1. Mutations in voltage gated Na or K channels: linked to epilepsy
1.1. Mutation in SCN1B (generalised epilepsy with ferbrile seizures plus)
1.1.1. Increased Na current
1.1.2. Increased spike frequency
1.2. Mutation in KCNQ2, KCNQ3 (benign familial neonatal convulsion)
1.2.1. Decreased K current
1.2.2. Increased spike frequency
2. Pain
2.1. Basic info
2.1.1. Nociception is the processing on noxious sensory inputs
2.1.2. Pain is a perception, at the brain levels
2.1.3. Nociceptors: respond to intense mechanical, thermal, or chemical stimuli
2.1.3.1. Quite unspecialised nerve endings
2.2. Sensory neurons
2.2.1. Are bipolar cell bodies in dorsal root ganglia (DRG), axonal projections peripherally and centrally to the spinal cord
2.2.2. Subtypes
2.2.2.1. Aβ (thick myelinated axons, fast conduction)
2.2.2.1.1. Touch
2.2.2.2. Aδ (thinly myelinated axons, slower conduction)
2.2.2.2.1. Fast acute pain
2.2.2.3. C (unmyelinated axons, very slow conduction)
2.2.2.3.1. Function
2.2.2.3.2. Two major classes
2.2.3. Primary sensory neurones
2.2.3.1. Primary sensory neurons relay in spinal dorsal horn and send projections to the forebrain
2.2.3.2. Nociceptive (pain) and non-nociceptive afferents terminate in different laminae of the dorsal horn
2.2.3.2.1. Nociceptors (C and Aδ fibres)
2.2.3.2.2. Non-nociceptors (Aβ fibres)
2.3. Mediation
2.3.1. Pain is mediated by several ascending pathways to the brain
2.3.1.1. Spinothalamic Tract
2.3.1.1.1. Somatosensory pathway through thalamus to somatosensory cortex
2.3.1.2. Spinoparabrachial Tract
2.3.1.2.1. Affective pathway through brainstem parabrachial nucleus to hypothalamus and amygdala (limbic area)
2.4. Chronic pain
2.4.1. Classifications of chronic pain states
2.4.1.1. Inflammatory, due to tissue damage:
2.4.1.1.1. Causes
2.4.1.1.2. Treatment
2.4.1.2. Neuropathic, direct nerve damage
2.4.1.2.1. Causes
2.4.1.2.2. Treatment
2.4.1.2.3. Additional problem
2.4.1.2.4. Examples
2.4.2. What does chronic pain involve?
2.4.2.1. Stimuli that are normally innocuous can evoke pain
2.4.2.1.1. e.g. sunburn hurts when you touch your skin
2.4.2.2. Pain is perceived from a wider region than the original damage
2.4.2.3. These changes are due to both:
2.4.2.3.1. Peripheral sensitisation
2.4.2.3.2. Central sensitisation
2.4.3. Functional changes in hypersensitive pain states
2.4.3.1. Increased excitability (sensitisation) of neurons in dorsal horn
2.4.3.1.1. Much greater excitatory responses to sensory stimulation of peripheral tissues after inflammation or nerve injury
2.5. Temperature sensing
2.5.1. Types of temperature sensing receptors
2.5.1.1. TRPV1 - hot > 42ºC
2.5.1.1.1. Is the capsaicin receptor
2.5.1.1.2. Identification
2.5.1.1.3. Activation and expression
2.5.1.1.4. Analgesic?
2.5.1.2. TRPV2 - hot > 52ºC
2.5.1.3. TRPV3 - warm > 33ºC
2.5.1.4. TRPV4 - warm > 27-42ºC
2.5.1.5. TRPM8 - cool 15-23ºC
2.5.1.5.1. Expression
2.5.1.5.2. Activation
2.5.1.5.3. Evidence for role as cooling mediator
2.5.1.5.4. Analgesic?
2.5.1.6. TRPA1 - cooling
2.5.1.6.1. Activation
2.5.1.6.2. Expression
2.5.1.6.3. Analgesic?
3. Synaptic Vesicles
3.1. Vesicle Life Cycle
3.1.1. 1. Filling
3.1.1.1. Mechanism
3.1.1.1.1. Neurotransmitter is accumulated into the nerve terminal cytoplasm by Na⁺ coupled transporters
3.1.1.1.2. All neurotransmitter uptake across the presynaptic plasma membrane is driven by sodium coupled co-transporters
3.1.1.1.3. Neurotransmitter uptake into synaptic vesicles is driven by a protonmotive force
3.1.1.1.4. Transporters sit in vesicles which allow uptake of specific neurotransmitters
3.1.1.1.5. All neurotransmitter uptake is powered by the proton motive force (sodium at plasma membrane, protons at vesicle membrane)
3.1.1.2. Evidence
3.1.1.2.1. Proton Motive Force
3.1.1.2.2. Glutamate uses a uni-porter
3.1.2. 2. Translocation
3.1.2.1. Mechanism
3.1.2.1.1. Once the vesiccle is full, how does it get to the plasma membrane?
3.1.2.1.2. The reserve pool is a large pool of vesicles which tends to be stuck to the actin cytoskeleton that sits close to the plasma membrane
3.1.2.1.3. The readily releasable pool is a smaller pool of vesicles that are already docked on to the membrane and are ‘ready to go’ and are awaiting calcium stimulus
3.1.2.1.4. During action potential stimulation, the readily releasable pool goes first and is replenished by the reserve pool
3.1.2.1.5. When synapsin is phosphorylated by CaMKII (on action potential stimulation) it loses affinity for actin, so synapsin is released from the cytoskeleton
3.1.2.1.6. PKA phosphorylates synapsin which releases it from the synaptic vesicle causing translocation
3.1.2.2. Evidence
3.1.2.2.1. Reserve pool of synaptic vesicles is missing in mice lacking synapsin I KO for gene encoding synapsin I Vesicles in readily releasable pool still present During reptitive stimuli, KO nerve terminals show reduced transmitter release as readily releasable pool can't be replenished
3.1.3. 3. Docking/Priming
3.1.3.1. Mechanism (Docking)
3.1.3.1.1. SNARE hypothesis
3.1.3.1.2. During docking, the V-SNARE associates with the T-SNARES forming a 7S SNARE complex. This causes docking
3.1.3.2. Mechanims (Priming)
3.1.3.2.1. NSF binds the SNARE complex via αSNAP
3.1.3.2.2. αSNAP binds to the assembled SNARE complex, bringing NSF with it. This makes a 20S complex
3.1.3.2.3. They may act as zippers pulling two sets of membranes together
3.1.3.2.4. After fusion, the SNARE complex is using energy from NSF hydrolysing ATP
3.1.3.2.5. Then, synaptobrevin can be put back on the synaptic vesicle
3.1.3.2.6. Complexin stops SNARE proteins fully going into fusion
3.1.3.3. Evidence
3.1.3.3.1. SNAREs are essential for vesicle fusion
3.1.4. 4. Exocytosis
3.1.4.1. Mechanism
3.1.4.1.1. Only occurs in active zones as Ca²⁺ channels bind to syntaxin
3.1.4.1.2. Exocytosis is fast, ATP-independent (SNARE proteins drive it themselves) And requires large amounts of Ca²⁺ for its stimulation because you don't want vesicles to fuse when they don’t need to
3.1.4.1.3. It can be assumed that the Ca²⁺ receptor must have a very low affinity for Ca²⁺ because high concentrations of calcium is required
3.1.4.1.4. The low affinity calcium receptor is synaptotagmin as it is an integral synaptic vesicle protein, binds at a low affinity and binds more than one ion
3.1.4.2. Evidence
3.1.4.2.1. Evidence for a low affinity Ca²⁺ trigger 1.
3.1.4.2.2. Evidence for a low affinity Ca²⁺ trigger 2.
3.1.4.2.3. Evidence synaptotagmin is calcium receptor
3.1.5. 5. Endocytosis
3.1.5.1. Properties
3.1.5.1.1. Endocytosis is slower than exocytosis
3.1.5.1.2. Endocytosis requires many essential enzymes and proteins, unlike exocytosis which only requires 3 (SNAREs)
3.1.5.1.3. Endocytosis is a calcium dependent process
3.1.5.2. Steps
3.1.5.2.1. 1. Activation
3.1.5.2.2. 2. Nucleation
3.1.5.2.3. 3. Invagination
3.1.5.2.4. 4. Fission
3.1.5.2.5. 5. Uncoating
4. Membranes
4.1. Volume regulation
4.1.1. How can you measure cell volume?
4.1.1.1. Introduce a molecule into the cells whose fluorescence depends on cell volume
4.1.1.1.1. How do you get a fluorescent molecule into a cell?
4.1.1.2. Dilute cell (swelling) signal falls, concentration cell (shrinking) signal rises
4.1.2. RVD
4.1.2.1. Pathways involved
4.1.2.1.1. KCL co transport
4.1.2.1.2. Coupled K+ and Cl- channels
4.1.2.1.3. Osmolyte channel
4.1.2.1.4. Activation leads to water loss and thus cell shrinking
4.1.2.1.5. Activation of RVD pathways can cause cell shrinking in iso-osmotic solutions
4.1.2.2. Response
4.1.2.2.1. Decreasing external osmotic pressure can cause cell swelling
4.1.2.2.2. Cells can respond by regulatory volume decrease (RVD)
4.1.2.2.3. Efflux/loss of osmolytes and water causes cell shrinkage back to normal cell volume
4.1.2.2.4. Hoffman E.K 1979
4.1.3. RVI
4.1.3.1. Pathways involved
4.1.3.1.1. Net movement of osmolytes into the cell
4.1.3.1.2. NHE exchanger coupled to Cl-/HCO3 exchange
4.1.3.1.3. NKCC cotransporter
4.1.3.1.4. NaCl cotransport
4.1.3.1.5. Activation leads to osmolyte and water gain and cell swelling
4.1.3.1.6. Activation of RVI pathways can cause cell swelling in iso-osmotic solutions
4.1.3.2. Response
4.1.3.2.1. Increasing external osmolarity can cause water loss and cell shrinkage
4.1.3.2.2. Regulatory volume increase (RVI) restores cell volume
4.1.3.2.3. Uptake of osmolytes and water causes cell swelling back to normlal volume
4.1.3.2.4. Cell shrinkage can also arise from efflux of osmolytes and water from e.g. cell metabolism in iso-osmotic conditions
4.2. Membrane transport
4.2.1. Passive permeation
4.2.1.1. Leak
4.2.1.1.1. Why are membranes leaky?
4.2.1.1.2. Basal Leak
4.2.1.2. Gap junctions
4.2.1.2.1. Structure
4.2.1.2.2. Disease
4.2.1.2.3. Characteristics
4.2.1.3. Ion channels
4.2.1.3.1. Characteristics
4.2.2. Transporters
4.2.2.1. Simple Transporters
4.2.2.1.1. Characteristics
4.2.2.1.2. Types
4.2.2.2. Secondary active transporters (SATs)
4.2.2.2.1. Function
4.2.2.2.2. Characteristics
4.2.2.2.3. Examples
4.2.2.3. Primary Active Transporters
4.2.2.3.1. Characteristics
4.3. Integrative cell physiology example
4.3.1. Glucose absorption across an epithelium
4.3.1.1. Na+ dependent and Na+ independent pathways
4.3.1.2. Na+ glucose cotransporter moves into the cell (ping pong)
4.3.1.3. Glucose then leaves the cell using uniport mechanism
5. Ion Channels
5.1. Measuring ion channels activity
5.1.1. Patch clamp electrophysiology
5.1.1.1. Keeping V constant allows determination of I (Voltage clamp)
5.1.1.1.1. Measurement of ionic currents
5.1.1.1.2. Flow of positive ions into the cell is denoted as a negative current
5.1.1.1.3. Flow of positive ions out of the cells is denoted as a positive current
5.1.1.2. Keeping I constant allows determination of V (Current clamp)
5.1.1.2.1. Measurement of membrane potential
5.1.1.2.2. Measurement of action potential properties and firing frequency
5.2. Pharmacological isolation of Na and K currents
5.2.1. Sodium is inward positive current, potassium is outward positive current
5.2.1.1. Can block K channels with TEA
5.2.1.2. Can block Na channels with TTX
5.3. Potassium channels
5.3.1. Purpose
5.3.1.1. 1. Sense transmembrane voltage (voltage sensor)
5.3.1.2. 2. Open in response to depolarisation (activation)
5.3.1.3. 3. Select between Na+ and K+ ions (selectivity)
5.3.1.4. 4. Allow very high flux of ions (high flux)
5.3.1.5. 5. Switch off on repolarisation (deactivation)
5.3.2. Structure
5.3.2.1. Basic structure
5.3.2.1.1. Composed on four subunits (polypeptides)
5.3.2.1.2. The four subunits assemble as a tetramer to form a pore
5.3.2.1.3. S4 transmembrane helix: positively charged amino acids (voltage sensor)
5.3.2.1.4. S5-S6 loop: line the ion channel pore
5.3.2.2. Structural classes
5.3.2.2.1. 6TM(P)
5.3.2.2.2. 2TM(P)
5.3.2.2.3. 4TM(2P)
5.3.2.2.4. 8TM(2P)
5.3.3. Activation
5.3.3.1. Repeating positively charged arginine amino acids in S4 causes high density on positive charge
5.3.3.2. Transmembrane potential causes S4 segment to move
5.3.3.2.1. This can be measured as a gating current or through molecular imaging
5.3.3.3. The movement causing a conformation change of the channel
5.3.3.4. The exact mechanism for the voltage sensor remains controversial but it causes S6 to move to open channel
5.3.4. Selectivity
5.3.4.1. Mechanism
5.3.4.1.1. Simple pore diameter cannot explain selectivity
5.3.4.1.2. K channel is 10000 x more selective for K+ than Na+ or Ca2+
5.3.4.1.3. Na+ ion is smaller than K+ ion, so how is the K channel pore so selective?
5.3.4.1.4. Hydration energy: the energy that is required to remove water molecules from an ion
5.3.4.1.5. It takes a larger amount of energy to remove water from a sodium ion than from a potassium ion
5.3.4.2. Selectivity filter
5.3.4.2.1. Conserved pore sequence: TVGYG
5.3.4.2.2. Selectivity is a result of K+ ion being dehydrated in the selectivity filter
5.3.4.2.3. The energy of hydration for K in K+ channel pore is less than for Na+ in a K+ channel pore
5.3.4.2.4. Carbonyl oxygens from residues in TYGYG act as binding sites for K+
5.3.4.2.5. K+ ions pass in single file, multiple ions in pore
5.3.5. Function
5.3.5.1. Physiological function depends on number and activity
5.3.5.1.1. Number of channels (N) depends on:
5.3.5.1.2. Open probability (Po)
5.3.5.2. Where are K channels important?
5.3.5.2.1. Endocrine cells
5.3.5.2.2. Neurones
5.3.5.2.3. Epithelia
5.3.5.2.4. Blood cells
5.3.5.2.5. Smooth muscle
5.3.5.2.6. Hair cells of inner ear
5.3.5.2.7. Cardiac muscle cells
5.3.6. Electrochemical gradients
5.3.6.1. Any permeant ion is subjected to two forces:
5.3.6.1.1. Chemical
5.3.6.1.2. Electrical
5.3.6.1.3. Nernst Equation to calculate electrochemical gradient
6. Reproduction
6.1. Follicles
6.1.1. Structure
6.1.1.1. Each follicle has one oocyte in it which is surrounded by somatic granulosa cells
6.1.1.2. These follicles are formed before the female is born
6.1.2. Growth
6.1.2.1. Oocyte grows
6.1.2.2. Somatic granulosa cells start dividing
6.1.2.3. At the beginning you get a mix of flattened granulosa cells and some that have started to divide
6.1.3. Types
6.1.3.1. Primordial follicle
6.1.3.1.1. Formed very early in development, before birth
6.1.3.1.2. By birth, there is a fixed supply that will gradually run out over time
6.1.3.1.3. Once growth is initiated, follicle cannot return to a resting state, it either dies or continues to grow
6.1.3.1.4. There is intense competition amongst growing follicles, with few developing fully
6.1.3.2. Primary follicle
6.1.3.2.1. All granulosa cells are rounded and undergoing mitosis
6.1.3.2.2. Rapidly increases population of granulosa cells
6.1.3.2.3. Also have thecal cells on the outside
6.1.3.2.4. Basement membrane is between granulosa and thecal cells
6.1.3.2.5. Once there is one complete layer of granulosa cells it is called a primary follicle
6.1.3.2.6. It won’t go into a resting phase again
6.1.3.3. Antral follicles
6.1.3.3.1. -More layers of granulosa cells grow
6.1.3.3.2. After third layer of granulosa cell, an antral cavity forms
6.1.3.3.3. Antral cavity is filled with antral fluid
6.1.3.3.4. A mature follicle has a large antral cavity and is ready to ovulate
6.1.3.3.5. Still has thecal cells, granulosa cells and basement membrane
6.1.4. Development
6.1.4.1. As a follicle develops, that development supports step-wise development of the oocyte in the follicle
6.1.4.2. As follicle stage progresses, oocytes become progressively able to support the processes of fertilisation and beyond
6.1.4.3. If you took an oocyte out of a...
6.1.4.3.1. ...primordial follicle, nothing will happen it will just die - it cannot be fertilised or even resume miosis
6.1.4.3.2. ...bigger follicle, it will get ready to resume miosis but then nothing else will happen
6.1.4.3.3. ...early-antral follicle it resumes miosis, can be fertilised and may undergo one cell division but then it stops
6.1.4.3.4. ...mid-antral-follicle it undergoes meiosis, fertilisation and cell division but it can’t implant
6.1.4.3.5. ...preovulatory follicle, it can produce a baby
6.2. Endocrine regulation of the ovary
6.2.1. Hormone secretion sequence
6.2.1.1. Hypothalamus produces GnRH
6.2.1.2. GnRH acts on the pituitary gland to release:
6.2.1.2.1. FSH
6.2.1.2.2. LH
6.2.2. FSH and oestrogen in follicle growth
6.2.2.1. Follicles produce oestrogen
6.2.2.2. Low levels of oestrogen produced in early stages of follicle growth has positive feedback on hypothalamus and pituitary gland to increase FSH production
6.2.2.3. FSH causes them to grow, as they grow they produce more oestrogen
6.2.2.4. High levels of oestrogen then have a negative feedback effect on the hypothalamus and pituitary to reduce FSH
6.2.2.4.1. So, final stage of follicle competition, setting up number of follicles ovulating, is dependent on oestrogen secretion by the mature follicles
6.2.3. Follicle competition
6.2.3.1. FSH induces antral follicle growth
6.2.3.2. A follicle that is slightly more ahead can react to FSH by producing more oestrogen
6.2.3.2.1. The high level of oestrogen will lead to a fall in FSH
6.2.3.3. More mature follicle copes with low FSH, surviving and growing in part by responding to LH
6.2.3.4. Less mature follicles still need FSH (can’t use LH yet) and so die when FSH levels fall
6.2.3.5. So, final stage of follicle competition, setting up number of follicles ovulating, is dependent on oestrogen secretion by the mature follicles
6.2.4. Oestrogen production
6.2.4.1. Two cell - two gonadotrophin hypothesis
6.2.4.1.1. Thecal cells have receptors for LH
6.2.4.1.2. So when LH acts on a thecal cell, it stimulates production of androgens from the thecal cell
6.2.4.1.3. Androgens cross over the basement membrane to granulosa cell
6.2.4.1.4. Granulosa cells have receptors for FSH
6.2.4.1.5. When FSH binds it secretes cyp 19 which turns androgen into oestrogen
6.2.4.1.6. Oestrogen can then be released
6.3. PCOS
6.3.1. Diagnosis criteria
6.3.1.1. Now usually defined as someone with at least 2 of the following 3 criteria
6.3.1.1.1. The appearance of polycystic ovaries
6.3.1.1.2. Irregular or absent ovulation
6.3.1.1.3. Hyperandrogenaemia (high levels of androgens)
6.3.1.2. So a woman can have PCOS without having polycystic ovaries
6.3.1.3. While these have been written as 3 individual criteria, they can and do affect the others making it harder to dissect what is happening
6.3.1.4. Other common symptoms
6.3.1.4.1. Insulin resistance/hyperinsulinaemia, hirsutism (excess hair), overweight/obesity, mood disorders/depression/anxiety
6.3.1.5. There are no symptoms of PCOS necessarily exhibited by all PCOS patients
6.3.2. Metabolic disorders
6.3.2.1. Insulin signalling
6.3.2.1.1. Insulin signalling maintains function of glucose transporter GLUT-4, and hence maintains low glucose levels in blood
6.3.2.1.2. If this system doesn’t work, blood glucose level will increase
6.3.2.1.3. In PCOS, it’s because of post-receptor signalling defect
6.3.2.1.4. In extreme cases, body will try to compensate by increasing insulin production (hyperinsulinaemia)
6.3.2.1.5. Cause of insulin resistance is unclear
6.3.2.2. Overweight/obesity
6.3.2.2.1. Women with PCOS have a higher rate of weight gain than those without PCOS
6.3.2.2.2. Obese PCOS patients will often have reduced post-prandial energy thermiogenesis
6.3.2.2.3. Likely linked to impaired insulin signalling
6.3.2.2.4. This will exacerbate the tendency to obesity
6.3.2.2.5. Overweight women with PCOS are more likely to also be anovulatory rather than just abnormal ovulation
6.3.2.2.6. Overweight women with PCOS can sometimes restore ovulation simply through diet
6.3.3. Causes
6.3.3.1. Fetal programming hypothesis
6.3.3.1.1. PCOS-like symptoms develop in animal models where there is experimentally induced or naturally occurring prenatal testosterone excess
6.3.3.1.2. This is well defined in animal models but less clear data indicating this is a cause in humans
6.3.3.2. Genetic regulation
6.3.3.2.1. Genetic component strongly suggested by frequency of family history
6.3.3.2.2. Several candidate genes were implicated, investigated, but not sufficient evidence to pursue
6.3.3.2.3. More likely an interaction of several genes
6.3.3.2.4. Genome-wide association studies in different populations have implicated overlapping set of genetic loci
6.3.3.2.5. Some of the genes identified have clear links to PCOS symptoms
6.3.3.2.6. Recent studies are also implicating epigenetic differences in PCOS patients
6.3.3.3. Treatment
6.3.3.3.1. Treatments based around affecting hormone interactions
6.3.3.3.2. If have anovulatory infertility, FSH levels are raised by reducing the feedback action of oestrogen:
6.3.3.3.3. - How does clomiphene citrate act?
6.3.3.3.4. Before treatment, if patient has high levels of oestrogen which causes too much of a suppression of FSH
6.3.3.3.5. Overall with either clomiphene citrate or letrozole
6.3.3.3.6. Problems:
6.3.3.3.7. Why is PCOS so prevalent?