Module 3: Ion Channels

Normal Function of Ion Channels

• Release, synaptic potentials, action potential → fast signals/process

• Electrical signal transmission across membranes allows multicellular organisms to operate in very short time scales.

  • Function determined by its structure

Channelopathies

• Disease caused by a dysfunction of ion channels.

  • Acquired(drugs, toxins, antibodies) or Genetic Cause

• e.g.

  • Skeletal: Myasthenia, myotonia,

  • Cardiac: dysrhythmias, LQT & SQT syndromes,

  • Neurones: ataxia, epilepsy, migraine, multiple sclerosis,

  • Retina: achromatopsia, retinitis pigmentosa, night blindness,

  • Cochlea: tinnitus, deafness,

  • Pancreatic  β-cells: neonatal diabetes, hyperinsulinemia,

  • Lungs: cystic fibrosis, asthma,

  • Kidney: Bartter syndrome,

  • Gut: irritable bowel syndrome.

Pore:

• A large protein that allows the aqueous phase to pass through it – allows water, macromolecules and ions to pass through

  • (molecules up to 45kDa) can pass through via diffusion

• E.g.       

  • porin (mitochondrial membrane),     

  • perforin (cytotoxic T lymphocytes), inserts into cells and causes them to burst and die   

  • nuclear pore complex (nuclear membrane),

  • aquaporin (cell membrane - water + small molecules)

Ion-Selective Channels

• Only certain ions/ charged species able to pass through

  • Sensor present to regulate process of gating

    • Exist in 2 states open and closed

Structural Determinants of Channel Function

• Extracellular opening: outer channel pore region/ vestibule

• Intracellular charge region/ vestibule – accessible from the intracellular side      

• Gates allow the channel to be freely conductive   

  • Allows specific ions to go through and is determined by whether channel is open or closed

Structure of Ion Channel: α (alpha) Subunit

• Main subunit of ion channels

• An integral transmembrane protein with multiple transmembrane passes.

  • simplest K+ channel is a tetramer with two transmembrane regions per subunit.

  • Na+ and Ca2+ channels are monomers with 24 transmembrane domains.

• Collectively forms the ion conductive/ pore region

K⁺ Channel Channel

• Tetramer, with 2 transmembrane regions per subunit

  • 8 hydrophobic transmembrane spanning regions

P-Loop

• Loops of polypeptide present in the lumen or pore region of the channel that determines ion selectivity

  • 4 loops forms the selectivity filter within the channel pore

Na Channel Structure

• Monomeric channel → made up of a single (pseudo)subunit.

  • This subunit has 24 transmembrane domain

Transmembrane Spanning Regions

• Hydrophobic alpha helices

• Cylinder Rod-like structure

  • Collectively coalesce to form the ion conductive or pore region

Structure of Inward Rectifier K⁺ Channels

• 4 × two transmembrane passes present

Structure of Voltage K⁺ Channels

• 4 x six transmembrane passes

  • 24 ‘rods’ that form the channel

Structure of Inward BK K⁺ Channels

• 4 x seven transmembrane passes

Gating

• Transition between the 2 different stable conformational states of the channel

  • Open and closed

Closed State of Channel

• ion not permitted through pore region – non-conductive and undergoes a process of activation to allow the pore to become permissive and ion can pass through

  • Dependent on concentration gradient and electrical gradients across the cell

Single Channel Recording

• Measure the flow of current through a single ion channel

  • Deactivated: no current flow – at zero   

  • Activated in response to voltage/ ligand: conformational shift causes the channel to open = current

    • If fully activated- constant change in the current shift

Mechanism of Generalised Gating: Activation/Deactivation

• Whole-cell shifting of the a-subunits to allow the channel to achieve an open configuration

  • Involves the shifting and re-organisation of the alpha-helical rod position around the pore region

• Twisting, tilting or bending of subunits and trans- membrane-spanning a-helices (much of α-subunit around the ion pore region)

  • generalised conformatonal change

Generalised Gating: Lignand Binding

• Mediated through extracellular (NT)/ intracellular (By- dimer) ligand

  • Fast neurotransmitters receptors such as ACh nicotinic, GABA_A, glutamate, ATP purinergic receptors.      

  • Intracellular binding of Ca²⁺,  By-subunits of G-proteins or nucleotides such as ATP, cAMP or cGMP.

• Bind to channel causing it to open    

• Modulation of ion channels by metabotropic ACh muscarinic, GABA_B, mGlu, 5HT or photo-receptors

Mechanism of Generalised Gating: Intracellular Modifications → Phosphorylation

• Phosphorylation or dephosphorylation – presence/ removal of phosphate can cause alpha helices to shift and modify the channel to open

• ↑ Opening of /leakage K⁺ channels (dephosphorylation) and gap junctions (phosphorylation).

Mechanism of Generalised Gating: Membrane Voltage

• Mediated through voltage (TM4) sensors in Ca²⁺, K⁺ & Na⁺ channels at a particular voltage

• Depolarization or hyperpolarisation of membrane potential shifts the position of the TM regions as they are charged and so sense membrane voltage

• One of the 4 alpha helices is a charged rod and is sensitive to voltage – depending on the voltage/ electric field generated the rod can change, leading to gating

Mechanism of Generalised Gating: Mechanical Distortion

• Opening of channel is mediated thorugh distortion of the plasma membrane (mechanosensitive)

  • Ion channels linked to the cytoskeleton- physical linkage- when membrane is distorted -pulls on ion channels to change its configuration from open to closed      

• ↑ opening induced either directly or through mechanical linkage to the cytoskeleton.

  • Form of sensory transduction

Where Can Sensory Transduction Be Sensed

• Organs e.g.

  • Cochlea and in skin by TRP channels.

• Also, some “leakage” K⁺ and Cl^- channels

Additional State of Na⁺ Channel in Gating

• Inactivation state

• Activated by voltage then after a period undergoes Inactivation (2^nd state change) and closes again

• The channel briefly conductive following activation by voltage then undergoes inactivation and the pore closes again

  • 2 gates: activation/ Inactivation gate

• Brief openings in Na+ channels due to this Inactivation

Mechanism of Inactivation/Deactivation State

• Either a region in the pore wall or close to it alters conformation physically occluding the pore.

• Localised form of gating: small shifts in sections of the polypeptide chain within the channel  - occurs within the pore region; associates with selectivity filter loop     

• Or the selectivity filter changes conformation, reducing ion transfer.

  • Involves a relatively short amino acid sequence

Particle ‘N-Type’

• A free intracellular region of the channel protein that plugs the pore; a.k.a. “ball and chain” gating.      

• Involves a relatively long amino acid sequence or subunit. 

• Polypeptide sequence form a plugging Particle- ball and chain arrangement – formed by large free polypeptide sequences within the cell

• Process of activation initiates movement 9f Particle to occlude ion channels

  • I.e ACTIVATION IS GENERALISED; INACTIVATION LOCALISED

Goldman-Hodgkin-Katz voltage (GHK_v) equation

• a modified Nernst equation (check by setting two of the P_ion= 0)

• Theoretical equation developed in 1940’s to determine the membrane potential of a system when more than one monovalent ion is able to permeate the membrane, where P_ion is the permeability function for each ion 

  • No accommodation of ion charge

Unquie feature of GHK Equation

• Chloride concentration ratio is flipped – negative ion looked at as a ratio from in to out.

Application of GHK Equation: Setting Ion Permeability to 0

• Allows the ion to be isolated and removed from the equation

  • Allows the return to a Nernst-like equation

Evidence For High K+ Permeability: Hodgkin and Horrowicz

• Aimed to determine how membrane potentials were set in biological systems and whether the theoretical predictions were applicable

• Intracellular recording from a frog skeletal muscle fibre @18°

• Record RMP at various [K⁺]_o

Evidence For High K+ Permeability: >10mM [K⁺]

• Can plot membrane potential against log[K⁺]_o values

  • data fitted by Nernst eq

    • Straight line fitted log10 version of Nernst

  • K⁺ channels contribute background ion permeability for RMP >10mM

Evidence For High K+ Permeability: <10mM [K⁺]

• Can plot membrane potential against log[K⁺]_o values

• Deviation from relationship - fitted by using a modification of the GHK_v eq.

  • Focused on [K+] [Na+]      

  • No longer fits Nernst's relationship 

• Intracellular [K+] 140mM – increasing concentration high enough to equal intracellular concentration, Nernst prediction = 0

Evidence For High K+ Permeability - Representation of Experimental Data

• Best fitted by accommodating [K+] and [Na+] with relative permeability ratios – ensure permeability of the system for sodium ions is 0.01 or 100 fold less than potassium

• Similar results/ processes seen for axons

Role of Na⁺ in Resting Membrane Potential (GHK)

• Involved in some way:

  • either a contribution from a small number of Na⁺ channels (hence low permeability)      

  • K⁺ channels do NOT show absolute selectivity for K⁺ - allow a small number of Na⁺ to pass through

Effect of Ion Selectivity on Ion Channels

• Ion channels exhibit a degree of selectivity

  • charge sign + or - (cations v. anions).      

  • charge density (ion size and amount of charge associated with the radius/ surface area)

    • e.g. Na⁺ v. K⁺ or Ca²⁺ v. Na⁺ & K⁺

      • Na+ is smaller has a greater charge density

Relative Selectivity of Ion Channels (GHK)

• Removing a specific ion from intra/extracellular fluid doesn't stop a class of ion channels from conducting charge.

• Channels still support ion flow/ current mediated by other ions

• K+ channels prefer K+ over Na+ and Ca2+, but will conduct Na+ if K+ is absent.

  • based on electrostatic attraction between channel and potential permeating ion

• In skeletal muscle, K+ channels are 100 times more selective for K+ than Na+

Generalised Concept For ALL Channels

• Have a preference for a particular ion that is relative

What does the proximity of RMP to E_K+ suggest about K+ channels in skeletal muscle and axons?

• It suggests that there is a single class of K+ channels with a high degree of selectivity for K+ over Na+ (100-fold).

  • Based off Hodgkin and Horowicz type experiment

K Channels of The Plasma Membrane

• Typical contains bot K_ir, K_2P channels

• Both have the four amino acid TVGYG sequences (one form each subunit) line up to form a cylinder in their selectivity filters

Variation of RMP

• Seen in some neurons until they become spontanoeusly active - i.e. dont have a RMP

  • Underlying systems suppourt their dynamic activity by engagning with many different ion channels e.g. inhibition of Na+ channels can show them finding a RMP above threshold

Two types of potassium channels contributing to RMP

• Inward rectifier and two-pore tadem domain channels.

• They contain a stereotypic sequence in the P-loop within individual subunits that form the selectivity filter.

• These sequences create a tube-like structure within the pore to select for K+ ions.

  • They can allow Na+ movement in hyperpolarised membrane potentials.

Explanation of Variability of RMP: Based on H&H Exp.

• One class of K+ channels with a given selectivity ratio, will not affect the RMP, regardless of the number present

  • A system with moderate or intermediate n.o channels will still have an E value of -90mVs – MP still finds an equilibrium value regardless  

• Variability can’t be explained through channel numbers – regardless if the same or different classes, will reach RMP of -90mV

K+ Channel Selectivity Filters

• Typically highly conserved amino acids in the P-loop in most ion channels

• Some have been lost or altered from the TVGYG sequence in some K⁺ channels such as I_h [HCN] or CNG

  • I_h/HCN - hyperpolarization-activated cyclic nucleotide-gated channel → Important in neuronal and cardiac tissue  

  • CNG - cyclic nucleotide-gated channels → found in photoreceptors of retina

Variation In Selectivity Filters

• Occurs in response to the loss of conserved sequence that would give rise to highly selective sequence

  • HCN and CNG have much lower K⁺ to Na⁺ selectivity only four-fold, so α = 0.25

Equilibrium position of a system that relies on Na+ and K+ channels

• -20mV.

  • This is true regardless of the number of channels.

• High levels of these channels present in spontaneously active neurons to maintain a depolarised membrane potential

• If only one class of channel is responsible for RMP, the value will be the same regardless of the number of channels.

Consequence of Combining Highly Selective And Non-Selective K+ Channels

• Significantly impacts the overall resting membrane potential.      

• High selectivity for potassium ions leads to a closer overall resting membrane potential of -90mV

• Conversely, less selective channels contribute less to the overall resting membrane potential. ( closer to -20mV).

  • If present at equal numbers RMP would be between -90mV and -20mV

Explanation for The Varaibilty of RMP

• It is due to the relative numbers of channels with different ion selectivity and associated driving forces.

  • Not the absolute number of ions type of ion channel that determines RMP, but the relative number of different channels – explains variability

What Determines Excitability

• The voltage difference between RMP and threshold

Implications of GHK_v

• Has generalised the number of different channels with different ion selectivity into a conceptualised measure

  • Membrane ion permeability