ion channels

what is the difference in intracellular and extracellular concentrations of postassium, sodium and calcium?

• potassium intracellular = 140mM and extracellular 4-5mM because posassium diffuses out of the cell down its concentration gradient to maintain rest

• sodium intracellular = 10-15mM and extracellular 145mM as sodium enters the cell when channel opens

• cakcium intracellular = 100nM and extracellular 2mM

mechanism of the Na/K ATPase pump

• N/K pump uses ATP directly to keep high potassium inside and high sodium outside to maintain rest

• for each ATP hydrolysde 3 sodium ions move out of cell and 2 potassium ion in 

• this contributes to the slightly negative restinf membrane potential 

how is resting membrane potential generated?

• postassium leak channels (inward rectifier channels) are always open and this allows potassium to move down its concentration gradient 

• this means the inside of the cell becomes negatively charged 

• when elecrtical force balances concentration force, postassium is at equilibrium (nernst)

what is the nernst equation?

• nernst equation tells you the equilibrium potential for a particular ion

• R = gas constant = 8.314

• T = absoulute temperature = 37

• z = valence of ion (e.g = +1 for sodium, +2 for calcium)

• F = faraday constant = 96485 c/mol

what is the goldman equation?

• extension of the nernst equation that calculates the RMP when the membrane is permeable to more than one ion 

• P = membrane permebaility for the ion 

• for anions the concentration ratio is in/out flipped because of the negative charge 

what is an IV plot?

• shows the relationship between current (I)through an ion channel and the membrane voltage (V) = helps to describe how ion channels behave under different voltages 

• inward current = flow of positive charge into the cell, ona  coltage clamp = downward deflection 

• outward current = flow of positive charge out of the cell = upward deflection

• for negtaive ions the sign of current is reversed (current is the movement of positive charge) 

how to interpret an IV plot

• the IV relationship for a leak potassium channel is linear, forming a straight line 

• current increases proportionally with voltage 

• there is no gating- the channel is always open 

• the line crosses the x-axis at equilibrium potential (EK) as this is when net potasssium current is 0

• if the membrane is more positive that EK the there is outward current (graph climbs upwards)

how can the slope of an IV polt be used to calculate channel conductance?

conductance (siemens) = change in I/change in V

but this is only true when the IV relation is linear such as open leak channels, ohmic channels and many passive conductances 

voltage gated channes haev curved IV relationships 

• steep slope = big change in current for a small change in voltage = high conductance 

how do we record membrane currents?

• two electrode voltage clamp

• uses two microelecrodes (voltage to measure membrane potential and current to hold membranr at commanded voltage)

• used xenopus oocytes as they are very large and have few endogenous ion channelss, can be injected with mRNA or DNA that encodes specific ion channels

how do we study individual neurons?

• extracellular recording aka cell-attached mode 

• place electrode just outside the neurons membrane, recording activit that leaks out when the neuron fires 

• you can thus measure the frequency and pattern of action potentials

• this is non-invasive and does not change the intracellular environment HE you cant measure membrane optential and synaptic currents

• cell attached mode = place pipette on membrane and form giga seal

what is whole cell mode patch anaylsis?

• after forming pipette seals membrane you rupture the membrane patch 

• pipette interior becomes continuous with the cytoplasm

• you can now measure full action potentials, synaptic currents etc 

• you can control membrane voltage and measure the current that flows in response to the voltage steps

• cell held at a specific voltage and small voltage step is applied, this small change produces a small current step due to membranr resistance

• we calculate resistance using ohms law (R=V/I)

what is the difference between picoamp and macro currents?

• picoamp are extremely small and typical size of single ion channel openings = found when doing single channel and cell attached patch clamp

• macrocurrents are much larger (nano and micro) recorded from entire cell = found when doing whole cell voltage clamp

general structure of ion channels

• ion channels ar eproteins embedded in the cell memrane 

• inside the channel is a water-filled tunnel (aqueous pore) that allows ions to pass through

• it if filled with water because the lipid membrane is hydrophobic 

• ions have a hydration shell (each ion surrounded by 4-8 water molecules) e.g sodium strongly binds to water so has a large hydration shell 

• this shell must be stripped or partially removed for ions to pass through the narrow channel pore 

how is ion flow facilitated by the pore susbstituting for water?

• ions cant cross the membrane with their hydration shell because water is charged and will get repelled by the lipid bilayer

• ion channels therefore have a pore lined with polar groups e.g carbonyl oxygens that can replace the water molecules

• this is esepcially true in potassium channels where the selectivity filter uses carbonyl oxygens to mimic water molecules

• this lets the ion move through the hydrophobic membrane, ensures that only the correct ion passes (selectivity) and speeds up ion flow dramatically

why are ion channels selective?

• for stabilisation to work, the ion must be exactly the right size

• e.g the spacing of the carbonyl oxygen is arranged to fit potassium perfectly when dehydrated, sodium is smaller than this 

• when sodium enters a potassium channel, it is too small to reach the carbonyl oxygens and therefore cannot make strong stabilising intercactions so the pre cannot replace its lost water shall and sodium would be energetically unstable 

what is a leak channel?

• channels that are open all the time

• they allow ions to move continuously across the membrane according to concentration/electrical gradients 

• this helps paintain resting potential 

• activity can change on relatively slow time scale via association with porteins or phosphorylation 

• provide baseline level of excitability 

what is a gated channel?

• open in response to a specific stimulus 

• voltage gated, ligand gated and mechanically gated 

• rapid, controlled ion flow to generate action potentials, synaptic transmission and sensory transduction 

• when closed they do not pass ions at all

what are the two main classes of potassium channels that generate and regulate resting membrane potential?

1. K₂P (two-pore domain) leak channels

2. inward-rectifier potassium channels

what is the structure of inward-rectifier potassium channels (kir)?

• made up of four subunits (tetramers) like most potassium channels 

• each subunit has 2 transmembrane domains and a pore forming loop between them (p-loop)

• p-loop contains TVGYG sequence which gives potassium selectivity 

• kir channels have large intracellular (cytoplasmic) domains that modulate channel activity by interacting with magnesium, polyamines and PIP₂

• do not have a voltage sensing S4 helix so are not vooltage gated 

what is the structure of K₂P leak channels?

• each subunit contains 4 transmembrane segments and 2 pore-forming domains

• two identical K2P subunits assemble a dimer so can form 4 pore loops

what are the possible ways to get 4 P-domains?

1. 4 separate proteins, each with 1 P-domain assemble to form a tetramer

2. 2 proteins each with 2 P-domains

thus to build a potassium channel pore, you always need 4 P-domains

how does inward rectification work?

• when the membrane is negative, kir channels allow potassium to flow into the cell

• when the membrane becomes positive, the channel blocks potassium from flowing out

• this helps stabilise resting membrane potential as potassium comes in when needed but cant leak out during depolarisation

how does K₂P work?

• current flows both ways fairly

• no strong pore blocked by magnsium or polyamines

• TWIK behaves more like a simple leak channel

what is the I/V plot curce for an inward rectifier?

• at negative voltages the channel passes large inward current

• at positive voltages, outward current is very small

anesthetics and TWIK channels

• TWIK channels are a subtype of K2P channels

• they are normally open at rest to help set the resting membrane potential

• when they open more, the membrane becomes even leakier to potassium

• volatile anesthetics like insoflurane and halothen can increase the activity of TWIK

• this causes hyperpolarisation therefore neurons become less excitable and there is global depression of neural activity

• this leads to loss of consciousness, reduced pain perception and muscle relaxation during anesthesia

what are two key factors that affect RMP?

1. many leak channels like K2P are modulated by second messengers e.g cAMP or by direct action of GPCRs e,g Gβγ subunits can open certain channels, GPCR activation can open or close some K2P channels

2. presence of persistent sodium currents (INaP) which does not fully inactive and provides continuous small sodium leak

what did hodgkin and huxley (1952) do?

• recorded currents from the squid giant axon using a voltage clamp, measuring how ionic currents depent on voltage, measured how these currents change over time and then use this to build a numerical model

how are sodium channels controlled?

• at 70mV (rest) sodium channels are closed, inactivation gate ioen but activation gate shut

• when the voltage rises to -45mV the activation gate opens and sodium rushes into the cell causinf the rising phase of the action potential

• at positive voltages even if the membrane stays depolarised the inactivation gate closes and this stops sodium flow because channels are now inactive

• during repolarisation the inactivation gate reopens

how does positive feedback make action potentials rise very fast?

• action potentials are incredibly fast, membrane goes from -70mV to +30mV in less than a millisecond and this comes from a positive feedback loop

• stimulus makes membrane less negative and this small depolarisation is the trigger

• this opens sodium channels

• this causes further depolarisation as the inside becomes more positive

• this means each step amplifies the next (self reinforcing process)

what is the I/V plot for a voltage gated sodium channel?

peak inward current (~ –20 to –10 mV):

• many channels open

• Na⁺ current becomes strongly inward

• the curve reaches its large negative peak

• above ENa (~ +60 mV):

  • driving force reverses sign

  • Na⁺ current becomes outward

what is the structure of a voltage gated sodium channel?

• one large alpha subunit (the main pore forming part)

• 4 auxillary beta subunits that do not form the pore but modulate kinetics, gating and localisation

• alpha subunit has 4 homologous domains and each domain contains 6 transmembrane helices (S1-6)

• S4 = voltage sensor as it contains positively charged residues (arginines)

• between S5-S6 the pore and selecitivity forms

• inactivation gate in intracellular loop betwee domain III-IV

what is the ball and chain model?

• explanation for fast inactivation of coltage gated ion channels

• a part of the channel prtein that acts like a ball = small peptide segment that is attached to the channel by a flexible chain (stretch of amino acids)

• when the channel opens, the ball swings in and plugs the inner mouth of the pore, blocking in the ion flow

what is the absolute refractory period?

• voltage gated sodium channels open during an AP then quickly inactivate (ball and chain style)

• when they are inactivated they cannot reopen no matter how strong the stimulus is, creating the absolute refractory period (a neuron cannot fire another action potential immediately after one has just happened)

• this sets a maximum firing frequency

how does the absolute refractory period stabilise the network?

• if neurons could reopen sodium channels instantly they might fire repetitively without control, form unwanted loops of excitation and generate pathological oscillations (e.g seizures)

• the combo of sodium inactivation and delayed potassium opening (repolarisation) stops the membrane from re-depolarising immeditely again

what does refractoriness prevent?

• prevent retrograde propagation of action potential

• refracttory regions behind AP cannot fire again as the sodium channels behind the spike are inactivated meaning the spike cannot travel backwards = one-way conduction

what are shaker potassium channels?

• voltage gated potasium channels

• part of the Kv1 family

• responsible for repolarising the membrane after an action potential

• 4 identical subunits, each with 6 transmembrane segments

• act as delayed rectifiers = open with a delay after membrane depolarisation and this helps repolarise the cell during the action potential = limit excitability

• without delayed rectifiers action potentials would be much longer

what are nicotinic acetylcholine receptors?

• non selective cation channels

• when acetylcholine binds, the channel opens and allows sodium to enter and potassium to leave

• the mixed ion flow drives the membrane toward 40mV and each ion has its own equilibrium potential: sodium wants to bring the membrane to about 60mV and potassium bring it down to about -90mV

• this -40mV is called the reversal potential

• this opens voltage gated sodium channels

• this is how neuromuscular junctions work