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IONIC  BASIS  OF  MEMBRANE  POTENTIALS  
 
Principles  of  electrostatistics  explain  why  aqueous  pores  formed  by  channel  proteins  are  needed  for  ion  diffusion  across  
cell  membranes    
 
Plasma  membranes  are  electrically  polarized,  as  evidenced  by  the  presence  of  membrane  potential
...
1V  
 
Brain,  Heart  and  Neuron  cells  uses  energy  for  signaling  purposes  
Brief  impulses  formed  by  the  cells  mentioned  are  called:  action  potentials  
 
Atoms  can  exist  as:    
1
...
NEGATIVLE  CHARGED  
3
...
6022  x  10^-­‐19  
Charge  of  single  ion=  valence  x  elementary  charge  
 
Let    
F=  electrostatic  force  between  ions  that  have  different  valences  
 
r  =  square  of  the  distance  between  two  ions  
 
d=  dielectric  constant  
 
KNOW  THAT:    
F  is  proportional  to  r    
F  is  inversely  proportional  to  d  
In  aqueous  solution,  the  positive  and  negative  charge  is  always  equal
...
   
Thus,  inorganic  ions  cannot  readily  pass  without  the  aid  of  a  protein  or  a  channel  
Channels  provide  a  favor  polar  environment  of  the  ion  to  move  across  the  membrane
...
Intracellular  recording  
Step  1:  tip  of  microelectrode  inserted  to  cell  
Step  2:  ground  is  measured  
 
Note:   Membrane   potential   is   directly   proportional   to   electrical   field,   and   inversely   proportional   to   the   distance  
across  the  membrane  and  other  parameter
...


Spectrophotometric  techniques:  for    measurement  of  membrane  potentials  of  inaccessible  cell  membranes  
Step  1:  labeling  of  cell  membrane  with  organic  dyes  
Step  2:  monitoring  of  the  absorption  
Step  3:  measurement  of  optical  signal  of  dye  molecule  (measurement  of  dye  molecule  is  the  membrane  
potential  

 
Resting  potential:  cell  is  not  undergoing  active  responses
...
impermeable  membrane  
2
...
conductance  pathway  
Impermeable  membrane:  barrier  to  inorganic  ions,  poor  electric  conductors,  highly  resistant  to  ionic  current
...
   
Diffusion  potential:  movement  of  an  ion  down  its  concentration  gradient  
Nernst  equation:  predicts  the  equilibrium  membrane  potential  of  a  particular  ion  across  the  membrane  
Membrane  potential:  determined  by  the  relative  permeabilities  of  the  cell  membrane  to  various  ions
...
compute  ionic  current    
a
...
   
2
...
 
Constant-­‐field  equation:  
Predicts  how  the  membrane  potential  will  respond  to  changes  in  ion  concentration  gradients  or  membrane  permeability
...
external  and  internal  ion  concentration  
2
...
permeability  coefficient  for  the  ion    
 
How  an  ion  behaves  in  the  membrane:    
1
...
electrical  field  is  constant  throughout  the  thickness  of  a  membrane  
a
...
what  we  call  the  Constand  field  assumption  
3
...
The  permeability  constant  of  an  ion  is  always  constant  
Permeability  constant  of  an  ion  describes  the  ability  of  an  ion  to  dissolve  thru  a  membrane  and  diffuse  from  
one  side  to  another
...
computes  the  current  carried  by  a  single  ion  across  the  membrane  
b
...
current  carried  by  a  single  ion  has  a  unit  of  amperes  
d
...
Movement  of  molecules  in  a  solution  
ii
...
Direct  proportionality  of  a  current  to  voltage  
Current-­‐Voltage  relationship:  current  carried  by  a  single  ion  depends  on  the  membrane  voltage  

e
...
   
 
Ions  that  generally  passes  thru  the  membrane,  pass  thru  distinct    pathways
...
   
 
Each  ion  carries  its  own  electric  current
...
 
The  ability  of  a  lipid  bilayer  to  maintain  a  separation  of  charge  across  its  4  nm  width  makes  the  lipid  bilayer  function  as  a  
capacitor
...
 1  uF/cm2  
The  thickness  of  a  cell  membrane:  4
...
Variable  resistor  
2
...
Capacitor  
 
The  separation  of  relatively  few  charges  across  the  bilayer  capacitance  maintains  the  membrane  potential  
 
Capacitance  of  a  cell  membrane  can  also  be  used  to  measure  the  amount  of  charge  that  the  membrane  actually  separates  
in  generating  a  typical  membrane  potential    
 
Cell  membrane  potentials  are  sustained  by  a  very  small  separation  of  charge  
 
Existence  of  membrane  capacitance  allows  the  total  membrane  current  to  have  two  compartments:    

1
...


carried  by  ions  thru  channels  
carried  by  ions  as  they  charge  the  membrane  capacitance  

 
Ionic  current  is  directly  proportional  to  the  electrochemical  driving  force  (Ohm’s  Law)    
 
WHEN   MEMBRANE   POTENTIAL   IS   MORE   NEGATIVE   THAN   THE   EQUILIBRIUM   CONSTANT,   THE   CURRENT   IS   NEGATIVE,  
THEREFORE  GOING  INWARD
...
   
 
Ionic  current  depends  on  the  difference  between  the  actual  membrane  potential  and  equilibrium  constant
...
   
 
The  larger  the  driving  force,  the  larger  the  current  produced
...
Ca  
2
...
K  
4
...
conductance  elements  
2
...
driving  force  
Membrane  potential  is  always  at  constant  when  in  resting  potential
...
   
When   membrane   potential   is   changes,   CAPACITATIVE   CURRENT,   due   to   the   membrane   capacitance   helps   shape   the  
electrical  responses  of  cells
...
impaling  a  cell  with  2  sharp  electrodes  
i
...
injecting  the  current  
Steps  of  voltage  clamping:    
1
...
set  membrane  voltage  to  a  value  different  from  the  resting  potential  
3
...
feedback  amplifier    injects  opposing  current  to  maintain  consntant  membrane  potential  
a
...
 
 
 
 

TOTAL  MEMBRANE  CURRENT  COULD  EITHER  BE  FROM  MULTIPLE  CURRENTS  OR  ONE  TYPE  OF  CURRENT  
   
1
...
Total  membrane  current  is  the  sum  of  the  capacitative  current  and  the  ionic  current  
Hyperpolarization:  negative  going  membrane  potential  change  
Depolarization:  positive  going  membrane  potential  change    
 
Time  dependent  current:  
1
...
due  to  the  opening  and  closing  kinetics  of    voltage-­‐gated  Na  channels  
3
...
   
 
Macroscopic  current:    ionic  current  due  to  the  activity  of  large  population  of  channels  
 
Sodium  channels  are  voltage  sensitive  ion  channels  that  are  activated  by  depolarization
...
   
 
The  patch  clamp  technique  resolves  unitary  currents  through  single  channel  molecules  
 
Patch  clamp  technique    is  when  the  area  of  voltage  clamp  membrane  is  reduced  to  a  very  small  fraction  of  the  cell  surface  
area
...
suction  to  patch  pipette  creates  a  high  resistance  seal  between  the  glass  and  the  cell  membrane    
2
...
Cell-­‐attached  recording:    
Recording  of  current  from  channels  within  the  patch  
...
start  at  cell  attached  configuration  
2
...
production  of  whole  cell  configuration  
4
...
resealing  of  membrane  
Extracellular  patch  surface  faces  the  bath  solution  
 
Single  channel  currents  sum  to  produce  macroscopic  membrane  currents  
 
When  two  or  three  channels  in  the  patch  are  open  simultaneously,  the  measured  current  level  is  an  integral  multiple  of  the  
single-­‐channel  or  unitary    transition
...
Conductance  measurments  are  close  to  the  theoretical  value  expected  for  ion  diffusion  through  a  cylindrical,  water  
filled  pore  that  is  long  enough  to  accept  an  ion  and  has  a  diameter  large  enough  to  accept  an  ion
...
   
 
 
Single  Channels  can  fluctauate  between  open  and  closed  states  

  
MOLECULAR  PHYSIOLOGY  OF  ION  CHANNELS  
 
Classes   of   ion   channels   can   be   distinguished   on   the   basis   of   electrophysiology,   pharmacological,   and   physiological  
ligands,  intracellular  messengers,  and  sequence  homology  
 
Electrophysiology:    
1
...
Characterization  of  channels  accdg  to:    
a
...
Voltage  dependence  
c
...
u-­‐conotoxin  
a
...
 Na  in  skeletal  muscles  
b
...
conotoxin  
a
...
Ziconitide:  treatment  of  neuropathic  pain  in  patients  
 
Physiological  ligands  
Channels  are  activated  by  binding  to  an  agonist  
Example:    Vertebrate  Neuromuscular  Junction  
1
...
Ach  binds  to  Ach  receptor  
3
...
Gramicidin  
a
...
Spans  the  membrane  
c
...
Porin  
a
...
Forms  a  large  pore  
c
...
16   staves   of   the   barrel,   formed   by   16   strands   of   protein,   each   of   which   are   beta-­‐sheet  
conformation  
 
Gap  junction  channels  are  made  up  of  two  connexons,  each  of  which  has  six  identical  subunits  called  connexins  
 
Gap  junction    
a
...
5  nm  in  diameter)  
b
...
provides  pathways  for  chemical  communication  and  electrical  coupling  between  cells  
Compostion  of  gap  junctions:    
1
...
each  connexon  bridges  gap  between  two  cell  membranes  with  a  distance  of  approx
...
 
Each  connexon  has  6    identical  subunits  surrounding  a  central  pore:  radial  hexameric  symmetry  
 
Each  subunit  is  an  integral  membrane  protein  called  connexin  
The  pore  formed  at  the  center  of  the  six  connexin  subunits  has  a  diameter  of  approx
...
2-­‐2  nm  
 
Apposition  of  two  identical  connexon  hexamers:  homotypic  channel  
Apposition  of  two  dissimial  connexon  hexamer:    
Heterotypic  channel  
 
REGULATION  OF  GAP  JUNCTIONS  
1
...
pore  is  an  open  configuration  
2
...
pore  closes  
2
...
 Voltage  difference  between  the  coupled  cells  as  well  as  by  phosphorylation  
 
Nicotinic  acetylcholine  receptor  channels  are  ______  pentamers  made  up  of  four  homologous  subunits  
 
Alpha,  beta,  gamma,  delta  
Alpha  subunit  represented  twice  
 
Location  of  Nicotinic  Ach:    
1
...
Post  synaptic  nerve  terminal    
Response:    
Responds  to  Ach  released  from  nerve  terminals  by  opening  and  allowing  cations  to  flow  thru  its  pore  
 
Hole  diameter:  2-­‐  2
...
rare  form  of  hereditary  neuropathy  

b
...
mutation  in  the  gap  junction  protein:  connexin  32,  which  is  located  in  the  X  chromosome    
 
 
 
 
Hydrophobic  domains  of  channel  proteins  can  predict  how  these  protein  weave  through  the  membrane  
 
Protein  superfamiliies,  subfamilies  and  subtypes  are  the  structural  bases  of  channel  diversity  
 
Major  aspects  of  the  molecular  physiology  of  human  ion  channel  families  
1
...
Voltage-­‐Gated  Na+  Channels  
a
...
Has  four  domains  
c
...
Associated  with  auxiliary  Beta  subunits  
3
...
Also  known  as  Cav  
b
...
Ligand  Gated  Channels  
a
...
pentameric  Cys-­‐loop  receptor  family  
2
...
Purinergic  ligand  gated  cation  channels  
5
...
HCN:  Hyperpolarization-­‐activated,  cyclic  nucleotide=gated  ion  channels:  electrical  automaticity  of  the  
heart  and  rhythmically  firing  neurons  of  the  brain  
b
...
 
c
...
TRPC  
ii
...
TRPM  
iv
...
TRPP  
vi
...
K+  CHANNELS  
a
...
Common  potassium  selective  pore  domain  
c
...
Kv  voltage  gated  K+  channels  
2
...
Large  conductance  Ca2+  and  voltage  activated  K+  channels  (BKCA)  
4
...
Dimeric  tandem  two  pore  K+  channel  (K2P)    
 



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