NEUR3001 Lecture Notes - Lecture 3: Protease, Bungarotoxin, Electric Eel
Ion Channels
1. Fundamentals of Bioelectrical Signalling Across Membranes
1.1. Summary of Ion Channels
- Act as conductive pathways for ion movement across non-conductive membrane
- Rate of ion movement very high
- Ion movement through channels produces ALL electrical activity in cells
- Many open/close in response to voltage, chemical, mechanical energy
- Selectively permeable
1.2. Cell Membrane as an Ionic Barrier
- Biplanar lipid structure is non-permeable to ions
- To generate membrane potential, ions must move across membrane
- Physio-chemical forces drive ion movement
o Chemical gradient of ion
o Electrical charge distribution across membrane
1.3. Ions
• Salt dissolves in water
o Solvent molecules attracted to salt
charge centres
o Salt dissociation into ions +
hydration of ions
o Hydrated ion complex becomes
larger, disguising true ionic radius
o Dehydration of ions require ATP
o Smaller/more charged ion require
more energy
• Ions with hydration prefer high polar environment (extracellular)
• To pass through membranes, need to undergo depolarisation by minimising energy barrier enter non-polar
environment
More -ve
More +ve
Strip hydration shell to
obtain true radius (green)
1.4. Membrane Potentials Rely on Ionic Diffusion through Ion Channels
- Ion channels = proteins with pores
- Ion permeability based on ion selectivity by ion charge & size
- Na+, K+, Cl- permeable in ion channels membrane potential
- Ca2+ permeable in ion channels Transduction of electrical signals to chemical
- Concentration & electrical gradients = ionic channel flow
1.5. Squid Giant Axon
- Hodgkin & Huxley used intracellular electrode to record potential changes during AP
- ~1mm diameter
- Evidence of ionic changes in AP
- Hypothesis: AP due to selective increase in membrane permeability to Na+
- Voltage clamp technique
o Determines changes in membrane conductance (g) during AP (ionic movement)
o Control membrane potential at set voltage
o Use feedback current to counteract dynamic current changes & keep MP at constant level
Separated Na+ & K+ conductance
o Transient early inward current close to ENa (Ion equilibrium for Na)
o Lowering ENa by removing Na+ reverses transient inward current polarity does not alter late sustained
outward current
o Late sustained outward current not Na, maybe K
1.6. Concept of Charge Movement
• Influence of membrane potential on Na & K conductance = evidence of role in movement of charged particles
into different positions due to changing electric gradient across membrane (depolarisation)
• Position of particles on membrane permits Na+ & K+ ion movement
• Rate of movement of charged particles rate of activation of Na & K conductance
1.7. Summary of H&H Results & Theory
• Na+ ions flow inwards while K+ ion flow outwards (separate conductive pathways)
• Each pathway closed @ resting membrane potentials, opens with membrane depolarisation
• Na conductance closes rapidly when depolarisation maintains
• No measurable initial gating current (small, can’t be measured) Na+ & K+ always at low density in membrane
• Further study on radioactive giant axons show that Cl- flux remains constant (not inward/outward current)
2. Voltage-Gated Ion Channels
2.1. Measuring Ion Channel Density
• Determines total amount of current/membrane area
• Determines unitary current information on mechanisms of ion movement
• Strategies to determine density:
o Lebel individual channels & count label intensity
o Measure total gating charge & divide by channel gating charge
o Measure total membrane conductance & divide by single channel conductance
2.2. Neurotoxins
• Block specific VG channels & AP with high affinity
• Tetrodotoxin (TTX)
o Binds to Na+ channels by fitting into outer “mouth”
o Quantifies channels
o Low density in squid giant axon
• Tetraethylammonium (TEA)
o Binds to K+ channels by fitting into outer “mouth”
• Shows that Na+ & K+ channels are independent of each other since specific toxins have no effect on the other
2.3. Gating Current
• H&H deduction that Na+ channel activation produced a
small gating current confirmed
• Clay Armstrong & Francisco Bezanilla measured in squid
giant axon
• Total gating current Na+ channel density
~300channels/μm2
• Similar measurement in K+ channel
TEA molecules block inside channel
↑ hydrocarbon chain = shows that channel gate
needs to be open first before being blocked by TEA
Inward flow of K+ ions could dislodge TEA from
blocked channel
Use-dependent block speed & level of
block depends on activation of channels
More frequent intervals of
activation faster block
Document Summary
Ion channels: fundamentals of bioelectrical signalling across membranes. Act as conductive pathways for ion movement across non-conductive membrane. Many open/close in response to voltage, chemical, mechanical energy. Ion movement through channels produces all electrical activity in cells. Biplanar lipid structure is non-permeable to ions. To generate membrane potential, ions must move across membrane. Physio-chemical forces drive ion movement: chemical gradient of ion, electrical charge distribution across membrane. Strip hydration shell to obtain true radius (green) Ions with hydration prefer high polar environment (extracellular: to pass through membranes, need to undergo depolarisation by minimising energy barrier enter non-polar environment. Membrane potentials rely on ionic diffusion through ion channels. Ion permeability based on ion selectivity by ion charge & size. Na+, k+, cl- permeable in ion channels membrane potential. Ca2+ permeable in ion channels transduction of electrical signals to chemical. Concentration & electrical gradients = ionic channel flow. Hodgkin & huxley used intracellular electrode to record potential changes during ap.
