Transition state theory: this is the state of the reaction when the molecule is neither a substrate or product (unstable). This reduces the activation energy and stabilizes it. What is Enzyme Kinetics? It is the rate at which enzyme reactions occur This is affected by temp, pH, and substrate concentration Variables Vmax= max velocity (when the enzyme is saturated). This means no matter how much substrate you add the rate of reaction will not increase because the enzyme is “done”. Km= the concentration of the substrate when half of the active sites are occupied. It is the affinity of the substrate Isoenzymes are enzymes that catalyze the same reaction (the only difference is they have different substrate affinities) Example= Hexokinase 1 & Glucokinase JOIN THE DARKSIDE Isoemzyme vs Isoform Isoemzymes perform the same function but differ in their properties (not apart of same family) Isoforms are products that have similar genes and families Example= Cytochrome P450 family Enzymes can be measured in micro moles of substrate Enzyme Inhibition 1. Competitive: when an inhibitor binds to the active site of an enzyme preventing the substrate from binding. In this case Km increases and Vmax remains unchanged. 2. Non-competitive: when an inhibitor binds to an allosteric site ( anywhere but the active site) of the enzyme changing its active site so that the substrate can no longer enter. In this case the Km stays the same but the reaction rate (Vm) decreases. Allosteric Regulation Allosteric enzymes have a active site and regulatory site ( this is where a modulator goes) This is when a molecule binds to an allosteric site altering the enzymes shape either activating or deactivating the enzyme. This is different from Non competitive inhibition In this case both Vmax and Km can be changed either positively or negatively Affinity VS Efficacy Affinity= Km (how strongly a ligand binds to a target) Efficacy= Vm (the ability of a ligand to make an effect) Regulation of Enzyme Activity 1. Feedback loops (positive and negative) 2. Feedforward activation (upstream enzyme activates downstream enzyme) 3. Phosphorylation/ dephosphorylation 4. Proteolysis (breakdown of proteins to amino acids by enzymes) 5. Changes in gene expression 6. Unspecific regulation (inhibition) 7. Allosteric regulation 8. Enzyme induction (increase in enzyme as a result of some stimulus) Enzyme induction is mostly done by drugs and is slowly reversible Example: chronic ethanol consumption induces CYP2E1 (a specific isoenzyme) JOIN THE DARKSIDE  Acids, Bases, & Buffering Systems Part 1: Fundamentals of pH, [H+], and Unit Modifiers - Acid base disorders are common S Blood analyses are done to assess acid base balance Some terminology - H= Hydrogen - [H+]= concentration of hydrogen ions in a solution P= potential/power ↑ - So pH= power of hydrogen & Mole= unit used to measure the amount of atoms of a particular substance Mol/L= amount of substance per unit of solution & So basically the amount of [H+] in a solution (usually blood) More hydrogen ions= more acidic - Measured in mol/L - - pH= -log[H+] that’s why the pH is lower even though there are more hydrogen ions The reason we use a logarithmic transformation is because it is easier - to interpret Note: In clinical settings [H+] is usually measured in nmol ~ Unit modifiers are just more manageable numbers (eg: using - nanomoles instead of moles) - In order to convert to pH the values must be in mol/L, to do that just multiply by the given number in chart Part 2: Acids and Bases at the Clinical Level- The Importance of the Bicarbonate Buffering System ! Urine is typically acidic so using pH is reasonable But for blood there are narrower values and using [H+] is preferred - Blood [H+] is kept in tight limits (35-45 nmol/l) which is pH 7.35-7.45 - [H+]> 120 nmol/l (pH7.69) IS DANGEROUS AND REQUIRES Bicarbonate buffering system - URGENT TREATMENT - Approx 60mmol of hydrogen ion concentration is produced through metabolism everyday (not good) and that’s why we need systems to maintain blood pH in limits 1. Buffers (temporary) / 2. Renal [H+] excretion H+ ions are secreted through urine and that’s why urine is acidic - Large amounts of CO2 are produced everyday and is excreted by the lungs so that it doesn’t affect acid base balance ↓ A buffer is a solution of a weak base and its salt (or weak acid and its salt). They can bind to H+ and resist changes in [H+] JOIN THE DARKSIDE - Changes in pH affect: enzyme activity, cellular uptake, use of minerals, and uptake and release of O2 & The body can only tolerate fluctuations in pH of +/- 0.04 If pH drops below 6.8 or above 7.8 you die : pH fluctuations are mostly a result of the body’s production of organic acids Example: acetic, acetoacetate, propionic, etc. PH is maintained by: Intracellular and extracellular buffers Alveolar ventilation that controls PaCO2 Renal H+ excretion that controls plasma [HCO3] Major Buffering Systems of the Body: 1. Bicarbonate buffering system (in lungs)- fastest, and primary one 2. Phosphate buffering system- 3. Ammonia buffering system (in kidneys)- 4. Proteins- can be acidic or basic (depends on side chain) If [H+] increases, CO2 increases, Respiration rate increases- HYPERVENTILATION Respiratory acidosis= failure to excrete sufficient CO2 by lungs Metabolic acidosis= failure to excrete enough endogenous acid by kidneys Henderson-Hasselbalch Equation Simplified Henderson-Hasselbalch Equation (Kassirer-Bleich Equation) Davenport Diagram JOIN THE DARKSIDE  Part 3: Transport and Removal of CO2 - CO2 is exchanged for O2 in lungs by RBC Oxygen Transport: 1. Oxygen taken to lungs 2. RBCs flow through alveolar capillaries 3. Gaseous exchange takes place Haemoglobin carries oxygen Gas transfer in Lungs: 1. O2 binds to deoxyhemoglobin forming oxyhemoglobin when H+ is released 2. H+ binds to bicarbonate to form carbonic acid 3. Carbonic acid dissociates to CO2 and water 4. CO2 is then expired Gas Exchange in Tissues: (reverse steps) 1. Oxygenated Hb dissociates to release O2 in tissues 2. Deoxygenated form of Hb is produced 3. CO2 generated in tissues is hydrated to form carbonic acid. This ionizes to form H+ and bicarbonate 4. Deoxygenated Hb accepts hydrogen When O2 is unloaded in tissues, BPG binds to Hb - Beta chains. This causes a change to all four chains and does not allow anymore O2 binding When BPG levels are low (in lungs), it dissociates & from Hb allowing O2 binding again Hb is created in mitochondria of immature red I blood cell Steps: 1. Iron is delivered to RBC by transferrin (protein) 2. Iron is translocated to mitochondria of RBC 3. ALA (delta aminolaevulinic acid) is made 4. Processed in cytoplasm then put back into mitochondria as protoporphyrin 5. Protoporphyrin binds iron to make Haem groups 6. Everything is combined to form Hb JOIN THE DARKSIDE  Part 4: Other Key Processes and Buffering Systems & Kidneys have to perform 2 physiological functions 1. Resorption of all filtered bicarbonate (happens in proximal tube) 2. Excretion of daily H+ load (happens in collecting duct) Phosphate Buffering System: - Act as a sink for H+ forming dihydrogen phosphate - We have high concentrations of phosphate inside of the cell because we need it for ATP and many signalling pathways that use phosphorylation Ammonia Buffering System Generated from amino acid glutamine & / Used to secrete H+ in urine Proteins are good buffering agents because they accept excess H+ S keeping pH in limits JOIN THE DARKSIDE  Neuromuscular Junctions Terminology & Sarcolemma: membrane of muscle cell - Sarcoplasm: cytoplasm - Sarcoplasmic Reticulum: specific form of ER, has calcium ion pumps (important for muscle function), and no ribosomes - T-tubule: connected to sarcoplasmic reticulum by protein complex Protein complex triggers release of calcium from SR - Action potential reaches down the T-tubule - Synapse: point of contact between a nerve cell and another OR between a nerve cell and target cell Types: Electrical Synapse: - Formed by gap junction between 2 neurons where electrical current can flow directly Chemical Synapse: Signal is transmitted via neurotransmitters y Function is to facilitate communication - Steps of Transmitter Release 1. Vesicles containing neurotransmitters are pre-docked to membrane 2. Action potential arrives at synapse 3. Voltage gated calcium channels open and calcium gets released 4. Calcium binds to synaptotagmin which is a protein 5. This initiates fusion with plasma membrane ↑ How do 2 membranes fuse together? SNARE proteins coil and fuse vesicles to plasma membrane They are anchored membrane proteins Tetanus toxin blocks release of neurotransmitters causing muscle - spasms JOIN THE DARKSIDE  Recycling of Membrane at Synapse After neurotransmitters are released there is lots of excess membrane ! 1. Kiss and run mechanism: when it’s time to send a message, the vesicle lightly touches the cell wall making a tiny hole and some chemicals go come out. The vesicle then pulls out and keeps the rest of the content inside to use again. 2. recycling via clathrin-mediated Endocytosis: clathrin coated vesicles are reusable “bags” that are filled up and brought back into the cell 3. Pool of docked vesicles can be exchanged and are quality controlled 4. Recycling via invagination (slower process): like phagocytosis but on a smaller scale Neuromuscular junction: chemical synapse between motor neuron and skeletal muscle cell 1 nerve fibre per end plate Steps: 1. Impulse arriving triggers calcium influx 2. Acetylcholine (neurotransmitter) is released into synaptic cleft 3. It binds to nicotinic cholinergic receptors (found on endplate) 4. Action potential released 5. Increase of calcium concentration in muscle 6. Contraction occurs Acetylcholine Receptor Nicotinic acetylcholine receptor (nAChR) Transmitter gated sodium channel 5 subunits Muscarinic acetylcholine receptor (mAChR) Muscle Action Potential 1. Acetylcholine sodium channel- influx of sodium 2. Calcium channels on Sarcolemma- influx of calcium 3. Calcium channels on sarcoplasmic reticulum- influx of calcium Motor Action Potential Important because muscle cells can be excited over and over again Ensures reliable and consistent transmissions Safety factor is the ratio between the amount of depolarisation and amount needed to depolarize It releases more acetylcholine than needed to make sure safety factor is reached Steps: 1. Ryanodine receptor is activated by voltage sensitive DHP receptor 2. Calcium released from sarcoplasmic reticulum 3. Influx of calcium to SR 4. Calcium binds to Troponin and triggers myosin contraction Calcium acts as second messenger JOIN THE DARKSIDE ~ Auto antibody is an antibody that binds to self and then the immune system starts attacking itself Pathophysiological conditions - Autoimmune disorders - Antibodies against AChR and MuSK in myasthenia gravis, VGCC in Lambert-Easton myasthenic syndrome , VGKC in neuromyotonia (Isaac’s syndrome) How Myasthenia Gravis occurs: 1. Acetylcholine released normally 2. Not enough receptors for it 3. Folds on motor endplate flatten 4. Action potential on endplate does not reach full potential 5. Autoimmune disorder Thymus is mostly involved (abnormal) Symptoms: Muscle weakness & Fatigue ↑ Cranial muscles affected j In smooth and cardiac muscles there are no neuromuscular junctions, & no recognizable end plates or postsynaptic specializations They have different membrane potentials & JOIN THE DARKSIDE