Cell Physiology of Disease

The Plasma Membrane

Two molecules thick consisting of amphipathic phospholipids with embedded proteins. It contains important features that can regulate cellular processes including cell-cell signaling, apoptosis, cell division, membrane budding, and cell fusion.

Phospholipid

Have two tails connected by a glycerol and then a phosphate head group. They are very nonpolar at the tail and quite soluble at the head group.

Glycolipid

Have two tails connected by a glycerol and then a sugar attached.

Cholesterol

Has a hydroxyl group connected to a non-polar tail. The hydroxyl group gives it orientation because of its polar nature.

Phospholipid Head, Base, and Tail Groups

Head Groups: amines, serine, inositol

Base Group: glycerol, sphingosine

Tail Groups: C16 for saturated and C18, 20, and 22 for unsaturated

What are the different areas in this diagram?

A. polar region where head groups influence packing

B. Non-polar region where fluidity is influenced by cholesterol and alky chain saturation

C. Fluid non-poar region

Unsaturated vs. Saturated Fats

Fatty acid tails often have double bonds (unsaturated) which put kinks in them to create more fluidity in the membrane.

Cholesterol Content in Membrane

• As cholesterol content increases the membrane will become more rigid because the cholesterol molecules stack together

• Normally it makes it more fluid though

Different Composition of Inner and Outer Leaflets

• There are more negatively charged lipids in inner leaflet

• Similar amount of cholesterol

• More phosphatidylcholine in the outer layer

  • Choline is bigger than ethanolamine so creates bigger gaps to create curvature

• More phosphatidylethanolamine in the inner layer

• Phosphatidylserine is found predominantly in the inner layer (it is negatively charged) this creates a charge imbalance so that it’s more negative on the inside 

• More polyunsaturated lipids on the inner layer

Different Composition of Leaflets Between Cell Types (Epithelial and Neurones)

• There is a lot more cholesterol in neuron leaflets which makes neuronal membranes are less fluid

• Different levels of saturation

Lipid Rafts

Areas of the cell membrane that have a different composition with less fluidity and more thickness and are involved in trafficking and signalling processes.

Features of Lipid Rafts

• Membrane proteins can’t move in rafts but can move in other sections

• Rafts only exist in the outer leaflet

• Rafts typically contain sphingolipids and cholesterol and more saturated fats

Signalling Differences in Lipid Rafts

Many proteins associate with rafts to facilitate signalling, whilst others may avoid rafts to maintain slower rates of signalling.

Function of receptors, ion channels, and pumps/transporters

For a cell to be able to control and respond to its environment.

4 Types of Transport Across Cell Membranes

Simple diffusion, carrior-mediated facilitated diffusion, channel-mediated facilitated diffusion, and osmosis.

Simple Diffusion

Lipid-soluble molecules diffuse directly through the cell membrane.

Carrier-Mediated Facilitated Diffusion

Lipid-insoluble solutes such as sugars and amino acids diffuse through the membrane via protein carriers specific for them which change shape upon binding.

Channel-Mediated Facilitated Diffusion

Small lipid-insoluble solutes travel through a channel protein. Most ions are transported this way and are selected on basis of size and charge.

Osmosis

Diffusion of a solvent such as water through aquaporins or through the lipid bilayer.

Two Types of Transporters

Passive and Active Transporters

Passive Transporters (Facilitated Diffusion)

Concentration gradient drives the diffusion of a substrate across the membrane and no energy is required.

Example of a Passive Transporter

• Glucose Transporter 2 (GLUT2)

• High capacity, low affinity (Km ~20mM)

• GLUT2 is upregulated after a meal and allows glucose to passively diffuse down its electrochemical gradient

• Defects in the SLC2A2 gene (that encodes GLUT2) are associated with a glycogen storage disease called Fanconi-Bickel syndrome

Active Transporters (Pumps)

Requires energy to move a substrate against a concentration gradient and a membrane protein assists in this process. Two types: primary and secondary

Primary Active Transporters

Energy for the transport process is derived from ATP hydrolysis performed by ATPases which are a arge family of enzymes that catalyse the breakdown ATP – ADP + Pi – release energy. 4 Types include ABC, P-type, F-type, and V-type.

Primary Active Transporter Example

Na+/K+-ATPase where the pump moves sodium and potassium ions in opposite directions, each against its concentration gradient. In a single cycle of the pump, three sodium ions are extruded and two potassium ions are imported into the cell.

Secondary Active Transporters

Couple the movement of solute to the co-transport and/or counter-transport of ions.

Two major types of secondary active transporters

Neurotransmitter transporters and Cl^- transporters

Neurotransmitter Transporters

• All Na+ - coupled transporters

• Can also be coupled to Cl-, H+, K+

• Glutamate, GABA, dopamine, noradrenaline, serotonin, glycine

• Important for clearing neurotransmitters from the synaptic cleft

• Associated with neurological disease and important drug targets

• Ion and water regulation

Cl^- Transporters

• Other Na+/K+ transport proteins

• Regulate ion and water movement across cell membranes

5 Types of Receptors and Channels

GPRCs, Ligand-gated ion channels, voltage-gated ion channels, mechanosensitive channels, and leak channels.

GPRCs

Activate a signal transduction cascade on the inside of the cell via coupling to G-proteins inside cell. Ex. beta-adrenergic receptors

Beta-Adrenergic Signalling

• Adrenaline binds to receptor, which induces a change in receptor association with the  subunit of the G-protein 

• GDP is released and replaced with high energy GTP 

• The alpha subunit (purple) and the beta-gamma subunits dissociate

• Alpha and beta-gamma subunits modulate the activity of intracellular enzymes or ion channels

Ligand-Gated Ion Channels

Ligand binding opens a channel to allow ions to move in or out of the cell. LGIC are a large family that allow for the flow of ions (Na+/K+/Ca2+/Cl-) down their electrochemical gradient.

Family of LGICs

Family of Cys-loop receptors

• Cationic (serotonin, nicotinic Ach)

• Anionic (GABA, glycine)

• Ionotropic glutamate receptors (NMDA, AMPA, Kainate)

Voltage Gated Ion Channels

Changes in voltage across the membrane activate the channel. Large family of proteins with a characteristic structure that allow for the movement of Na+, K+, and Ca2+.

Mechanosensitive Channels

Channels open and close in response to mechanical stimuli. All membrane proteins can be affected by pressure exerted from the membrane, Msc are gated by changes in membrane pressure or ‘stretch’.

Leak Channels

Non-gated channels that are always open. Most are leak K+ channels that permit K+ to flow out of the cell to maintain membrane potential (-ve inside).

Cystic Fibrosis

The most common single gene disease that primarily affects the lungs, but also affects the bowel, sweat glands, pancreas and liver. It is usually diagnosed in the first two months of life, often during the newborn screening process that babies undergo at birth.

Incidence of CF in Australia

• 3,738 people in Australia living with CF

• 2,124 adults + 1614 kids

• 57% males

Main Symptoms of CF

• Respiratory Dysfunction: infections, obstruction, and destruction

• Digestive problems: pancreatic enzymes don’t reach GIT as duct blockage and they also get cysts

• High NaCl sweat

Causes of Symptoms of CF

• CF patients have reduced Cl- flux/currents conductance in their cells

• CF patients also have altered Na+ flux/currents in their cells

Inheritance of CF

Autosomal recessive disease:

• No mutations: 100% protein function

• One gene allele with F508del mutation: 50% protein function

• Both alleles with F508del mutation: <1% function

• One gene allele with F508del + one allele with other mutation: generally ~<1-5%

CF Gene

Encodes a protein called CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) which functions as a transmembrane Cl^-/HCO3- channel.

Structural Domains of CFTR

• Two transmembrane domains (TMD)

• Two nucleotide binding domains (NBD)

• One Regulatory domain (R)

How does CFTR open?

Phosphorylation by cAMP/PKA (R) allows ATP hydrolysis (NBDs) to open the channel (TMDs).

Disease-Causing CF Mutations

• Over 2500 mutations known. 700 documented as disease-causing

• F508Del most common which impacts how the two NBDs interact, so the protein doesn’t fold properly and hence not trafficked to the membrane. Hence, expressed but trapped in Golgi.

• Other mutations affect expression and/or function

6 Classes of Disease-Causing CF Mutations

• Class I: Early termination of CFTR

• Class II: Defective trafficking, doesn’t reach plasma membrane

• Class III: reaches plasma membrane but no function

• Class IV: reaches plasma membrane but defective function/Cl transport

• Class V: normal CFTR but less of it

• Class VI: less CFTR as protein is unstable

Absorption of Fluid Across Respiratory Epithelia

1. Start with Na+ pump (basolateral)

2. Na gradient enables Na+ influx (apical)

3. Water follows salt by osmosis

Secretion of Fluid Across Respiratory Epithelia

1. Start with Na+ pump (basolateral)

2. Na gradient enables Cl- influx (NKCC, basolateral)

3. Enables Cl efflux (CFTR, basolateral)

4. Water follows (osmosis)

Normal Fluid Balance in Respiratory System

1. Cl - / HCO3- secretion through CFTR drives osmotic flow of water

2. Absorption of Na + through ENac balances this osmotic gradient and amount of water. ENaC usually inhibited by CFTR

CF Fluid Balance in Respiratory System

There is less fluid in the airways and thick mucus means less clearance, and more bacterial infection, inflammation & degradation

1. Less Cl - / HCO3- secretion with less osmotic flow of water

2. Excess absorption of Na+ and water further dehydrates airways.

Normal Sweat Production

• Secretory Gland Component: secreted as iso-osmotic NaCl (~100 mM, plus some urea and lactate)

• Absorbing duct Component: epithelial re-absorb Na+ and Cl- so that ([NaCl] is reduced to 10-30 mM)

• Both controlled by sympathetic nerve control – activates the transporters

Secretory Gland Component

1. Na pump establishes Na+ gradients (basolateral membrane)

2. NKCC co-transporter (symport) brings Na+ in with Cl- (and K+) (basolateral)

3. Cl- diffuses across apical membrane, K+ diffuses back into ISF

4. Na+ and water follow through gap junctions

Absorbing Duct Component

1. CFTR Cl - channel opens and Cl - flows across apical membrane

2. Na+ channel (called ENaC) opens and Na + also flows across apical membrane

3. Cl - diffuses back across basolateral membrane, Na+ pumped across

4. This part of epithelia has no water channels and tight gap junctions (so water not re-absorbed)

CF Disfunction of Sweat Glands

CF causes sweat glands to produce sweat with excessively high salt (sodium chloride) levels because faulty CFTR proteins cannot reabsorb chloride from sweat as it moves up the duct. Leads to dehydration…

Two Main Aspects of Ion Channel Function

• Gating = opening and closing

• Permeation = ion passage

Gating of CFTR

CFTR opens when PKA phosphorylates the R domain (via cAMP signaling) and ATP binds and is hydrolyzed at the NBDs, enabling channel gating.

Channel Open Probability (P_o)

= (open time)/(open + closed time)

Open and Closed Ion Channel States

Open (ion-conducting) and closed (non-conducting)

Opening and Closing Rate of an Ion Channel

• β (s⁻¹) = rate the channel opens (C → O)

• α (s⁻¹) = rate the channel closes (O → C)

Mean Open and Closed Time

The rates determine how long the channel stays in each state:

• Mean open time = 1 / α

• Mean closed time = 1 / β

F508del Mutation

• This mutation means that CFTR does not express in the plasma membrane

• Significantly reducing its ability to transport chloride ions even when it reaches the plasma membrane. This mutation results in a severely reduced open probability (P_o), which is approximately one-third or less than that of wild-type CFTR.

Aims of Drug Application for Cf

• Prevent the misfolding of mutant CFTR proteins = Correctors

• Improve the gating of mutant CFTR proteins that do get to the plasma membrane = Potentiators

• Increase the number of stable CFTRs at the plasma membrane = Amplifiers and Stabilizers

1989 Cf Dug Development

CFTR gene is cloned. Disease mutations identified and can be characterized using patch clamp recordings and other models.

2000 Cf Drug Development

Aurora Bioscience (later acquired by Vertex) partners with CF Foundation to develop CF drugs that can counteract.

Current Cf Drug Developments

Extended approvals of Trikafta & 1/day Alyferek for younger CF patients and in other countries. Research for improved drugs and ways to target the remaining 10% of patients not helped by Trikafta.

Key Cf Drugs

• Kalydeco (ivacaftor, 2012) - approved for 4% of CF patients (G551D mutation).

• Orkambi (lumacaftor/ivacaftor, 2015) - approved for 35% of homozygous F508del mutation patients.

• Symdeko (tezacaftor/ivacaftor, 2018) - introduced with fewer side effects.

• Trikafta (elexacaftor/tezacaftor/ivacaftor, 2019) - approved for 90% of CF patients aged over 12

High Throughput Screening for CFTR Modulators

In HTS assays, cells engineered to express CFTR (often mutant forms such as F508del CFTR mutation) are exposed to thousands of compounds in multi-well plates. Automated robotic systems, fluorescence-based reporters, and imaging technologies measure changes in CFTR activity, typically by detecting chloride or iodide ion movement across the cell membrane. Potentiators give a bigger fluorescent response.

What does ivacaftor (VX-770) do for Cf patients?

• Increases the hormone stimulated lumen volumes ie, mucous becomes less thick and sticky

• Increases cilia beat frequency

• Rapid, significant & sustained improved lung function

• Sweat Cl- decreases (below CF cutoff)

• BMI (nutrition) increases

• Better scores, less hospital visits

What does trikafta a corrector + potentiator do for Cf patients?

It is a highly effective, prescription triple-combination therapy used to treat cystic fibrosis (CF) in patients aged 2 and older with at least one F508del mutation. It significantly improves lung function, reduces coughing, and boosts quality of life, acting as a disease-modifying treatment.

Neonatal Epilepsy

Caused by genetics, is generalized, and can have severe impact.

Late Onset Epilepsy

• Cerebrovascular, trauma, neoplasms, neurodegenerative

• Mostly focal onset

• Higher mortality

Epileptic Seizure

Transient (short) occurrence of signs and/or symptoms due to abnormal excessive or synchronous neuronal activity in the brain.

Epilepsy

A disorder of the brain characterized by an enduring predisposition to generate epileptic seizures, and by the neurobiologic, cognitive, psychological, and social consequences of this condition.

Epilepsy Syndrome

A specific, identifiable type of epilepsy characterized by a cluster of features, including specific seizure types, EEG patterns, and age of onset.

Focal Onset Seizure

• Starts in a specific brain region and may be associated with a specific thought, sensation, memory or even action

• Can spread to adjacent (“1”), cross-hemispheric (“2”) or subcortical regions (“3”)

• Symptoms associated with region

• Focal seizure can “generalize”

Generalized Onset Seizure

• Results from abnormal electrical activity emerging rapidly across both sides of the brain simultaneously, typically causing consciousness loss

• Seizure involves thalamocortical and/or broad cortical changes very early in seizure

• Four types include: absence, myotonic, atonic, tonic-clonic

• Always unaware

EEG for Seizures

• Non-invasive diagnostic tool

• Uses electrodes placed outside of the skull (on skin)

• Begin as focal or generalized

• Ictal vs Interictal

How do seizures start?

Arise from excessive action potentials in neurons (too much excitation) where different Action Potential (AP) firing patterns in response to the same set level of stimulation are seen across the brain.

What do excessive APs in seizures result from?

1. increased intrinsic excitability, or

2. increased levels of input at synapses.

Neuronal Connections

Neurons are connected in local and distant circuits through a dense network of synapses. These synapses are either excitatory or inhibitory.

Excitatory Signals

• Activates a nerve cell

• Increases chance nerve cell fires an action potential

• Moves the nerve cell Vm towards voltage threshold

• Depolarizing

Inhibitory Signals

• Switches off a nerve cell

• Decreases chance nerve cell fires an action potential

• Moves the nerve cell Vm away from voltage threshold

• Hyperpolarizing

Excitatory Synapses

When open allow cations to permeate (Na+ and/or Ca2+) and the postsynaptic response includes cation influx which results in depolarization and an excitatory post synaptic potential.

Excitatory Synapse Neurotransmitter

Glutamate which is detected by ionotropic receptors which include either AMPA (mostly Na+ influx) or NMBA (mostly Ca2+ influx).

Inhibitory Synapses

When open allow anions to permeate (Cl- ) and the postsynaptic response includes anion influx which causes hyperpolarization and an inhibitory post synaptic potential.

Inhibitory Synapse Receptor

GABA is the neurotransmitter in the brain, and glycine is also common in the spinal cord. They are detected by ionotropic receptors. Permeant ion for both receptors are chloride ions (Cl - ).

Causes of Epilepsy

Genetics is the most common cause of epilepsy (70-80%), followed by structural (eg cancer, neurodevelopmental, strokes) and then metabolic.

Genes that Cause Epilepsy

Ion channel genes, synaptic support protein genes, mTOR pathway regulator genes, and chromatin remodelling and transcription regulator genes. Include more than 140 genes identified.

3 Typical Genetic Patterns of Epilepsy

1. De Novo Mutations (not inherited) are the most common

2. Polygenic/Complex (complex inheritance of risk variants) are less common

3. Autosomal Recessive (tranditional inheritance patterns) very infrequent

Dravet Sydrome (an epileptic encephalopathy) Symptoms

Prolonged and frequent seizures, behavioral and developmental delays, movement and balance issues, orthopedic conditions, delayed language and speech issues, growth and nutrition issues, sleeping difficulties, chronic infections, and sensory integration disorders.

Causes of Dravet Syndrome

Many genetic causes, but typically (80%) a de Novo loss of function mutation in the voltage-gated Na+ channel gene, SCNA1. SCN1a will be (NaV1.1) is expressed only in inhibitory interneurons (GABA neurons). Therefore, less APs in GABA neurons = less inhibition therefore more excitation.

Drug to Treat SN1a Loss of Function

Rescue of the cellular defect using Hm1a, a specific activator of SCN1a channels. A specific drug derived from Australian funnel web venom.

Factors Affecting Nerve Excitability

• Type of synaptic inputs (EPSPs & IPSPs)

• Number and activity of synapses

• Intrinsic Excitability (how easily a set input causes an AP or increase in APs)

• The type of on channels and shape of neurons contributes to intrinsic excitability

What cellular factors cause seizures?

• Too little inhibition

• Too much excitation

• Altered synapse number or efficacy

• Increased intrinsic excitability

• Changes in APs

GABAA Receptor

• Mediates brain inhibition

• Target of many anti-epileptic drugs

• Seems to fail in severe seizures

• 100s of gene mutations impacting GABA

What does KCC2 do?

KCC2 (K+ Cl- co-transporter subtype 2) is a transporter that keeps chloride low inside neurons so GABA stays inhibitory, but during seizures KCC2 membrane expression decreases, chloride builds up, and GABA inhibition fails making seizures worse.

KCC2 as a viable therapeutic strategy for epilepsy

In vitro Cl- transport was increased, there were no effects on normal behaviour, and no effects on normal GABA inhibition in isolated nerves.

Ascending Pain Pathways

1. Detection of stimulus by sensory nerve fibre endings

2. Processing of signals in the dorsal horn of the spinal cord

3. Transmission of response to CNS

4. CNS response

Ascending Pain Pathway Processing in the Brain

• Primary afferent nociceptive neurones synapse in the superficial dorsal horn

• Axons of nociceptive neurones (second order) cross the midline and ascend the spinoreticular and spinothalamic tracts and project to the brain stem and thalamus

• Third order neurones project to the somatosensory region of the cerebral cortex, thalamus and limbic system