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22.416 SensoryMotor PhysiologyLecture 5
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Which properties of pore determine its ionic selectivity? a. ... ve aas repel cations, attract anions anion selectivity - whereas -ve aas in neck cation selectivity ... – PowerPoint PPT presentation
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Title: 22.416 SensoryMotor PhysiologyLecture 5
1
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)
7
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)
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