Intro to Ion Channels

Question 1

What is membrane transport?

Answer 1

The selective movement and redistribution of solutes across biological membranes.

Question 2

What are examples of membranes involved?

Answer 2

Plasma membranes.
Vacuoles.
Mitochondria.
Chloroplasts.
Endoplasmic reticulum (ER).
Sarcoplasmic reticulum (SR).
Lysosomes.

Question 3

What is the importance of membrane transport?

Answer 3

Osmoregulation / turgor control.
Nutrient acquisition.
Waste excretion.
Compartmentalisation of metabolism.
Energy transduction.
Signal transduction.

Question 4

What are the 3 classes of transport system?

Answer 4

Primary pumps.
Carriers.
Ion channels.

Question 5

How do primary pumps work?

Answer 5

Use a primary source of energy to pump ions against electrochemical gradients

Primary active transport

Ions = usually H+ or Na+

Pumps are electrogenic.

Establish electrochemical gradients for ‘driver’ ions.

Question 6

How do carriers work? (general)

Answer 6

Energised by ‘driver’ ion electrochemical gradients

Secondary active transport.

Question 7

What are the classes of carriers?

Answer 7

Symporters / co-transporters
= move solute against gradient

Antiporters / counter-transporters
= get rid of waste solute in opposite direction

Facilitators / uniporters
= cannot be energised, allow facilitated diffusion passively down electrochemical / chemical gradients

Question 8

How do ion channels work?

Answer 8

Transport is always passive down electrochemical gradients.

Usually highly regulated with defined open and shut kinetics = gating.

Usually selective for specific ions.

Question 9

What are the 3 distinct components of signalling networks?

Answer 9

Perception of signal.

Intracellular signalling.

Cellular responses.

= ion channels involved in all 3

Question 10

What are the 4 basic properties of ion channels?

Answer 10

Permeation.

Selectivity.

Gating.

Modulation.

Question 11

What is permeation?

Answer 11

When a channel opens = permeant ions move through.

Direction governed by electrochemical gradient.

Rate governed by electrochemical gradient / intrinsic properties of channel pore.

Question 12

What is selectivity?

Answer 12

Simple = distinction between cations and anions.

Other channels more complex = can distinguish between size

Non-selective = permeable to anions and cations.

Question 13

What is gating?

Answer 13

The process of transition between the open and closed states is gating.

Question 14

What is the frequency or opening / closing directly influenced by?

Answer 14

Changes in membrane voltage (voltage gated).

Binding of intracellular or extracellular ligands (ligand gated).

Mechanical stress (mechano-sensitive).

Changes in temperature.

Question 15

Which substances can modulate gating of channels?

Answer 15

Ca2+, H+, ATP, Fatty Acids, Phosphorylation, G proteins.

= they influence gating following activation (indirectly)

Question 16

What are accessory proteins?

Answer 16

Usually present the pore forming unit.

Most channel possess tightly associated accessory subunits.

Question 17

What are some functions of accessory proteins?

Answer 17

Specifying their location and abundance of ion channel
= Rapsyn clusters AChR at excitatory synapses

Modulate ion channel gating
= SUR subunit of KATP channels has key role in hydrolyzing ATP to stimulate channel opening

Fine tuning sensitivity to physiological and pharmacological agents
= SUR is target of drugs used to treat diabetes

Question 18

How to study ion channels?

Answer 18

Electrophysiology
= properties / activity

Cloning and determining primary sequence
= physiological role

Topology
= structure / function

Question 19

How to clone ion channels?

Answer 19

Size fractionate on a gel to form different pools of mRNA and inject into oocytes (e.g. from Frog)

Electrophysiology.

Generate cDNA library (used to generate large amounts of mRNA).

Repeat fractionation and expression to isolate single mRNA.

Sequence mRNA - gene identified

e.g. anoctamins

Question 20

What is another example of expression cloning?

Answer 20

Heterologous expression and complementation of yeast mutant growth phenotype

e.g. trk1D, trk2D mutant

Question 21

What are the 6 principles of hydropathy analysis? (Topology)

Answer 21

(1) Assume hydrophobic polypetides in a hydrophobic environment (membrane) form apha helices.

(2) Membrane span is 6nm (equivalent to 20 a.a. in a helix)

(3) Each a.a. is given hydropathy index (reflects hydrophic/hydrophilic nature)

(4) Stretches of a.a. sequence are analysed by computer using a moving segment approach (20 a.a.)

(5) Hydropathy index is plotted

(6) Values over +1 are scored as forming transmembrane spans

Question 22

What are the ion channel families?

Answer 22

Pore-loop channels.

S4 family.

Non-pore loop channels.

Question 23

What are the 3 pore-loop families?

Answer 23

Kir family
= K+ inward rectifiers
= e.g. ATP sensitive, G-protein coupled
= K+ influx, regulate membrane voltage

Two pore K+ channel family
= tandem duplicate of Kir
= 15 members
= represent ‘leak’ channels

Glutamate receptor family
= all cation permeable
= pore loop in inverse direction
= extracellular binding domain

Question 24

What are examples of the S4 family?

Answer 24

Voltage gated family
= S4 voltage sensor (depolarisation)
= Kv subfamily = K+ efflux, repolarisation of AP
= NaV subfamily = Na+ INFLUX, AP depolarisation
= CaV subfamily = Ca2+ signalling

SK + BK families
= Ca2+ activated K+ channels (efflux), neuron repolarisation
= SK subfamily = small G , voltage insensitive, activated in nM range of Ca2+
= BK subfamily = large G , activated by depolarisation and uM range of Ca2+, 7TMS

CNGC family
= photo and olfaction reception
= activated by direct binding to CNs, modulated by voltage

HCN family
= activated by hyperpolarisation, CN modulated
= identified in cardiac pacemaker cells

TRP family
=promiscuous activation
= voltage insensitive
= roles in sensory perception

Question 25

What are the non pore-loop families?

Answer 25

Cys-loop receptor family
= pentamers, pore formed by TMS2
= bind neurotransmitter extracellular (N-terminus)
= large family, members according to agonist, synaptic transmission
= e.g. AChR, GABA,GlyR, 5HT

IP3 and RYR receptor families
= major intracellular Ca2+ release channels
= endomembrane location, large structures
= IP3 - a2+ signalling roles
= RYR - muscle contraction

CLC family
= anion selective
= complex structure / function relationships - channels and transporters
= located in plasma membrane (CLC1) and endomembrane (CLC5)

CFTR (ABC transporter)
= ABC family
= ATP hydrolysis used to drive protein between open and closed
= epithelial transport in humans
= e.g. cystic fibrosis

Bestrophin family
= anion selective, Ca2+ activated
= likely to form tetramers (TMS2 forms pore)
= possible PM / endomembrane location
= physiological function unclear
e.g. eye diseases - BEST disease (macular degeneration)

Anoctamines (TMEM16)
= Cl- selective (Ca2+ activated), 10 family members, 8 TMS
= thought to be ubiquitous cells found in all animal cells

Question 26

Ion Channels are complex because they exibit:

Answer 26

Selectivity amongst ions.

Tight control over opening and closing = gating

Fine control or adjustment of gating = modulation

Complex structures (composed of 100s a.a)

Accessory proteins

Wide variety of structures, loosely arranged into distinct families based on secondary structure

Complex structure / function relationships