22.416 SensoryMotor PhysiologyLecture 5

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22.416 Sensory-Motor Physiology Lecture 5

• FUNCTIONAL AND STRUCTURAL STUDY OF ION CHANNEL
  PROTEINS
• - general characteristics
• - classification by gating stimulus
• - structure-function relationships
• 1. Characterization of Ion Channels ...

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1. Ionic selectivity of channels pore

• - determines channel's effect on VM (? reversal
  potential)
• Which properties of pore determine its ionic
  selectivity?
• a. diameter of pore must be gt ions diameter
  when open
• Note hydration of ion (by H-bonded H2O) ?? ion
  diameter
• - Na ion is smaller than K, but has larger
  hydration shell (H2Os get closer tove nucleus
  so more can be held)
• -so one pore may pass hydrated K but not
  hydrated Na
• - while a smaller pore that can strip off
  hydration shells may pass bare Na ions but not
  bare K ions

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b. chemical nature of amino acids lining pore

• i. hydrophilic aa residues (charged or very
  polar) - may strip off ion's hydration shell by
  H-bonding to H2O
• (if pore is narrow, only small bare ions
  squeeze through)
• - but if pore lining is mostly hydrophobic,
  hydration shells stay intact so pore must be
  larger
• ii. net charge of aa residues in pores narrow
  neck
• - ve aas repel cations, attract anions ? anion
  selectivity - whereas -ve aas in neck ? cation
  selectivity
• iii. aromatic rings (e.g., Tyr, Phe) favour
  cation selectivity through cation-? interaction
  with electrons in rings ? bonds
• Note that cation channels can often be converted
  into anion channels ( v.v.) by substituting a
  few key aas in the pore

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2. Gating and modulation

• Most ion channels are gated (opened or closed)
  by specific stimuli or switches, including
• - ?VM (depoln, hyperpoln)
• - ec ligands
• - ic ligands (? messengers)
• - ?T (warming, cooling)
• - mechanical force
• Many require combinations of gating
  stimuli (e.g., ligand depoln)
• Many are gated by a primary stimulus, but are
  modulated in their function by other stimuli, to
  adapt to circumstances

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3. Pharmacological (drug) effects

• Foreign drugs or toxins may
• block channels (antagonists),
• open them (agonists), or
• modulate their activity
• These permit experimenters to
• turn a channel on or off to see how it affects
  function
• label tightly-binding toxins to count ion
  channel proteins, purify them, or study their
  structure within the membrane
• 4. Single channel conductance, ?
• measured by patch clamp as described earlier
• may reveal channel states between fully "open"
  "closed

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5. Molecular structure

• a. Primary (1o) structure ( aa sequence)
• - isolate, clone and sequence cDNA for the
  protein
• b. Higher order protein structure
• 2o - ?-helices, etc. 3o - how these fold 4o -
  subunits
• Techniques
• i) X-ray crystallography
• - purify enough protein to crystallize
• - place crystals in X-ray beam
• - analysis of diffraction pattern reveals
  positions of atoms within protein
• Electron diffraction is used similarly (e.g., on
  nAChR)

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ii) probes of ec vs ic access to specific aas by
...

• - antibodies (Abs) against specific aa sequences
• physiological ligands (nts, ...), agonists,
  antagonists,...
• proteolytic enzymes
• glycosylation (always on ec side)
• iii) hydropathy profile
• assign hp index to each aa acc. Kyte Dolittle
  (p.45) -4.5 (most hydrophilic) to 4.5 (most
  hydrophobic)
• plot hp index vs aa position (e.g., Fig. 3.5)
• e.g., 20-aa-long hydrophobic sequence may form
  a membrane-crossing ?-helix, interacting with
  lipid tails(sometimes called a TransMembrane
  Segment, TMS)

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• e.g. aa seq. for nAChR subunit with hydrophobic
  regions M1-M4 highlighted

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... as Fig. 3.3 suggests for thenAChRs tertiary
structure (recent evidence on this in L.8)

• or a TMS may form part of a pore lining if every
  3rd or 4th aa is hydrophilic (see overhead)
• Mainly hydrophilic aa stretches, on the other
  hand, may contact water (ec, ic or pore)

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iv) site-directed mutagenesis

• delete or replace specific aas (by recombinant
  DNA techiques) to test suspected functions and
  locations, e.g., selectivity, gating,...
• v) expression in oöcytes (large cells, easy to
  work with)
• inject (modified) mRNA into oöcyte, which
  translates it and incorporates product into its
  membrane, where...
• electrophysiology can assay modified proteins
  function

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Major Classes of Ion Channel Proteins and their
Relationships

• 1. Voltage-gated channels
• often tetramers (of 4 subunits) or of 4 similar
  domains
• many (Na, K, Ca families) belong to same
  superfamily
• 2. IC ligand (Messenger)-gated channels
• gated by ic Ca, cAMP, cGMP, G proteins, ATP, etc.
• many belong to V-gated superfamily and are
  V-sensitive
• 3. EC ligand-gated channels
• often pentamers, with larger single channel ?
• several superfamilies (more follows below on 1 -
  3)

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4. Gap junction channels (connexons)

• hexamers, very large ? (100 pS)
• may rectify (pass ve current only in 1
  direction)or be non-rectifying
• 5. Mechanically gated channels
• gated by mechanical tension or deformn in
  membrane
• important in mechanoreception cellular
  osmoregn

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6. Temperature-activated cation channels

• e.g., some are opened by noxious or painful heat
  ...
• and also by capsaicin (hot chemical in hot
  peppers)
• PCa 12 x PNa, so chronic capsaicin ? large Ca
  entry ? death of cells (e.g., pain receptors in
  arthritis, etc.)
• others respond to cold and cooling chemical
  menthol
• 7. Constitutively open (non-gated) channels
• - for K may help establish VMR
• - for Na epithelial Na channels regulate
  passive transport of NaCl and water across
  epithelia

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Voltage-gated ion channels

• 1. Physiological groupings of V-gated channels
• a) V-gated Na channels - familiar from role in
  ap
• densities in electrically excitable membranes
  2,000/?m2 (nodes of Ranvier), 500/?m2 (squid
  axon), but only 2/?m2 in neonatal rat optic
  nerve
• Distinctive properties
• selectivity for Na (12-fold over Ca)
• gating depoln ? transient ? in opening
  probability gating current suggests depoln
  moves charged gate inactivation blocks channel
  if depoln maintained
• single channel ? 18 pS
• most (but not all) are blocked by TTX STX

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b) V-gated Ca channels

• depoln ? ?PCa ? ? Cainside (to act as
  messenger),
• ? ? depoln (regenerative, may ? ap)
• i) L-type Ca channels (in vert. heart, T-tubules,
  ....)- open -10 mV inactivate slowly
  (Long-lasting current)
• - dihydropyridines (DHPs) may ? opening
  (nifedipine) or ? it hence called DHP
  Receptors or DHPRs
• - also blocked by verapamil diltiazem
  (non-DHPs)
• ii) T (Transient current)-type Ca channels- open
  around 70 mV then inactivate rapidly
• - e.g., add to cardiac pacemaker potential
  after their inactivation is removed by hyperpoln
  following last ap
• iii) N (Neither), iv) P (Purkinje), v) Q all
  ? nt release

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c) V-gated K channels Electrophysiological
groupings

• i) outwardly rectifying K channels
• Delayed rectifiers (as in ap downstroke)(let K
  current out during depoln when open, but not in
  during hyperpoln when closed ? outward
  rectification)
• open slowly upon depoln no fast inactivation
• A-type or transient K channels (pass A
  current, IA)
• open more quickly, often at or below VMR
• show fast (N-type) inactivation, often below
  VMR
• an enabling hyperpoln will remove
  inactivation then, as VM ? to VMR, IA turns on
  K efflux slows recovery
• used to slow a depoln, or space out spikes or
  bursts
• Ca-gated K channels depoln ? Cas gating
  effect

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ii) inwardly rectifying K channels

• all close upon depoln (preventing K outflow),
  and open upon hyperpoln (allowing K in) ? inward
  rectification
• e.g., KIR channels of cardiac working cells
• recall, their closing during depoln permits
  very long ap,
• opening during repoln ? rapid regenerative
  downstroke
• e.g., Ih (hyperpolarization-activated) K
  channels
• have PNa ¼ PK, so rev potl ? 30 mV
• opened by hyperpoln in heart pacemaker cells(?
  Na entry ? beginning of pacemaker potential)
• also opened by hyperpolarizing SRP in rods (see
  later)
• Note ic cAMP enhances Ihs opening upon
  hyperpoln
• others, HERG K channels (human), KAT1 AKT1
  (plants)