L2: Voltage gated ion channels ADD ppt!

what are ion channels

• membrane-spanning proteins

• with aqueous solution in a central pore

How do they open and close (gating)

• conformational changes of protein strucutre

• within the plasma membrane

  • rapidly flux ions

Hodgkin and Huxley did what

• showed the squif giant axon generates the action potential waveform

• using only two types of voltage-dependent conductance

  • Na+ and K+

• used the voltage-clamp method

Voltage-clamp method

• uses a differential amplifier

• to inject a currnet into the cell

• in order to counteract any differeence between the voltages of its two inputs

1. The measured cell membrane potential

2. command potential

Why is this way useful?

• current necessary t maintain the cell membrane at a precise value is generated by

• the circuit

and

• this can be measured

When it is measured, it shows that…

• it is equal and opposite to the current flowing across the membrane

How did researches get to understand the strucuture of ion channel proteins:

• sequence the DNA encoding the primary amino acid sequence of ion channel proteins

  • using recombinant DNA technology

exmaples:

1. HYdropathy analysis

2. mRNA things?

What did hydropathy analysis do?

• determines the hydrophobivity of the amino acids

• allowed putative transmembrane sequences to be identifed

  • enabling the prediction of the secondary strucutre of the polypeptide chain

    • i.e which parts were in the membrane and which parts were in the extracellular and intracellular space

What to do next?

Make models:

• developed of tertiary and quaternary arrangements of the channels

• their accessory subunits in the membrane

what techniques has 3D strucutre been elucidated by

• high-resolution electron microsopy

• electron and X-ray diffraction

Once an ion channel DNA sequence was identified, mRNA can be…

1. mRNA made

2. injected into expression systems e.g Xenopus oocyte or mammalian cell lines

3. causing it to be expressed in the cell membrane

4. properties of the cloned channel were investigated using electrophysiology

How are the cloned channels investigated using electrophysiology

• use two-electrode voltage clamp (in oocytes) of macroscopic currents

or

• patch-clamp recordings of single channel and whole cell currents in cell lines

What do site-directed mutagenesis help do?

• site-directed mutagenesis applied to change individual amino acid residues

• in regions thought to be functionally important

Ionic conductances underlying the action potential: in the squid giant axon

• a depolarisating voltage change from rest (-65mV) to 0mV 

• caused by an early fraction of current carried by inward movement of Na+ ions (Na+ conductance)

What happened if Na+ was reduced?

• this depolarising voltage change disappeared

If the voltage step is maintained

• It activates then inactivates

• over several ms

A later fraction of current is carried by…

• outward movement of K+ ions (K+ conductance)

What does this do?

• activates more slowly than Na+ current

however, with maintained depolarisation

• does not inactivate

On repolarization

• it deactivates 

What do the size of the Na+ and K+ currents depend on

• the size of the voltage step

  • the IV relationship

What did H&H find

• they could describe the time course of the Na+ and K+ conductances

• The full model of the action potential 

  • which incorporates the resting leak conductances

• Can be expressed in a differential equation for the voltage across a patch of membrane

What is this equation called

• Hodgkin-Huxley  equation

By solving equation, H&H were able to

• reproduce action potential waveforms of exactly the right shape

  • this was significant because thei on channel strucutre has not been eluciated and recordings of current through single ion channels not yet been possible

Voltage-gated ion channels (VGIC) are gated by…

• changes in membrane potential (voltage)

When open, VGICs tend to be

• highly selective for a particular ionic species

• named for this permeabbility:

  1. Voltage-gated Na+ channels (Nav)

  2. Voltage-gated K+ channels (Kv)

1. Voltage-gated Na+ channels (Nav): how identified and studied

1. partially purified from electric ell

   1. with aid from puffer fish selective toxin (tetrodotoxin TTX)

2. Messenger RNA was then used to make complementary DNA libraries

3. cDNA clone encoding the electric eel voltage-gated Na+ channel (Nav) was isolated

1. Voltage-gated Na+ channels (Nav): What was found out about the strucutre of the channel

• single large (260 kDa) pore-forming peptide of 1832 amino acids→ alpha subunit

1. Nav: isolation of the primary amino acid sequence meant predictions could be made about the stucture of the channel…

Hydropathy analysis

• 4 similar domains (I-IV) each composed of 6 transmembrane (TM;hydrophobic) segments, S1-6

1. Nav: the initial proposed secondary strucuture

• needs to incorporate intracellular N- and C-termini

includes:

1. a pore loop between S5 and S6

   • believed to line the entry to the pore

   • with S5 and S6 forming the remainder of the pore

2. S4

   • Although sufficiently hydrophobic to be membrane spanning

   • charged amino acids were also noted 

   • S4 initially placed in the cytoplasma

1. Nav, but where is S4 now thought to be?

• thought to be trans-membrane TM

• forms a voltge sensor

1. Nav how many S4 voltage sensors are there per channel?

• 4

1. Nav: upon depolarisation, S4 helix…

• changes its conformation in the membrane

  • represents activation

1. Nav: alpha subunit in different tissues

• have many different isoforms

  • brain→ Nav 1.1-1.3

  • preipheral nervous system (Nav1.6-1.9)

  • skeletal muscle (Nav1.4)

  • cardiac muscle (Nav 1.5)

however:

• their properties are similar

1. Nav: what does an associated auxillary beta subunit do?

• modified key properties of the channel

2. Voltage-gated K+ channels (Kv)

• part of the larger family of potassium channels

  • also includes partially voltage-sensitive K+ channels and voltage-insensitive K+ channels

2. Why were voltage-gated K+ channels types named what they are

• according to their properties

  • delayed rectifiers, inward rectifiers, A-tpyse

2. However, with out better understanding we now know that…

• different functional properties can arise from various combinations of subunits

  • → it is perhaps better to view voltage-gated K+ channels from a subunit perspective…

2. The many alpha subunits

• Kv1-Kv12

2. Although, 4 families…

• Kv1,Kv2,Kv3,Kv4)→ form the core voltage-gated K+ channels

2. Classical ‘delayed rectifer’ K_ channels are formed by…

• e.g the slowly sctivating Kv in the squid giant axon

• Kv1, Kv2, Kv3 and Kv4 families in mammalian neurons

  • each family contains several subtypes

2. What are A-type K+ channels formed from

• Kv4 proteins

The very first K+ channel gene isolation…

• Drosphophila nad called ‘Shaker’ gene

• wildtype Drosophila will shake when exposed to 4-aminopyridine (4-AP)

  • a drug that blocks A-type K+ current

• mutant drosophila line shakes under normal conditions in a similar way to wildtype flies under 4-AP conditions

This lead to the idea that ‘Shaker’ mutant might have a mutation…

• in a K+ channel 

  • because that is what 4-AP blocks

This is correct:

• Shaker flies do not have a normal A-type K+ currents

Researchers took advantage of this and…

• isolated the DNA for the mutant’Shaker’ Kv in 1987

• → provided the first amino acid sequence for a voltage-gated K+ channel

2. Kv channel strucutre

• forms from 4 alpha subunits

  • each hoomologous to one domain of a much larger Na+ channel alpha subunit

• form tetramers of alpha subunits (along with auxiliary subunits)

2. Kv alpha subunits are described as 

• 6TM proteins

  • each alpha subunit has the S1-S6 transmembrane segments

  • including S4 coltage sensor

  • and a pore loop

    • as seen in Nav channels

• THEREFORE: each voltage-gated K+ channel would have 4 S4 segments for voltage sensing

Structurally related K+ channel family members include…

1. Calcium-activated K+ channel (Kca)

   • similar membrane topology to Kv channels

   • but sytoplasmic doomain has calcium-binding sites which mediate gating

     • in addition to voltage-gating in some Kca subtypes)

2. K_IR channels are strongly inwardly-rectifying channels

   • as a result of intracellular polyamine and magnesium block

   • have different topology:

     • tetramers of subunits, but

     • each subunit has only two membrane-spanning segments (2TM)

       • analagous to S5 and S6, with the pore loop between 

3. ‘Two pore domain’ K+ channels (K_2P)

   • have 4 TM domains

   • 2 pore loops

     • essentially, two units of the 2TM channels

     • usually open, not gated by voltage

     • contribute to the resting membrane potential

K+ channel and information coding?

• little information can be coded in the size of the axon potential

• continuous depolarising current at the axon initial segment

  • repeated action potentials are fired

The bigger the current…

• the higher the frequency of the action potentials

  • ‘spike frequency coding’

'Frequency and pattern of action potential firing is influenced by…

• types of voltage-gated ion channels expressed in the axon

Example 1→ ‘delayed rectifier’ K+

• conductance activates relatively slowly

• shows little inactivation

compared to…..

Example 2→ ‘A-type K+ conductance

• activates and then inactivates relatively rapidly

• and required hyperpolarization to relieve inactivation

Other voltage-gated K+ conductanecs …

• keep the action potential waveform narrow enough

  • to allow repetitive action potential firing

  • at very high frequencies

Certain voltage-gated K+ channels activate at…

• relatively hyperpolarised membrane potentials

• are very effective ‘brakes’ on excitability

Membrane depolarisation during the action potential rising phase can…

• activate voltage-gated Ca2+ conductances resulting in

  1. Ca2+ ion influx

  2. activation of calcium-dependent K+ conductances

Large calcium-activated K+ conductance (BK) contributes to…

• Repolarisation

• → Making the action potential width narrower

Small calcium-activated K+ current (SK) contributes…

• to longer-lasting after-hyperpolarisation (AHP)

• → broadens the action potential

  • slows the firing frequency

  • can help to keep the firing pattern regular

Strucuture-function studies of VGICs:

1. Inactivation

2. Selectivity filter

1. Strucuture-function studies of VGICs: Inavtication

1. suggested that the cause of Na+ channel inactivation was the N-terminal intracellular  region of the ion channel protein

• → ‘ball and chain’ to block pore

2. another paper: critical sequence N-terminal amino acids

   • when mutated→ disrupt inactivation in Drosophila ‘A-type’ K+ channels

2. Selectivity filter: what determineds which ions can permeate

1. size of the pore

2. charges of amino acid groups in the pore

3. perhaps some other things (water)…

Although K+ is larger than Na+, how do channels allow K+ in but not Na+ by a factor of more than 1000?

• mutations of amino acids in the pore loop between S5 and S6 altered ion permeation and ‘open channel block’

  • by the drug, TEA

What did this suggest?

• the pore loop contributes to the selectivity filter

  • in the outer (extracellular) part of the pore

MacKinnon published X-ray crystal strucutre of bacterial K+ channel (not voltage gated but selectively permeable): 

• 2TM spanning regions

  • analagous to S5 and S6 in a 6TM Kv

• shaker mutations that affect ion selectivity were mapped onto the crystal strucutre of the pore

The ion selectivity filter was deduced to be…

• short, narrow segment bind a dehydrated K+ ion

• effectitly mimic the hydration shell of the hydrated K+ ions in the rest of the pore

Why does this allow K+ in but not Na+?

1. K+ ions are close to eachother in the region of the pore

2. repel eachother and facilitate the passage of a line of K+ ions though the selectivity filter

3. into the cental region of the pore

4. Amino acids i the channel signature sequence are unable to bind a dehydrated Na+ ion

This signature sequence is…

• highly conserved amongst different K+ channels