4BMC Lecture 6: Spinal Cord - PDF

4BMC Lecture 6 Spinal cord Dr Katia Mahn [email protected]  Topic Contents Sensory input, integration, motor output, CNS/PNS, neuron (multipolar),...

4BMC Lecture 6 Spinal cord Dr Katia Mahn [email protected]  Topic Contents Sensory input, integration, motor output, CNS/PNS, neuron (multipolar), structural and functional classes of neurons, L1: Building blocks of a nervous system microglia L2: How does the signal work? Action potentials, membrane potentials, refractory periods, synapses, neurotransmitters Brain development, hemispheres, regions and organisation, brainstem, diencephalon, cerebellum, consciousness and L3: How does the brain do what it does? sleep, brain waves, memory and language, circuits L4: How is it protected? Meninges, cerebrospinal fluid, skull, blood brain barrier L5: Brain functional organisation II Hemispheric lateralisation, language, motor and sensory areas, homunculi L6: Spinal cord and reflexes Spinal cord anatomy, protection, neuronal pathways, reflex arcs and spinal reflexes, trauma L7: The peripheral nervous system PNS, sensory receptors, nerves and ganglia, cranial and spinal nerves, motor periphery, ANS L8: When things go wrong Alzheimer’s, Parkinson’s, Huntington’s, BSE, schizophrenia, diagnostics L9: Do we see what we think we see? Vision, eye and accessory structures, optics, retina, visual processing L10: All ears! Hearing and balance, structure of the ear, hearing, balance, imbalances L11: Sensing chemicals Chemosensation, olfaction, taste buds, olfactory epithelium, imbalances of chemosensation Types of cartilage, growth of cartilage, bone structure and classification, gross and microscopic anatomy, bone L12: Standing up and holding it together development and repair Smooth, skeletal and cardiac muscle, gross and microscopic anatomy, sliding filament theory, force, velocity and L13: Move it, move it duration of contraction, Duchenne’s muscular dystrophy Cerebral cortex: gray matter surface  The brain stem The brainstem consists of the Midbrain midbrain, pons and medulla 1. Sensory and motor 2. Reflex movement of head, eyeball and trunk Pons varolii (full name for pons) 1.Connects spinal cord with brain 2.Connects parts of brain with each other 3.Helps control breathing Medulla oblongata 1.Connect spinal cord with the brain 2.Ventilation 3.Cardiovascular centre – symp+ parasymp NS 4.Vasomotor centre – baroreceptors 5.Reflex centres: vomiting, coughing, sneezing, swallowing. 6.Motor fibres 1. Protective structures: the vertebral column 7 Cervical 12 Thoracic 5 Lumbar 5 Sacrum Fused 4 Coccyx 10  Atlas (topmost vertebra) and axis form a joint connecting the skull and spine. Atlas and axis are specialized to allow a greater range of motion than other vertebrae. 11 Vertebrae along the spinal column 12  Lordosis (swayback:) excessive anterior curvature of the lumbar vertebral column region  Protective structures: the meninges Distally, meninges form a strand of fibrous tissue, the filum terminale. Filum terminale attaches to the vertebral bodies of the coccyx Anchor for the spinal cord and meninges. Dura mater: extends from the foramen magnum to the filum terminale Spinal nerves pierce the dura mater Dura mater surrounds the nerve root Fuses with the epineurium.  Spinal cord external anatomy SC begins at the occipital bone SC ends as cauda equina at vertebral junction L1/L2 Cervical enlargement C4 to T1, max circumference 38mm Nerves serving arms emerge here Lumbar enlargement T11 to L2, max circumference 33mm Nerves serving legs emerge here Functions: provides 2-way communication between CNS and PNS Contains the spinal reflex centres 15  Cauda equina The collection of nerve roots at the inferior end of the vertebral canal Q What is cauda equina syndrome? Cauda equina = ‘horse’s tail’ Symptoms: Low back pain, radiating down the legs Numbness around the anus, loss of bowel or bladder control. Erectile dysfunction in men Onset rapid or gradual. Cause: usually a disc herniation, or cancer, trauma, epidural hematoma. 16  SC internal anatomy Grey matter inside, white matter outside Two lengthwise grooves divide cord into right and left halves Ventral (anterior) median fissure Dorsal (posterior) median sulcus Gray commissure connects left and right grey areas  Spinal Cord – Grey Matter Dorsal horns (posterior horn) – interneurons Ventral horns (anterior horn) – somatic efferents Lateral horns: part of symp ANS, present in thoracic and L1&L2; motor neurons to SM and glands Dorsal Horn Dorsal root ganglion (DRG) Lateral horn Ventral Horn Not all tracts are shown! 23 Simplified-spinal reflex 27 Even more simplified- spinal reflex Afferent cell body is in DRG !! This reflex arc is bi- or disynaptic. 28  Spinal nerves 31 pairs of spinal nerves: Note: 8 cervical nerves, but 7 cervical vertebrae. 8 cervical nerves Spinal nerves pass in and out superior to the 12 thoracic nerves corresponding vertebrae via intervertebral foramen Travel to the body region 5 lumbar nerves they serve 5 sacral nerves 1 coccygeal nerve 29  Example spinal nerves Iliohypogastric nerve: from L1, some fibres from T12 Saphenous nerve – largest cutaneous branch of femoral nerve Subcostal nerve – branch of T12 Pudendal nerve – S2 to S4 nerves Sciatic nerve – longest and widest single nerve in human body – L4 to S3 nerves Femoral nerve – largest branch of lumbar plexus – L2 to L4 nerves Peroneal nerve – fibular nerve Iliohypogastric nerve abdominal muscle and skin Saphenous nerve sensation in medial leg Subcostal nerve gluteal skin, abdominal wall Pudendal nerve genitalia, bladder, rectum Sciatic nerve skin of leg, muscles: back of thigh, lower leg and foot Femoral nerve quads, hamstrings Peroneal nerve hamstrings, muscles controlling ankle and foot  Spinal Cord - White Matter Contains bundles of nerve axons (tracts or fascicles) Grouped into 3 pairs of columns (funiculi) Allow communication within the CNS May be ascending or descending Ascending = sensory Descending = motor  Spinal Cord - White Matter Transverse Commissural fibres - Cross from one side to the other Longitudinal White matter is organised into tracts, in three white columns (funiculi) on each side dorsal (posterior) lateral ventral (anterior) 33 Each spinal tract is composed of axons with similar functions Descending Pathways and Tracts Deliver efferent impulses from the brain to the spinal cord Direct pathways—(pyramidal tracts) Serve to provide targeted volitional movements Parkinson’s disease Indirect pathways— (extrapyramidal tracts) Prevent unwanted movements Huntington’s chorea Controls tone e.g. rubrospinal tract red nuclei (shoulder flexor) 34 Spinal cord injury Injuries can be complete or incomplete Quadriplegia: Injury in cervical region All 4 extremities affected Paraplegia: Injury in thoracic, lumbar or sacral segments 2 extremities affected  Incomplete injuries Central Cord Syndrome Anterior Cord Syndrome Posterior Cord Syndrome Brown – Sequard Syndrome Cauda Equina Syndrome ▪ RTAs ▪ Falls ▪ Assault ▪ Sport ▪ Degenerative causes ▪ Tumours ▪ Spinal cord haemorrhage ▪ Spinal cord stroke ▪ Infections ▪ Aortic aneurysm ▪ Central cord syndrome ▪ Brown-Sequard Syndrome Age: 29 (1970s) 40 (2000s) Male 81 % Female 19 % Please take the time to have a look at the anatomy model and the atlas pages 104-107 (spinal cord) and 45 ff (vertebrae). Dr Katia Mahn [email protected] 39 Protein Sorting/Translocation/Trafficking Dr Dawn Jones  Intended Learning Outcomes By the end of this lecture you should be able to describe The pathways involved in protein translocation How proteins are ‘addressed’ to the correct location The effect post translational modification has on protein translocation How proteins that are no longer required are marked for destruction How the proteasome functions to remove unwanted proteins BSc in Clinical Pharmacology | Motivated by curiosity  Protein ‘address labels’ Amino acid sequences within a polypeptide specify its destination: signal sequences. These sequences are typically at the amino terminal end of the protein – the first part to be synthesised. Enzyme systems within the cell recognise the signal sequence and transport the protein to the correct destination. Signal sequences are usually removed when or before the protein arrives at its final destination. BSc in Clinical Pharmacology | Motivated by curiosity  A major sorting decision is made early in protein synthesis when specific proteins are synthesised either on free or membrane bound polyribosomes  Ribosomes are free! And bound! Proteins are made on ribosomes Cytosolic Ribosome pool proteins Used to make cytosolic proteins An ER signal sequence on growing protein directs Ribosome pool ribosome to continue protein synthesis on ER --rough ER Ribosome becomes attached to cytosolic side of Organelle* membrane and protein is translocated into ER whilst proteins being synthesised! Two fates of ER protein Membrane bound or lumen  Cotranslational import The first pathway is utilized by ribosomes synthesizing polypeptides destined for export from the cell These ribosomes become attached to ER membranes early in translation, and polypeptide chains are transferred across the ER membrane as synthesis takes place This is called cotranslational import  Signal sequence on protein binds to signal recognition particle while protein is being synthesised. SRP blocks further translation until it binds to the SRP-receptor BSc in Clinical Pharmacology | Motivated by curiosity  Signal recognition particle binds to receptor on ER membrane. BSc in Clinical Pharmacology | Motivated by curiosity  Translocon- (a protein conducting channel) opens Protein synthesis resumes Protein passes through a pore into the ER lumen. BSc in Clinical Pharmacology | Motivated by curiosity  Signal peptidase (attached to membrane) removes the signal sequence. If transmembrane signals are present within the sequence stop- start BSc in Clinical Pharmacology | Motivated by curiosity  GTP unblocks translation Translation continues (cotranslational insertion). When the protein is complete, it is released into the ER. BSc in Clinical Pharmacology | Motivated by curiosity  Compare the two mechanisms Synthesis passes whole protein Synthesis passes part of protein through and signal sequence through but then stopped and cleaved signal sequence cleaved ER membrane protein Lumen protein (water soluble) (transmembrane)  Protein Folding and Quality Control Take Place Within the ER After polypeptides are released in the ER lumen, they fold into their final shape Helped by Hsp70 a chaperone. Proteins that repeatedly fail to fold properly activate various quality control mechanisms One mechanism is the unfolded protein response (UPR), in which sensor molecules in the ER lumen detect the misfolded proteins The ER-associated degradation (ERAD) mechanism recognizes misfolded or unassembled proteins and exports them to the cytosol Here they are degraded by proteasomes – covered in second part of lecture BSc in Clinical Pharmacology | Motivated by curiosity Proteins Released into the ER Lumen Are Routed to the Golgi Complex, Secretory Vesicles, Lysosomes, or Back to the ER Most proteins synthesized on rough ER are glycoproteins The initial glycosylation takes place in the ER as the polypeptide is being synthesized In the Golgi complex, further glycosylation and processing of carbohydrate side chains occurs, and the proteins are sorted and distributed to other locations  Protein glycosylation Protein glycosylation can serve several different biological purposes Glycosylation is important for sorting secreted proteins For example, the phosphorylation of Man on N-glycan creates a recognition signal for sorting lysosomal proteins to lysosome Sugars are added to proteins as they enter the ER. Protein Protein N-acetylglucosamine Mannose Glucose Original oligosaccharide is This is usually trimmed by (Glc)3(Man)9(GlcNAc)2 removal of (Glc)3Man Finally proteins must be sorted and transported to their final destination BSc in Clinical Pharmacology | Motivated by curiosity  Pathways of vesicular transport Secretory pathway in red Endocytic pathway in green Vesicles transport proteins: From the Golgi apparatus to lysosomes and the plasma membrane From the plasma membrane to lysosomes. From endosomes to the plasma membrane. BSc in Clinical Pharmacology | Motivated by curiosity  Vesicular Transport Soluble proteins move from the Golgi Vesicles fuse to the Golgi and deposit their complex to secretory vesicles for secretion cargo inside the complex. from the cell. Cargo proteins are modified in the Golgi Transport vesicles from the ER carry their Stages: budding – movement – fusion cargo to the Golgi complex. Stages: budding – movement Rough ER Rough ER Golgi cisterna BSc in Clinical Pharmacology | Motivated by curiosity  Off to the Golgi apparatus Transport vesicles bud from one cisterna to fuse to the next Further oligosaccharide modifications on proteins Proteins are sorted on signal sequence ER retention signal – returned to ER To endosomes – for lysosome degradation For secretion from cell BSc in Clinical Pharmacology | Motivated by curiosity  Off to the cell surface There is a continuous stream of vesicles Regulated exocytosis pathway budding off Golgi constitutive exocytosis Only operates in specialised secretory pathway cells Function is to allow cell to grow and These cells produce many specialised secretory vesicles containing..Hormones, expand… mucus, digestive enzymes etc This pathway delivers lipids and proteins to the cell membrane And proteins that diffuse into matrix between cells It is the default pathway BSc in Clinical Pharmacology | Motivated by curiosity  Vesicles bud by forming a temporary coat Golgi budding has been very well studied Protein must bind to a receptor in the ER EM shows bud associated with outer region associated with the proteins thickening destination Coat of the protein clathrin Various characteristics of the cargo protein are recognised i.e. aa sequence or added CHO This coating occurs on cytosolic side of membrane Bud formation is facilitated by binding of Coat proteins (COPs) BSc in Clinical Pharmacology | Motivated by curiosity  Once transport vesicle is formed and released COPs removed revealing v-SNARE (vesicle) an integral protein. v-SNARE binds to t-SNARE (target) in the target membrane This binding leads to fusion of transport vesicle to the target membrane Cargo delivered BSc in Clinical Pharmacology | Motivated by curiosity  Posttranslational import An alternative mechanism is employed for polypeptides destined for the cytosol or mitochondria, chloroplasts, peroxisomes, or nuclear interior After translation is complete, the polypeptides are released from ribosomes and remain in the cytosol, or are taken up by the appropriate organelle Special targeting signals are required for this posttranslational import  Posttranslational Import Allows Some Polypeptides to Enter Organelles After They Have Been Synthesized Proteins destined for the nuclear interior, mitochondrion, chloroplast, or peroxisome are imported into these organelles after completion of translation These are synthesized on free ribosomes and released into the cytosol BSc in Clinical Pharmacology | Motivated by curiosity  Nuclear import Each protein released to the cytosol has localization signals specific to the destination E.g. import into the nucleus requires nuclear localisation signals that target proteins for transport through nuclear pores BSc in Clinical Pharmacology | Motivated by curiosity  Importing Polypeptides into Mitochondria and Chloroplasts Nearly all polypeptides encoded by mitochondrial or chloroplast genes are subunits of multimeric proteins with one or more subunits encoded by nuclear genes Most mitochondrial and chloroplast polypeptides are synthesized on cytoplasmic ribosome, released into the cytosol, and taken up by the organelle within a few minutes The targeting signal for mitochondrial and chloroplast polypeptides is a transit sequence located at the N-terminus of the polypeptide The mechanism is similar to cotranslational with the transit sequence transit peptidase. BSc in Clinical Pharmacology | Motivated by curiosity  Most mitochondrial proteins are imported Complex diffuses laterally in membrane. from cytosol Finds inner translocator protein and lines Transported across both membranes in one up step at sites where the two membranes These move cargo across with help of come close chaperone proteins in matrix and Mitochondrial signal sequence protein unfolds as it moves across detected by outer membrane Signal sequence is cleaved by signal import receptor protein which is peptidase inside, and folds again – mature linked to outer translocator form protein  Summary Signal sequences direct proteins to ER. In ER, signal peptides are removed and proteins are glycosylated. Proteins move to Golgi apparatus in vesicles. As proteins move through the Golgi apparatus, glycosylation is modified. From the Golgi apparatus, proteins are directed in vesicles to the lysosomes or to the cell surface. BSc in Clinical Pharmacology | Motivated by curiosity 4BMC Lecture 1 Building blocks of a nervous system Dr Katia Mahn [email protected] Topic Contents Sensory input, integration, motor output, CNS/PNS, neuron (multipolar), structural and functional classes of Building blocks of a nervous system neurons, microglia How does the signal work? Action potentials, membrane potentials, refractory periods, synapses, neurotransmitters, circuits Brain development, hemispheres, regions and organisation, brainstem, diencephalon, cerebellum, How does the brain do what it does? consciousness and sleep, brain waves, memory and language How is it protected? Meninges, cerebrospinal fluid, skull, blood brain barrier Spinal cord and reflexes Spinal cord anatomy, protection, neuronal pathways, reflex arcs and spinal reflexes, trauma The peripheral nervous system PNS, sensory receptors, nerves and ganglia, cranial and spinal nerves, motor periphery, ANS When things go wrong Alzheimer’s, Parkinson’s, Huntington’s, BSE, schizophrenia, diagnostics Do we see what we think we see? Vision, eye and accessory structures, optics, retina, visual processing All ears! Hearing and balance, structure of the ear, hearing, balance, imbalances Sensing chemicals Chemosensation, olfaction, taste buds, olfactory epithelium, imbalances of chemosensation Types of cartilage, growth of cartilage, bone structure and classification, gross and microscopic anatomy, bone Standing up and holding it together development and repair Smooth, skeletal and cardiac muscle, gross and microscopic anatomy, sliding filament theory, force, velocity Move it, move it and duration of contraction, Duchenne’s muscular dystrophy The mind “If the human brain were so simple that we could understand it, we would be so simple that we couldn’t”  Lecture learning outcomes By the end of this session, you should be able to: 1. Name some major functions of the nervous system 2. Understand inputs and outputs 3. Describe the organisation of the nervous system: Sensory vs. motor division Somatic vs. visceral division Sympathetic and parasympathetic autonomic nervous system 4. Describe a multipolar neuron and know its organelles 5. Understand structural classes of neurons 6. Know the glial cells  Related module ILOs 1. Understand the structure and function of nerve, bone and muscular cells and tissues in health and disease 3. Recognize and apply subject-specific theories, paradigms, concepts or principles (for example concepts of physics and chemistry that underly the function of excitable tissues, mechanical concepts to explain bone function and different theories about the underlying pathology of neurodegenerative diseases) What is the nervous system for? What does it do? What can we do to it?  What could possibly go wrong? Diseases Injuries Developmental problems  Is life possible without a brain? Central and peripheral nervous system CNS: Brain and spinal cord Control centre Integration PNS: Everything else Cranial nerves, spinal nerves Communication between CNS and body CNS PNS  Name some inputs! What outputs are there?  Inputs and outputs Afferent = to carry towards from the latin verb afferre (ad + ferre) Efferent = to carry away from the latin verb efferre (ex + ferre) Inputs travel from sensors to the brain along afferent nerves Outputs travel from brain to effectors along efferent nerves  Somatic vs. autonomic nervous system Somatic nervous system Autonomic nervous system Voluntary activity under Involuntary physiological conscious control processes Inputs and outputs Levels of organisation in the human nervous system CNS (brain, SC) PNS Somatic Somatic Visceral ANS motor sensory sensory Sympathetic Parasympathetic  Detour: neurotransmitters in the PNS somatic efferent motor nACh nACh NA Sympathetic ANS Parasympathetic ANS nACh mACh CNS ACh: acetylcholine, NA: adrenaline, n: nicotinic, m: muscarinic  Tissues and cells in the nervous system Neurons Neuroglia Connective tissue Blood vessels Structure of a neuron (motor neuron) Dendrites carry incoming signals towards cell body Axons carry signals away ‘Dendrites towards, axons away’ applies in many but not all cases The unipolar (pseudo-bipolar) sensory neuron is an exception as it has an axon to do both  Neuron processes Dendrites carry incoming signals towards cell body Axons carry signals away from the cell body Dendrites: Axons: Rough surface (dendritic spines) Smooth surface Usually many dendrites Usually 1 axon Ribosomes No ribosomes Cannot have myelin Can have myelin Branch near cell body Branch further away from cell body Exception: sensory cell dendrites may be myelinated, really extension of axon! Neuron body (soma) Soma= body Prominent nucleolus Nissl bodies- granular ER Activity: Write down what it is and its function for each of these 8 organelles: 1. Cytosol 2. Nucleus 3. Nucleolus 4. Golgi apparatus 5. Polyribosomes 6. Mitochondria 7. Rough ER 8. Nissl bodies  Organelles 1. Cytosol: K+-rich solution inside the cell containing enzymes responsible for the metabolism 2. Nucleus: DNA maintenance, RNA transcription, control of cell activities 3. Nucleolus: Production of pre-ribosome 4. Golgi apparatus: Sorting, packaging, processing and modification of proteins 5. Polyribosomes: Several free ribosomes attached to a single strand of mRNA. The ribosomes make multiple copies of the same protein. protein 6. Mitochondria: Energy production from the oxidation of glucose substances and the release of adenosine triphosphate 7. Rough ER: Translation and folding of new proteins 8. Nissl bodies: Granules of rough ER with free ribosomes, the site of protein synthesis, unique to neurons  Functional classification of neurons Sensory neuron Soma always outside CNS Examples: touch, pain, proprioceptors Motor neuron Soma within CNS Example: Somatic motor neurons for locomotion. Association neuron (interneuron) Complete neuron within CNS Connect sensory and motor neurons  Functional classification of neurons Sensory Neuron Interneuron Motor Neuron Long ‘dendrite’, short Short dendrites, short Short dendrites, long Length of fibres axon or long axon axon Cell body and ‘dendrite’ Dendrites and cell body outside spinal cord; cell located within spinal Location Entirely within the CNS body in dorsal root cord; axon outside the ganglion spinal cord Interconnect the Conduct impulse to an Conduct impulses to sensory neuron with Function effector (muscle or CNS appropriate motor gland) neuron  Structural classification of neurons Multipolar Unipolar Bipolar Many processes, one axon, multiple One process, dendrites axon only Two processes, one axon, one dendrite  Classification of neurons Multipolar Bipolar Unipolar Secretory region Major neuron in CNS In eyes, ears, Dorsal root ganglia, Most abundant neuron Conductive olfactory mucosa cranial nerve ganglia region in humans Rare in humans PNS Receptive region Examples of neuron types  Neuroglia (glial cells) CNS PNS Astrocyte Satellite cell Functional equivalents Oligodendrocyte Schwann cell Microglia Ependymal cell 24  Astrocytes (astroglia) - CNS Not blood-brain barrier – BBB is due to tight-junctions between capillary endothelial cells and basal membrane of capillary. Satelite cell - PNS Schwann cell - PNS Myelin: Fatty white substance surrounding the axon Surrounds long and large diameter axons Protects Insulates Increases speed of signal Oligodendrocytes - CNS 28 Microglia - CNS Similar to macrophages Migrate to injured neurons Phagocytes: phagocyte microorganisms and neuronal debris 29  Ependymal cell - CNS Can be different shapes (squamous or columnar, like epithelial cells) May be ciliated  Essential activities at home 1. Read about the levels of organisation of the nervous system: CNS vs. PNS Afferent vs. efferent Sensory vs. motor Somatic vs. autonomic 2. Describe a multipolar neuron 3. Remember the organelles of a nerve cell, including their function 4. Know the structural classes of neurons 5. Understand the functional classes of neurons 6. What are the functions of the different neuroglia? 7. Myelination- why is it useful? Next week: How does the signal work?  ? ? ? ? ? ? ? ? ? ? Time for questions ?  4BMC Lecture 7: The peripheral nervous system Dr Katia Mahn [email protected]  Topic Contents L1: Building blocks of a nervous system Sensory input, integration, motor output, CNS/PNS, neuron (multipolar), structural and functional classes of neurons, microglia L2: How does the signal work? Action potentials, membrane potentials, refractory periods, synapses, neurotransmitters Brain development, hemispheres, regions and organisation, brainstem, diencephalon, cerebellum, consciousness and sleep, brain L3: How does the brain do what it does? waves, memory and language, circuits L4: How is it protected? Meninges, cerebrospinal fluid, skull, blood brain barrier L5: Brain functional organisation II Hemispheric lateralisation, language, motor and sensory areas, homunculi L6: Spinal cord and reflexes Spinal cord anatomy, protection, neuronal pathways, reflex arcs and spinal reflexes, trauma L7: The peripheral nervous system PNS, sensory receptors, nerves and ganglia, cranial and spinal nerves, motor periphery, ANS L8: When things go wrong Alzheimer’s, Parkinson’s, Huntington’s, BSE, schizophrenia, diagnostics L9: Do we see what we think we see? Vision, eye and accessory structures, optics, retina, visual processing L10: All ears! Hearing and balance, structure of the ear, hearing, balance, imbalances L11: Sensing chemicals Chemosensation, olfaction, taste buds, olfactory epithelium, imbalances of chemosensation Types of cartilage, growth of cartilage, bone structure and classification, gross and microscopic anatomy, bone development and L12: Standing up and holding it together repair Smooth, skeletal and cardiac muscle, gross and microscopic anatomy, sliding filament theory, force, velocity and duration of L13: Move it, move it contraction, Duchenne’s muscular dystrophy Inputs and outputs Levels of organisation in the human nervous system CNS (brain, SC) PNS Somatic Somatic Visceral ANS motor sensory sensory Sympathetic Parasympathetic  Types of sensory receptors Smell Taste Vision Auditory Temperature Mechanoreceptors Chemical receptors (oxygen, pH) Nociceptors  Mechanoreceptors Respond to pressure, stretch, vibration, distortion Merkel: sustained pressure, static stimulation Meissner corposcule: light touch, flutter, slip Pacinian: rapid vibrations of about 200–300 Hz Free nerve endings: touch, pressure, stretch, tickle, itch Hair follicle: distortion of body hair 7 Proprioception – Muscle Spindle Muscle spindles – measure length Tendon organs measure tension Important in spinal reflexes, e.g stretch reflex, or knee-jerk reflex – stretched quadriceps Brain – keeps muscles at a set length, e.g. quadriceps when standing if a knee bends then a stretch reflex contracts the quadriceps  Muscle spindle activated when stretched 1. By external forces, e.g. lifting a weight, contraction of antagonistic muscles 2. By internal forces – activation of gamma motor neurones to contract ends of intrafusal muscle fibres Stretch ↑ rate of AP in sensory neurons 3-10 special muscle fibres – intrafusal muscle fibres Extrafusal muscle fibres – usual muscle fibres Only the ends of intrafusal fibres are contractile  Maintaining Muscle Length 1.Sensory neurons in spindle receptor send ↑AP to SC 2.Sensory neuron synapses with alpha motor neuron – excitatory - monosynaptic – stretched muscle contracts to maintain length 3.Sensory neurons also synapse interneurons – inhibitory – disynaptic – inhibits alpha motor neuron of antagonistic muscle so this muscle relaxes whilst the agonist in 2 contracts Muscle stretch reflex – maintains muscle length/tone All monosynaptic and ipsilateral  Types of Pain Fast Pain Slow Pain Acute, ‘sharp’, e.g. a pin-prick Chronic, more intense, e.g. intense ‘ache’ Removes part of the organism from Debilitates the organism to prevent harm’s way to prevent further damage over-activity until injury repairs Like most sensations, fast pain adapts Hyperalgesia (↑ sensitivity to pain), – pain subsides allodynia (normal stimulus → pain) A C myelinated Unmyelinated axons axons 11  Nociceptors- what do they detect? Nociceptor: detects a noxious stimulus, e.g. tissue damage, extreme temperature Free nerve endings in tissues: branching dendrites → extensive network of very fine dendrites Nociceptor activation: the following cause Na+ influx Physical distortion of membrane, damage to nociceptor Changes in pH Chemicals released from damaged cells: K+, ATP, mast cell secretions (e.g. histamine) Substance P: peptide released by damaged nociceptors Prostaglandins Arachidonic acid (cell membranes) → arachidonate Cyclo-oxygenase (COX) Prostaglandins  Pain! Cerebral cortex Thalamus Noxious Substance P ST pathway stimulus Nociceptor ATP, Prostaglandins Sensitisation Activation Substance P K+, Bradykinin Serotonin(5HT) Histamine Damaged Tissue Platelets Mast Cells ST = spinothalamic Noxious stimulus 13  Pain is Vital! Congenital analgesia: inability to sense or perceive pain Why do patients with congenital analgesia usually die young? 14  Pathological Pain (neuropathic pain) Chronic pain: Chronic pain is particularly debilitating and can destroy quality of life Often inappropriate: pain persists in absence of tissue damage Sometimes after healing the CNS fails to rest itself; pain persists! Like any system, pain can ‘go wrong’ and serve no useful purpose E.g. following an injury, pathways in the CNS ‘rewire’ – synaptic sensitivity increases so that the injured part is hypersensitive to pain hyperalgesia 15  Neuropathic pain Caused by damage or disease affecting the somatosensory system Associated with dysesthesia (burn, prickle, ache) or allodynia Continuous or episodic Episodic: stabbings or electric shocks. Includes sensations of burning or coldness, "pins and needles“, itching, numbness Diagram: selection of peripheral and central mechanisms contributing to neuropathic pain Neuropathic pain  Spinal nerves 31 pairs of spinal nerves: Note: 8 cervical nerves, but 7 cervical vertebrae. 8 cervical nerves Spinal nerves pass in and out superior to the 12 thoracic nerves corresponding vertebrae via intervertebral foramen Travel to the body region 5 lumbar nerves they serve 5 sacral nerves 1 coccygeal nerve 19  Cranial nerves 06/11/2024  The Olfactory Nerve (I) Originates from the olfactory bulbs. Passes through skull at the cribriform plate Terminates in olfactory mucosa of the nasal cavity. Entirely sensory. Related to smell.  The Optic Nerve (II) Originates in the diencephalon. Passes through skull at the optic foramen in the sphenoid bone. Terminates in the retina. Entirely sensory. Related to vision. The Oculomotor (III) Trochlear (IV) Abducens (VI) Nerves Originate in the ventral areas of the brainstem. Pass through skull at the superior orbital fissure in the sphenoid bone. Related to eye movements  The Trigeminal Nerve (V) Ophthalmic Division Three branches: Opthalmic Maxillary Mandibular Maxillary Division Mandibular Division All originate from the ventrolateral surface of the pons (brain stem).  The Trigeminal Nerve (V) Ophthalmic division Maxillary division Exits; superior orbital fissure, runs forward roof of orbit, Exit: foramen rotundum, runs through floor of emerges through supraorbital notch/foramen. orbit & emerges through infraorbital foramen. Mandibular division Exits foramen ovale, enters mandibular foramen  The Facial Nerve (VII) Originates from the lateral part of pontomedullary junction. Passes into internal acoustic meatus and exits through the stylomastoid foramen. The facial nerve give 5 branches within parotid gland Bell’s Palsy  The Vestibulocochlear Nerve (VIII) Consists of two branches Vestibular branch (balance) Also called the acoustic nerve. Cochlear branch (hearing) Cochlear Part Vestibular Part Semicircular canals  The Glossopharyngeal Nerve (IX) Originates from the medulla oblongata. Exits the skull through the jugular foramen. Motor fibres innervate the stylopharyngeus muscle (of the pharynx) Sensory to oropharynx and posterior tongue.  The Vagus Nerve (X) Arises from medulla oblongata Exits the skull at the jugular foramen Sensory to external auditory meatus & tympanic membrane Motor fibres innervate the muscles of the larynx, respiratory passages, lungs, heart, oesophagus, stomach, small intestine, most of the large intestine & the gallbladder  The Accessory Nerve (XI) Originates from both the medulla oblongata and from the spinal cord. Emerge out through the jugular foramen. Supplies : Trapezius & Sternocleidomastoid muscles  The Hypoglossal Nerve (XII) Originates in the medulla oblongata. Exits the skull through the hypoglossal canal Innervates the muscles of the tongue.  The Cranial Nerves I. Olfactory – sensory - olfaction II. Optic – sensory - vision III. Oculomotor – motor – proprioceptive, eye muscles, lens, iris IV. Trochlear – motor – proprioceptive, eye muscles V. Trigeminal – mixed – eye, face, head, chewing VI. Abducens – motor – lateral rectus muscle of eye VII. Facial – mixed – taste, facial muscles, salivary & lacrimal VIII. Vestibulocochlear – sensory – hearing & equilibrium IX. Glossopharyngeal – mixed – taste, mouth & pharynx X. Vagus – mixed – external ear, taste, larynx, organs XI. Accessory – motor – trapezius, sternocleidomastoid XII. Hypoglossal – motor – tongue muscles 33  The Cranial Nerves – name mnemonic I. Olfactory I. On Oh II. Optic II. Occasion Oh III. Oculomotor III. Our Oh IV. Trochlear IV. Trusty To V. Trigeminal V. Truck Touch VI. Abducens VI. Acts And VII. Facial VII. Funny Feel VIII. Vestibulocochlear VIII. Very Very IX. Glossopharyngeal IX. Good Good X. Vagus X. Vehicle Velvet XI. Accessory XI. Any Ah XII. Hypoglossal XII. How Heaven! 34 The Cranial Nerves – modality mnemonic I. Olfactory I. Some II. Optic II. Say III. Oculomotor III. Marry IV. Trochlear IV. Money V. Trigeminal V. But VI. Abducens VI. My VII. Facial VII. Brother VIII. Vestibulocochlear VIII. Says Starts with an… IX. Glossopharyngeal IX. Big S = Sensory X. Vagus X. Brains M = Motor XI. Accessory XI. Matter B = Both XII. Hypoglossal XII. More  Thalamus II Thalamus Hypothalamus III Midbrain Pons IV Medulla V Brain stem Pons VI VII VIII Pyramid IX XII Olive X Ventral root of 1st cervical nerve XI Spinal cord 06/11/2024 Motor periphery- mostly in muscle lecture Muscle stretch reflex – e.g. knee-jerk (patellar) reflex 1.Tapping patellar ligament stretches ligament – pulls on patella – stretches quadriceps (extensors) 2.Muscle spindle in quads excited 3.Monosynaptic excitatory reflex arc contracts quads 4.Inhibitory disynaptic reflex arc inhibits hamstrings (flexors) 5.Quads contract, extending knee 38  Question: What does the autonomic nervous system do? 39 Sympathetic nervous system: Fight and fright 1. Blood vessels in GI tract 2. Blood vessels in skeletal muscle 3. Blood vessels in the heart 4. Bronchioles in the lung 5. Cardiac muscle 6. Pupils 7. Urinary sphincter 8. Gastrointestinal peristalsis 9. Sex 10. Salivary glands 11. Sweat glands 06/11/2024  Sympathetic nervous system: Fight and fright 1. Blood vessels in GI tract 1. Vasoconstriction 2. Blood vessels in skeletal muscle 2. Vasodilation 3. Blood vessels in the heart 3. Vasodilation 4. Bronchioles in the lung 4. Bronchodilation 5. Cardiac muscle 5. Force and rate up 6. Pupils 6. Dilation 7. Urinary sphincter 7. Constriction 8. Gastrointestinal peristalsis 8. Inhibition 9. Sex 9. Ejaculation/ orgasm 10.Salivary glands 10.Thick saliva 11.Sweat glands 11.Activation 06/11/2024 Parasympathetic nervous system: rest and digest 1. Blood vessels in GI tract 2. Blood vessels in skeletal muscle 3. Blood vessels in the heart 4. Bronchioles in the lung 5. Cardiac muscle 6. Pupils 7. Gastrointestinal peristalsis 8. Sex 9. Salivary glands 06/11/2024 Parasympathetic nervous system: rest and digest 1. Blood vessels in GI tract 1. Vasodilation 2. Blood vessels in skeletal muscle 2. No effect 3. Blood vessels in the heart 3. No effect 4. Bronchioles in the lung 4. Bronchoconstriction 5. Cardiac muscle 5. Heart rate down 6. Pupils 6. Pupil constriction 7. Gastrointestinal peristalsis 7. Accelerates 8. Sex 8. Erection 9. Salivary glands 9. Thin saliva Read about these: Parts of the PNS Sensory receptors types Pain (nociceptive and neuropathic) Mechanosensation Cranial and spinal nerves Sympathetic and parasympathetic ANS Membrane Transport - Ion channels and pumps Dr Dawn Jones  Ion channels Ion channels are integral membrane proteins Contain an aqueous pore or hole through the membrane They allow flow of ions down the electrochemical gradient (passive transport) They are usually gated or controlled – not always open Often specific for a particular ion such as Na+, K+, Ca2+, Cl-  Potassium Channels K+ channels have been well studied Many studies focused on bacterial homologues of the channels, which are simpler versions of the proteins found in humans A bacterial potassium channel called KcsA was the first Side view ion channel to have its structure solved by x-ray crystallography (in 1998) Pore A homotetramer (4 identical subunits) Each subunit has 2 transmembrane a-helices and one shorter a-helix that folds into the membrane PDB ID = 1K4C  Pore through the potassium channel The path through the channel narrows to about 3 Å near the extracellular side of the membrane This narrow part is called the selectivity filter Contains the amino acid sequence TVGYG – conserved in all K+ channel proteins This allows the channel to selectively transport ONLY K+ ions  Potassium channel selectivity filter Ions dissolved in water are surrounded by a ‘shell’ of water molecules To pass through 3Å region in pore K+ must give up its shell of water molecules This is called desolvation and is energetically unfavourable Must make up for this loss of energy by making new interactions with the atoms in the channel – called resolvation  Potassium channel selectivity filter Backbone C=O groups of the amino acids TVGYG in selectivity filter point towards the pore K+ ions make favourable polar interactions with the carbonyl groups of the backbone of the selectivity filter residues Note only 2 of 4 subunits are shown here!  Ion selectivity in K+ channels K+ passes through channel 100 times more efficiently than Na+ Na+ is smaller and does not form such strong bonds with the selectivity filter carbonyl groups Hence Na+ gets less resolvation energy – not enough to overcome the loss from desolvation  Rapid ion movement through K+ channels K+ must be transported very quickly in order for action potentials to propagate But tight binding of K+ ions to selectivity should slow the movement down How is rapid AND selective movement achieved? There are 4 binding sites for K+ in the selectivity filter Binding of 2 K+ ions in adjacent sites causes electrostatic repulsion Hence the ions are rapidly pushed through the channel Potassium channel movie Opening of most ion channels is regulated or gated  Most channels are “gated” Stimulus to transiently open gate and allow transient (brief) ion flow Does not need a conformational change therefore very rapid Does not need ATP nor second ion Therefore only used down a concentration gradient – in PASSIVE transport Gated ion channels open in response to different stimuli Voltage-gated K+ Acetylcholine channels receptor  Action Potentials Signals are passed down neurons by transient changes in voltage across the plasma membrane The transient depolarisation and repolarisation of membranes is called the action potential Changes in voltage are caused by movement of Na+ and K+ through voltage-gated, or ligand- gated ion channels  Voltage-gated ion channels Action potentials propagate along the neuron by opening of voltage-gated ion channels Voltage-gated K+ channels are structurally similar to the bacterial K+ channel KcsA, but each subunit contain 4 extra transmembrane (TM) a-helices One of these helices, called S4, contains many positive charged residues The extra TM helices form a “voltage sensing paddle” on each of the 4 subunits Voltage gated K+ channel Bacterial KcsA channel Tetramer of 6 TM helices Tetramer of 2 TM helices Helix S4 contains many positive charges  How do voltage gated ion channels open in response to voltage change? Not fully understood as we still do not have complete structures of the open and closed states Thought to involve movement of voltage sensing paddles, causing change in structure of pore region  Channel inactivation About 1 ms after opening, the channel spontaneously inactivates, by binding of an “inactivation ball” This blocks the channel so the membrane can repolarize  A key experiment in proving this mechanism involved mutating the channel to delete the inactivation ball Wild-type channel Mutant channel with no inactivation ball Mutant channel with high concentration of a peptide of the inactivation ball Voltage-gated Na+ channels are important in action potentials in neurons Action potential movie  Clinical insight: Tetrodotoxin inhibits a Na+ channel Tetrodotoxin is a toxin found in pufferfish It is a very potent inhibitor of a Na+ channel, and therefore inhibits action potentials Pufferfish is considered a delicacy in Japanese cuisine (sushi) Several people die each year from eating this fish Lethal dose is only 10 ng! Ligand-gated ion channels are important at synapses Ligand-gated ion channel (open) Ligand-gated ion channel (closed)  Acetylcholine receptor - A ligand-gated ion channel Acetylcholine is a common neurotransmitter It can open a ligand-gated ion channel called the acetylcholine receptor  Acetyl choline receptor – a ligand-gated ion channel Pentamer – 5 subunits a2, b, g, d outside membrane inside 5 subunits have similar structure Side view Each have 4 TM helices 2 molecules of acetylcholine bind in the extracellular domain Bind at the interfaces between a-g and a-d  Opening of the acetylcholine receptor Each of the 5 subunits has 4 TM helices called M1-M4 Acetylcholine binds in the extracellular domain Causes a conformational change in the TM helices A rotation of M2 helix causes opening of the pore  Stress–gated ion channels Auditory hair cells Present in the inner ear in the organ of Corti and allows us to hear… What’s this? Organ of Corti?  Activating stress channels in inner ear Hearing involves converting sound waves to fluid motion in inner ear Leads to deflection (movement) of stereocilia – apical (surface) projections from hair cells Mechanical movement opens a variety of ion channels – allows influx of ions 3 Ion influx – initiates an action potential–a nerve 2 impulse to auditory (hearing) centre in brain …to be Continued  4MCB Cell Specialisations and Specialised Cells Dr Dawn Jones  After the session you should be able to: Discuss the types of junctions between cells and their function Discuss the extracellular membrane and its role Discuss stem cells  Most of the cells of multicellular organisms are in contact with other cells The immediate environment of many of these cells is other cells The nature of the physical connection between a cell and its neighbours in a tissue determine what that tissue will be like Cell junctions allow activities of individual cells to be coordinated i.e. enables each system to function as an integrated whole They are fundamental to the interactions between cells Those connections and interactions are INTERCELLULAR JUNCTIONS CELLS ALMOST NEVER WORK IN ISOLATION  Cell Adhesion Molecules (CAMs) WHAT ARE THEY? Mainly glycoproteins Located at the cell surface Form different types of complexes and junctions to join Cells to cells Cells to extracellular matrix (ECM) ECM to cell cytoskeleton WHAT DO THEY DO? AID… Adhesion of cells to each other to form tissue Transmission of signals from outside the cell to inside the cell Migration of cells  CAMs Four main families 1. Cell-cell junctions – mainly Cadherins Cadherins rely on Calcium ions to function (Ca-adhesion!) Transmembrane glycoproteins Link cytoskeleton of one cell to the cytoskeleton of another 2. Cell-matrix junctions- large family of CAMs called integrins Integrins are found in focal adhesion and hemidesmosome type junctions Transmembrane proteoglycans – adhesion to extracellular matrix linkage to cytoskeleton 3. Immunoglobulin superfamily 4. Selectins – special CAMs that bind cell-surface CHO involved in inflammatory response  Cell adhesion in animal cells Joins involve interactions between transmembrane proteins in neighbouring cells. The transmembrane proteins may be anchored inside the cell and associated with the cytoskeleton. Note: the extracellular matrix is commonly linked to the underlying cells by plasma membrane proteins called integrins.  Intercellular Junctions CLASSIFICATION Occluding/organising Junctions Tight junctions Adhering/anchoring Junctions Actin filament attachment sites Cell-cell junctions (adherens junctions) Cell-Matrix junctions (focal adhesions) Intermediate filament attachment sites Cell-cell junctions – Desmosomes Cell-matrix junctions – Hemidesmosomes Communicating junctions Gap junctions Chemical synapses Tight Junction Multiple strands of protein form the tight junction More strands = more impermeability Each strand formed from proteins Claudins Occludins ZO Proteins  Tight Junctions Tight Junctions as barriers to solute diffusion Electron-dense tracer is added in the apical (B) and basal sides of the cells (C).  Anchoring junctions Connecting cytoskeleton 1. Adherens junctions (cell-cell) 2. Desmosomes (cell-cell) 3. Focal adhesions and hemidesmosomes (cell-ECM)  Adherens Junctions They are composed of... Cadherins – bind to the catenins that are connected to the actin filaments Function Provide strong mechanical attachments Cadherins mediate cell-cell adhesion between adjacent cells Cadherins are linked to F-Actin via a They serve as a bridge connecting the diverse set of adaptor proteins actin cytoskeleton of neighboring cells Cadherins form Ca2+-depended through direct interaction homophilic interaction (Ca-adhering)  Desmosomes Also called “Anchoring Junctions” Arranged randomly on the lateral side of cells membranes The adhesion protein bridges the space between the cells  Desmosomes Desmosomes provide mechanically strong connections between epithelial cells. Plaque on the cytoplasmic side of the plasma membrane is attached to transmembrane adhesion proteins and to intermediate filaments (keratin fibres) that span the cell. 13  Function of desmosomes Fasten cells together into strong sheets Attach muscle cells to each other in a muscle Muscle tears can involve rupture of desmosomes  Desmosomes contain specialized cadherin molecules and interact with intermediate filaments The autoimmune disease pemphigus vulgaris disrupts adhesion of epithelial cells mediated by desmosomes. Caused by auto antibodies against desmoglein Patients suffer from severe blister formation  Focal Adhesion Focal adhesions are contact points for the cell with the extracellular matrix. These complex structures regulate communication with the surrounding extracellular environment, signalling regulates diverse cellular processes. The principal components are integrins, which are αβ heterodimers that regulate cell–matrix and cell–cell interactions.  hemiDesmosomes Similar in form to Desmosomes. Desmosomes link two cells together Hemidesmosomes attach one cell to the extracellular matrix and therefore use a different adhesion protein. Gap Junctions Connexons: assembly of six proteins that create gap between two plasma membranes 6 connexin transmembrane proteins form a connexon hemichannel Hemichannels of two adjacent cells form a gap junction with a central pore of 14Å Gap junctions allow passive transport (diffusion) of small molecules and ions  Gap Junctions Functions Rapid communication between neighboring cells, e.g. cardiomyocytes need synchronized actions Communication beyond nerve system, e.g. hepatocytes beyond sympathetic nerves need to be informed to produce glucose from glycogen Embryogenesis, form specific tissue with coupled group of cells Summary of cell junctions  The Cytoskeleton Operational definition: Intracellular network of protein filaments insoluble in non-ionic detergents. Comments: The cytoskeleton gives the cell strength, rigidity and shape; and is also responsible for cell motility and intracellular movements.  Cytoskeleton Functions Cells have specific shapes and internal organisation and carry out co-ordinated and directed movements. All of these properties are controlled by cytoskeleton – which is in effect the cellular cytomusculature. It is a characteristic feature of all eukaryote cells and was probably crucial to evolution of large complex single and multicellular organisms. Components of the cytoskeleton Intracellular Traffic Cell shape and locomotion Mechanical strength  1.Microtubules. These are hollow tubules ca. 25nm in diameter composed of a globular protein calledtubulin. It is heterodimer composed of two closely related globular polypeptides: α & β tubulin that polymersise to form a tubule up to several microns in length. At least 20% of vertebrate brain is composed of tubulin!  Microtubules are dynamic structures. Microtubules are polar – with plus (fast growing) and minus (slow growing) ends. The half-life of a microtubule is ~10 minutes. Dynamic instability – continues unless the + end is stabilised by attaching to molecule or cell structure. i.e attach a cap protein Summary of microtubule structure. Grow from central structure – centrosome or other microtubule organising centre: the spindle pole or basal body Generates system of tracks where… Organelles, vesicles and other cell components anchored Guides intracellular transport of these and cytosolic macromolecules Make beating structures – cilia and flagella  Inhibitors of Microtubules. Because of their dynamic nature microtubules are susceptible to drug action. Colchicine (used to treat gout since Egyptian times). This binds to tubulin subunits and prevents polymerisation. Used to inhibit spindle formation & arrest mitosis. Taxol – binds to tubulin subunits and prevents disaggregation. Used as a treatment for breast cancer.  Microtubules and their associated motor proteins Dyneins: Dumbell like globular molecules which move to negative end of microtubules towards centrosome Involved in organelle transport and mitosis. Ciliary dynein is motor protein responsible for bending.  Microtubules and Motor proteins Kinesins – Double stranded proteins composed with helical coil and small globular heads. Move towards the positive end of the microtubules away from centromere. Involved in meiosis, movement of synaptic vesicles along nerve axons. Cargo molecules are shuttled along microtubules to destinations.  Motor protein function Hydrolysis of ATP – gives energy for a cycle of conformational change of the head domain 41 Release – movement - and binding of head ATP-dependent “walking” along microtubule Cycling through 3 conformations ATP binding ADP + Pi ADP release Mechanism of Kinesin walking  Microtubules and movement of cilia/ flagella Cilia – small hair-like projections of apical cell membrane Consists of a bundle of stable microtubules that grow from a basal body Role is to move fluid over cell surface Respiratory tract epithelium – to move dust and dead cells up to throat in mucus from lungs/ bronchi Oviducts - to move eggs along oviducts to uterus Midline of embryo - to establish left-right axis  Beating of cilium - two different strokes Power stroke: Fully extended stroke to move maximum amount o fluid Recovery stroke curls back into position with minimal disturbance Difference in strokes ensures one direction to movement  Microtubular organisation in cilia and flagella Different organisation to the microtubule tracks Arranged as microtubule doublets arranged in a ring in around two single microtubules in a 9 + 2 pattern Additional proteins associated that project at regular intervals: Cross linking protein nexin holds microtubules together Motor protein to generate force – 2 rows of ciliary dynein Membrane transport Dr Dawn Jones  Membrane transport in biology Cells and organelles are surrounded by highly impermeable membranes Eukaryotic cell Prokaryotic cell  Membrane transport in biology Various membranes must develop and maintain concentration gradients and electrical gradients Molecules must be pumped into and out of cells or organelles High [Ca2+] in ER/SR Nutrients pumped in, e.g. sugar High [K+]i low [K+]o Toxins pumped out, e.g. antibiotics Low [Na+]i high[Na+]o Membrane potential -60 mV Transport of pyruvate and ADP in and ATP out of mitochondria Electrochemical gradient of H+ Low pH in lysosomes  Membrane permeability and diffusion Molecules diffuse from a high concentration area to a low concentration, i.e. down the concentration gradient But not all molecules can easily cross membranes  Active and passive transport 1 – Transport down a 2 – Transport up a chemical chemical gradient is called gradient is called active passive transport or transport. facilitated diffusion. This is energetically Energetically favourable unfavourable i.e. ΔG > 0 i.e. ΔG < 0 1 membrane 2 Will only occur if an However, may not energy source is happen at a significant provided rate without a transport protein  DV Most membranes also have an electrical membrane potential or voltage difference (DV) Movement of charged molecules (ions) is also affected by the membrane potential The effect of the membrane potential and the chemical gradient together is called the electrochemical gradient  Transporters and channels Proteins that facilitate membrane transport Transporters: Sometimes called pumps Have specific binding site for substrate Alternate between two conformations Can allow active or passive transport Channels: Form an aqueous pore allowing specific molecules through ONLY allow passive transport Often gated to control access How molecules cross membranes  Passive transport depends on concentration and electrical gradients UNCHARGED solutes e.g. glucose transporter in a liver cell Transporters like the glucose transporter undergo conformational change when molecule binds Reversible After a meal, blood glucose levels high, glucose binds to external sites of glucose transporter and taken into liver cell and released where concentration lower After fasting, blood glucose low enzyme glucagon in liver releases glucose from store glycogen increases intracellular concentration, binds internal sites and moved across out  Passive transport depends on concentration and electrical gradients CHARGED solutes Many small organic and inorganic molecules are charged Most cell membranes have a small voltage across them creating a membrane potential This potential tends to pull the opposite charge down Net driving force (green arrow) is sum of the two forces – electrical and chemical concentration gradients – “electrochemical gradient”  Electrochemical gradients are important in neurons [Na+] = 145 mM [K+] = 5 mM outside + -60 mV inside - [Na+] = 5 mM [K+] = 140 mM An action potential occurs when ion channels open, causing depolarisation of Electrochemical gradient the membrane across nerve membranes  Electrochemical gradients and energy (DG) Movement of molecules down an electrochemical gradient is energetically favourable So an electrochemical gradient is a store of potential energy How much energy is stored in an electrochemical gradient? The free energy change for moving an uncharged molecule across a membrane is: cin ΔG = RT ln cout where cin and cout are the concentrations of the molecule on the inside and outside R is the gas constant, 8.315 J mol-1 K-1 T is the temperature in K (37° C = 310° K)  Electrochemical gradients and energy (DG) The free energy change caused by the membrane potential acting on an ion is ΔG = ZFΔV where Z is the charge on the ion (can be negative or positive) F is the Faraday constant, 96500 J V-1 mol-1 DV is the membrane potential in V  Electrochemical gradients and energy (DG) The free energy of an electrochemical gradient consists of the chemical energy plus the electrical energy: cin ΔG = RT ln + ZFΔV cout Electrochemical gradient of Na+ across a cell membrane [Na+] = 145 mM outside + cin ΔG = RT ln + ZFΔV -60 mV cout inside - [Na+] = 5 mM Z = 1 (charge on Na+ is +1) DV = -60 mV = - 0.06 V  Electrochemical gradient of Na+ across a cell membrane DG is large and negative so this is VERY energetically favourable Na+ has a strong force pulling it into the cell The electrical and chemical gradients are working in same direction - both are driving the Na+ inside the cell BUT the Na+ cannot diffuse through the membrane – will only actually move if there are open Na+ ion channels or transporters What generates the membrane potential across the plasma membrane?  What generates the membrane potential across the plasma membrane? Distribution of Na+ and K+ across the cell membrane is generated by the Na+-K+ pump (more next lecture) What generates the membrane potential across the plasma membrane? An ion channel called the K+ leak channel allows some of the K+ to flow back out of the cell K+ will flow out until the chemical gradient driving it out balances the electrical force driving it in The movement of K+ out of the cell causes the outside to become positive compared to the inside  When the K+ ions are in equilibrium (chemical force balances electrical force), DG=O cin ΔG = RT ln + ZFΔV cout cin 0 = RT ln + ZFΔV cout RT cin V =− ln ZF cout The Nernst equation  Nernst equation The Nernst equation can be used to calculate the equilibrium potential for a certain distribution of ions For K+ ions, Z = 1 At 37 °C, the Nernst equation becomes: RT cin cin V =− ln V = −0.0267ln ZF cout cout  Resting membrane potential The actual resting membrane potential of most cells is around -60 mV Slightly less than the theoretical equilibrium value of -89 mV K+ leak channels are not fully open K+ leak channels do not quite achieve equilibrium of the K+ ions  Summary – membrane transport Many molecules are impermeable to biological membranes Channels and transporters are membrane proteins that allow movement of molecules across the membrane Movement of a molecule down its electrochemical gradient is passive (DG0) and requires an energy source An electrochemical gradient is made up of a chemical gradient and a membrane potential (DV) The membrane potential across the plasma membrane is caused by a Na+-K+ pump and K+ leak channels Membranes- Structure and Function  Membrane Structure & Function The plasma membrane encloses the cell and defines the boundary of ‘life’. The plasma membrane maintains the essential differences between the cytosol and the external environment. Interior cell membranes compartmentalise cell, enabling different parts of cell to develop specific functions. Membranes form a relatively impermeable barrier – which facilitates controlled access to cell interior. Ion gradients are established due to activities of membrane bound transporter molecules. These gradients drive: The synthesis of ATP, The movement of selected solutes, The production and transmittance of electric signals  Membrane Structure & Function The plasma membrane contains proteins which act as sensors to external signals – allow cells to respond to changes in environmental cues. These protein sensors/receptors transmit information across the membrane (signal transduction). The surface chemistry of the membrane is important in cellular recognition and processes like adhesion. Membrane chemistry can play a key role in either resisting pathogens or allowing them access to the cell.  Cell membranes: Composition & structure Membranes are composed of phospholipids and proteins (50:50). The hydrophilic head groups face outwards and hydrophobic tails facing inwards. The lipid molecules form themselves into a bimolecular leaflet consisting of a 5nm thick double sheets of phospholipid molecules. Cell membranes are plastic and deformable. It is now known that the cell membranes are fluid dynamic structures and the lipid molecules move freely about in the plane of the membrane (2 µms-1) although rarely flip across it.  Membranes are fluid The protein molecules form complexes which in effect float in a sea of lipid. Proteins also serve as structural links with the underlying cytoskeleton – which can anchor components in position and stabilise and rigidify membrane.  Phospholipid component of membranes Phospholipids are most common membrane components. Polar head group consists of -choline linked via phosphate group to glycerol (C3) Two hydrophobic hydrocarbon tails fatty acid chains 12-34 carbons in length. One chain fully saturated (straight) and the other unsaturated with 1 or more cis double bonds– (giving the molecule a kinked profile). Constitute 50% of mass of membrane – 5 x 106 molecules per square µm. Lipid is amphipathic – with hydrophobic and hydrophilic ends.  Properties of phospholipids If placed in water phospholipid molecules will aggregate with their hydrophobic tails buried inwards and their hydrophilic heads exposed to water (micelles). They typically form biomolecular sheets – which form ‘liposomes’ separating two aqueous phases. On outside Glycolipids Phosphatidyl choline Sphingomyelin On inside Phosphatidylethanolamine & Phosphotidylserine (negatively charged). Results in charge asymmetry.  Membrane asymmetry Enzyme – flippase 27 Flippase move phospholipids from inner (lumen) to outer facing monolayer cytosolic Different flippases for different phospholipids - help establish asymmetry Second group of enzymes – floppases also found in cell membrane - Move from cytosolic facing to inside facing Scamblases ensure even numbers of phospholipids on either side of the bilayer by random transfer from one monolayer to the other. ER lumen cytosol Nature Immunology 12, 373–375 (2011)  Other membrane components - Glycolipids Sugar containing lipid molecules which constitute around 5% of the plasma membrane lipid. The sugar groups are orientated towards outside of cell – and are exposed at cell surface. The most complex are so called gangliosides – which have attached oligosaccharides. These are important components of surface of nerve cells (10% lipid). Ganglioside GM1 acts as surface receptor for the bacterial toxin which causes cholera.  Membrane manufacture Glycolipids use a different mechanism to be asymmetrically localised… there are no glycolipid flippases Enzyme that adds sugar group on phospholipids is on inner face of Golgi Fusion of vesicle budded off Golgi keeps internal face outside cell – the cytosol face is maintained!  Other membrane components - Cholesterol The eukaryote cell plasma contains large amounts of cholesterol. Makes membranes less permeable. Cholesterol molecules orientate themselves in bilayer with hydroxyl heads (red) close to polar groups of lipid, partially immobilising phospholipid molecules. Makes membrane less deformable. Also acts as a spacer preventing phase transitions (‘freezing’ of membranes).  Fluid properties of phospholipid membranes. The fluidity of cell membranes is dependent upon their biochemical composition. Shorter chain lengths of fatty acids reduces tendency of hydrocarbon tails to interact with each other. Cis-double bonds produce kinks which make molecules less likely to pack closely together (& allow cholesterol to fit snugly between). Enables membranes to remain fluid at lower temperatures. Organisms adjust composition of their membrane lipids to maintain a constant fluidity with changing temperature. As temperatures fall proportion of lipids with cis bonds increase.  Membrane Proteins The amount and types of proteins associated with membranes are highly variable. In myelin membrane which serve to insulate nerve cells less than 25% of mass is protein. Whereas in membranes involved in energy transduction processes – such as the inner membrane of the mitochondrion nearly 75% is protein. Membrane proteins often have oligosaccharides chains attached to them – particularly the plasma membrane. The outer surface is coated by a layer of carbohydrate – forming the glycocalyx.  Glycocalyx A carbohydrate rich zone on the cell surface. Oligosaccharides mainly associated with proteins although some are glycolipids. Selectins are cell surface binding proteins that mediate cell- cell adhesions. These sugars provide surface markers used to identify cells (e.g. by potential pathogens or opposite mating types).  Functions of integral membrane proteins Lipid bilayer provides a selective barrier - membrane proteins are needed to Transport nutrients, ions, metabolites Anchor membrane to macromolecules Detect external signals Enzymes to carry out specific reactions  Structural proteins associated with inside of red blood cell membrane. Spectrin is associated with cytoplasmic side of membranes. Ankyrin links spectrin to transmembrane proteins and binds ‘skeleton’ to inner face of the membrane. Gives red blood cells their characteristic shape. Protein – membrane associations  Part 2 Getting into and out of cells Transporter molecules  Membrane Permeability Protein-free lipid bilayers are highly impermeable to ions. The smaller the molecule is and the more soluble it is in oil – the more rapidly it will diffuse across such bilayers. Small non polar gas molecules such as O2 and CO2 rapidly diffuse across lipid bilayers (i.e. can move easily into and out of cells). Uncharged polar molecules can also diffuse quickly if they are small enough – e.g. water, ethanol, urea. Larger molecules such as glucose diffuse hardly at all. Membranes are virtually impermeable to charged molecules irrespective of size – ions such as K+ and Na+ hardly move across membranes.  Proteins in membranes faciliate movement of molecules that cannot diffuse passively through membranes: Carrier vs Channel Proteins Carrier protein: alternates between two conformations – so binding site is subsequently available on one side of membrane and then other. Channel protein: forms a water filled pore across bilayer through which ions can diffuse. Can be opened and shut by variety of mechanisms. Opening and closing of channel proteins  Mechanisms of movement across membranes (passive – down electrochemical gradient, active – up an electrochemical gradient). Molecules bind to carrier protein and cause it to change conformation. 3 types of carrier proteins To Be Continued…………. Membrane Transport - Ion channels and pumps 2 Dr Dawn Jones  Ion pumps Transporters working in active transport are called pumps Transport is energetically unfavourable, so an energy source is required  Transporters switch between two different conformations Pumps work by alternating access to the substrate binding pocket from one side of the membrane and the other, driven by energy input How is the energy provided?  Energy sources for pumps Secondary Primary transport transport  Primary transporters Driven by ATP hydrolysis Substrate moving up ATP ! ADP+ Pi electrochemical Energetically favourable reaction gradient (ATP hydrolysis) is coupled to an energetically unfavourable reaction (movement of a substrate up its electrochemical gradient) ATP-driven (primary) transporters The Na+-K+ pump Also called Na+-K+ ATPase Pumps Na+ out of and K+ into cells Sets up Na+ and K+ gradients needed for action potentials This pump uses a large proportion of our cells’ energy needs! Belongs to a family of proteins called P- type pumps, because they are phosphorylated during transport  Cyclic nature of Na+/K+ pump 1. Na+ ions bind activating the ATPase 2. ATPase splits off terminal PO4 group from ATP and transfers it in a high energy bond to the pump itself – the pump is phosphorylated 3. Conformational change releases Na+ to outside and exposes K+ binding site 4. Extracellular K+ binds triggering (i) loss of the high- -energy PO4group--dephosphorylated pump and (ii)conformational change to starting form 5. K+ ions now released into cytosol, K+ site is lost and Na+ site reforms and pump is ready to work Cycle takes ~10ms! 3 binding sites for Na+ only 2 for K+. again.  Clinical insight Digitalis inhibits the Na+-K+ pump Digitalis, derived from the foxglove plant, is a potent inhibitor of the Na+-K+ pump It prevents dephosphorylation of the pump during the reaction cycle Blocking the pump raises the intracellular [Na+], which increases intracellular [Ca2+], causing the heart to pump harder Traditionally used to treat congestive heart failure Has been used as a treatment for hundreds of years in the UK!  Ca2+ ATPase (SERCA) is another P-type ion pump Pumps Ca2+ into the sarcoplasmic reticulum (ER of muscle cells), driven by ATP hydrolysis Mechanism is fairly well understood, as we have structures for the 2 major conformations of the protein (solved by x-ray crystallography) called E1 and E2 E1 E2 Ca2+ ATPase in E1 conformation Transmembrane domain – 10 TM a-helices with 2 Ca2+ ions bound A domain – actuator domain P domain – phosphorylation domain. Aspartate 351 is phosphorylated in reaction cycle N domain – nucleotide binding domain. ATP binds and hydrolyses here Ca2+ ATPase in E2 phosphorylated (E2-P) conformation Structure solved with aspartate 351 phosphorylated Conformation has switched to E2 Mechanism of the Ca2+ ATPase ATP-driven (primary) transporters  ABC transporters Contain domains called ATP-binding cassettes (ABC) 2 transmembrane domains + 2 ABC domains  ABC transporters Switching between 2 conformations causes substrate transport Binding and hydrolysis of ATP at ABC domains causes conformational change Substrate released on other side of membrane Substrate binds  Clinical insight Multidrug resistance in cancer Tumour cells often develop resistance to anti-cancer drugs Due to expression of an ABC transporter called MDR or P-glycoprotein Pumps a wide range of substrates, including drugs out of cells Structure of the mouse MDR protein was solved in 2009 Knowing the structure may help