importance of ion channels

• bilayer impermeable to Na, K, Ca, Cl but these mediate:
• nerve impulse
• secretion
• muscle contraction
• cell volume changes

ion channels allow regulated permeability to these ions

• highly selective
• bidirectional
• gated: three types
• voltage
• ligand
• signal

ion channels can be considered to be a superfamily

• evolutionarily related but divergence yielded the wide array of properties

K+ channel structure

• 6 pass membrane protein TMDs=S1-S6, cytoplasmic N & C termini
• 4 homologous subunits
• compare to homologous Na and Ca channel:
  one subunit containing 24 TMDs

K+ channel gating: S4 helix

• S4 TM is amphipathic sensor, charged residues respond to membrane voltage
• depolarization->helical sliding of S4 toward outer suface
  demonstrated by flourescent labeling of S4
• S4 movement-> releases ball opening channel
• open channel conducts 10e6 ions/sec

K+ channnel selectivity: P segment

• pore formed by one alpha helix from each subunit plus H5 segment (see fig)
• pore opening at narrowest site= 3A
• K+ ion w/out waters of hydration = 1.3A
• 3 K+ binding sites on H5
• electrostatic repulsion responsible for K conductance through channel
• channel conformation favors dehydration of K (1.3A) over Na (0.9A)-> selectivity

K+ channnel inactivation:

• N-terminus forms "ball" that closes cytoplasmic opening of channel

K+ channel regulation

• wide array of different genes with different elictracal responses ranges
• phosphorylation (eg in response to hormone stimulation) alters gating

AchR experimental history

• chemical transmission demonstrated by heart experiment (vagusstuff)
• Ach chemically identfied as the transmitter
• importance of electric organ for AchR study
• assay for AchR by bungarotosin binding
• purification of the AchR by Ach affinity chromatography->
• 5 subunits by SDS-PAGE
• reconstituted in liposomes-> demonstrates central activities in single molecule
• receptor has channel activity
• channel activity gated by Ach
• site directed mutagenesis
• mapped TMDs
• mapped Ach and toxin binding sites
• mapped ion channel
• EM
• determined pore size 5-8A
• pore lined with 5 alpha helixes (one from each sub)
• quick freeze +/- Ach
• gate near middle of passage
• (kink in closed conformation by leucines)
• studies of aggregation
• diffuse in developing muscle
• upon innervation -> clustered to 0.1% of PM
• extracellular factor required purified and named agrin
• agrin hypoth
• motoneuron syn, tansports and secretes agrin
• agrin binds agrin receptor triggers muscle cell cascade
• AchR is phosphorylated (beta sub) causing clustering
• anti-agrin Abs label moto neurons and synaptic cleft
• anti-agrin blocks clustering
• agrin KOs die, and have no clustering prior to death