Transcript Genomics of sensory systems - University of Maryland

Lecture 10: Membrane
potential and ion channels
Fain ch 3 end
10/5/09
Telomere - protects chromosome
ends
Chromosomes degrade w/o telomere
Telomerase adds telomeres
Questions
1. How do you follow membrane
potential?
2. What can you learn from evolutionary
relationships of ion channels?
Example - Membrane
potential in hair cells
 Important
systems
Auditory
Vestibular
Lateral line
 Hair
cell responds to mechanosensation
Bending causes electrical response
Bullfrog inner ear
 Very
accessible
 Use the sacculus
Large hair cells
Responds to head
movement (slow
frequency)
May respond to
sound
Frog sacculus

Maculus is sensory
epithelium
(location marked by
| | |)
Hudspeth and Corey 1977
Hair cells of inner ear (bull
frog)
Bundles
Kinocilium
Stereocilia - microvilli
HC = hair cell
SC = supporting cell
Arrows point to kinocillium
Hudspeth and Corey 1977
Remove otolithic membrane
(OM) to reveal hair cells
Use stimulus probe (SP) to
perturb hair cell
Record intracellular potential
with microelectrode (ME)
Hair cell motion
Towards kinocilium
Depolarize
Inside cell less
negative
Away from kinocilium
Hyperpolarize
Inside cell more Sideways motion had
no effect
Depol >> Hyperpol
Fain fig 3.11
How can we explain this
result?
 Are
channels opening or closing?
 What
ions are moving?
Cell membrane contains ion pumps and
channels - create concentration gradients
Inside cell
Outside cell
Na/K ATPase
Na+
141 mM
K+
3.3 mM
Na+
K+
15 mM
120 mM
Pump sends Na+ out
Channel lets Na+ in
Inside cell
Outside cell
Na/K ATPase
Na+
141 mM
Na+ pumped out
Na+
15 mM
Na+ flows in
through open
channel
K+
3.3 mM
K+
120 mM
Pump sends K+ in
Channel lets K+ out
Inside cell
Outside cell
K+
pumped in
Na/K ATPase
Na+
141 mM
Na+
K+
3.3 mM
K+
15 mM
120 mM
K+ flows out
through open
channel
Possible mechanisms
Na+ channel
Motion
rel kino
Away
Toward
Cell
Hyperpol
Depol
Channel
Na+
Possible mechanisms
Na+ channel
Motion
rel kino
Away
Toward
Cell
Hyperpol
Depol
Channel
Close
Open
Na+
Pump out
Flow in
Possible mechanisms
Na+ channel
Motion
rel kino
Away
Toward
Cell
Hyperpol
Depol
Channel
Close
Open
Na+
Pump out
Flow in
K+ channel
Motion
rel kino
Away
Towards
Cell
Hyperpol
Depol
Channel
K+
Possible mechanisms
Na+ channel
Motion
rel kino
Away
Toward
Cell
Hyperpol
Depol
Channel
Close
Open
Na+
Pump out
Flow in
K+ channel
Motion
rel kino
Away
Toward
Cell
Hyperpol
Depol
Channel
Open
Close
K+
Flow out
Pump in
Which is it?
Na+
K+
Voltage clamping
Hold cell at
fixed voltage
 Measure current
flow across
membrane

Direction
Size
Fig 3.13
Ohm’s law

V=IR
Voltage = current *
resistance
V
I  Current = voltage /
resistance
I=V/R
R

But conductance,g is 1/R
I=V g
Cell is a resistance / conductance
Resistance and
conductance
depend on how
many channels
are open
 Measure current
to learn about
conductance

Fig 3.13
Voltage clamping

Current flow
i  g(Vm  E rev )

Erev is potential at which no current flows
Potential which balances ion concentration gradient


E rev
RT Nao  K o

ln
F
Nai  K i
Vm is membrane potential during stimulation

Calculate Erev for hair cells
equally permeable to Na+ and K+
Outside cell
Na+
Inside cell
141 mM
Na+
15 mM
Na/K ATPase
K+
=1
3.3 mM
E rev
K+
120 mM
140 3.3mM
 59mVln
 1mV
15120mM
For hair cells, because Erev~0
 Ion
current is proportional to
conductance
i  gV m
 As
stimulate hair cell, conductance
changes
im
g 
Vm

Voltage gated current is prop to conductance

Current flow direction
Displace toward kinocillium
Depolarization
Vm positive, current is positive
Current flows out
Vm
Vm negative, current is negative
Current flows in
Fig 3.14
Hair cell stimulus
 Conductance
change
im current
g 

 positive
Vm
60m V
 So
movement towards kinocillium
 increases conductance
Hair cell stimulus
 Conductance
change
im current
g 

 positive
Vm
60m V
 So
movement towards kinocillium
 increases conductance
Channels open
Na channels!
Evolution of ion channels
 How
are different ion channels related?
 What are structural similarities?
K+ channel
Simplified 2TM channel
Roderick Mackinnon used the
Streptomyces lividans channel in
his Xray crystallography studies
Found it was similar to
vertebrate K+ channels because
both are blocked by neurotoxins
Only need 2 transmembrane TM
regions and the pore region
K+ ion pore formed from 4
subunits
Ion selectivity determined by
S5, S6 and pore
S1-S4 adds channel gating
How channels are gated by voltage
Nature 423 (2003) 42-8
Voltage sensitive paddles - move
to open and close channel
Large motion of S4 helix in
response to charge : Arginines (+)
Family of
ion
channels
Label
Ion :
K, Na, Ca
How
channel is
gated:
voltage
Ca
Root is
likely the
2TM
channels
TM channels
Verts+inverts
Bacteria group C
Verts+inverts
Bacteria group B
Bacteria group A
Gain of S1-S4 enables
voltage gating
Verts+inverts
Bacteria group C
Verts+inverts
Bacteria group B
Bacteria group A
Some
species
have
4x6TM
regions
Voltage gated sodium channel
Result of gene duplication and fusion
Multimeric channels
Verts
Na+
Jelly, cnidarians,
inverts
Verts+inverts
Ca+
Yeast
Na+
Bacteria
Phylogenies of different channels
What would be difficult about building a tree comprised
of these kinds of genes?
CNG
channels
are
important
for vision
and smell
Ion channel summary
 Structure
and function reasonably well
understood
 Domain and gene duplications followed
by fusions played role
 Diversity of ways to gate channels
Crystal structure of Kv channel
in open state