What do you notice?
Sula et al. Nature Comm., 2016 Chen et al., PNAS, 2010 Kanai et al., Nature, 2013
Colors? First impressions? Interpretations?
Amino acids: the building blocks of proteins
All 20 amino acids contain:
1. a central alpha carbon
2. an amino group (NH3+)
3. carboxyl group (COO−)
4. an R group
Interactions between R groups cause proteins to have unique shapes (e.g. hydrophobic clustering and disulfide bonds).
Examples:
Hydrophobic R group Hydrophilic R group
Cysteine
Valine Asparagine (can form disulfide bonds)
(propensity for alpha helix) (propensity for beta sheet)
Peptide bonds and polypeptides
1° - a chain of linked
amino acids linked by
peptide bonds: covalent bonds between amino acids
FOUR LEVELS OF PROTEIN STRUCTURE
peptide bonds
polypeptides: proteins composed of a single chain of amino acids
Four levels of protein structure
2° - chain coils into a
3D structure (e.g.
alpha helix, beta
sheet) tertiary structure
3° - further bending
and folding into a
complex 3D shape primary structure
(e.g. subunits)
secondary structure
4° - multiple folded
protein subunits (e.g. quarternary structure
K+ ion channels)
Neuroscience 4th Edition, Bear, Connors, Paradiso
Protein composition influences its structure and function
The general structure of ion channels in the membrane
Ion channels are suspended in the membrane with:
1. Hydrophobic portion inside the membrane
2. Hydrophilic portion exposed to water-based
intracellular/extracellular environment
They often contain the following properties:
1. 4-6 subunits that form a pore between them
2. Selective permeability to specific ions (dictated by
the size of the pore and the R-group composition
of the amino acids lining the pore)
3. Gating (selective opening and closing) depending
upon changes in the local intracellular or
extracellular space.
Neuroscience 4th Edition, Bear, Connors, Paradiso
Ion Channel Gating
So far, we have been showing examples where an ion channel suddenly appears in the membrane…
That framework was meant to imitate the selective opening and closing of ion channels that can occur through the
following example mechanisms:
ligand-gated ion channels (aka ionotropic receptors) voltage-gated ion channels
opens after neurotransmitter binding opens after reaching a membrane potential threshold
(may also require neurotransmitters)
Protein composition influences its structure and function
Selective permeability of potassium channels
(example)
How does a potassium channel selectively allow
K+ ions to cross the membrane (and not smaller
Na+)?
Protein composition influences its structure and function
Selective permeability of potassium channels
(example)
How does a potassium channel selectively
allow K+ ions to cross the membrane (and
not smaller Na+)?
Some history:
Chris Miller found that charybdotoxin bound
to the external end of Ca2+–activated
potassium channels.
Charybdotoxin (CTX) is a 37-amino acid toxin
from scorpions that is named after
Charybdis, the daughter of Poseidon, who
was turned into a whirlpool generating sea
monster by Zeus
This toxin plugs the K+ channel pore, and is released after increasing intracellular K+, due to an ion binding site inside
the pore that expels the toxin
Swartz, eLife, 2013
Protein composition influences its structure and function
Selective permeability of potassium channels
(example)
Charybdotoxin was also found to block Shaker in fruit flies which was partially sequenced and thus genetically modifiable
By inducing select mutations, they
were able to localize regions
(amino acid sequences) that form
the K+ selective pore.
Top right: shows the structure of
the Shaker protein weaving in and
out of the membrane, and the site
of mutations induced in one study.
shaker (Sh) gene mutation in Drosophila
melanogaster: when anesthetized using Bottom Right: Characterizing the
ether displayed a shaking of the legs, physiological responses of cells
wings, and abdomen containing mutant protein after
the application of the toxin. Some
mutants were more resistant to
the toxin.
MacKinnon and Miller, Science, 1989
Protein composition influences its structure and function
Selective permeability of potassium channels
(example)
x-ray crystallography map of Sh mutation locations
Portions in RED are absolutely
required for K+ selectivity
Doyle et al., Science, 1998
What differences might exist between two different ion channels to give them unique (or
altered) functions?
Learning outcome: Describe structural features of amino acids and proteins that determine
the unique functions of proteins (e.g. ion channels and their ability to selectively transport
specific ions across the neuronal membrane).
Summary
• The amino acid composition of a protein determines its structure and function.
• Ion channels are often consist of multiple subunits inserted into the membrane that surround a
pore for specific ions to travel across the phospholipid bilayer.
• Chemical (e.g. toxins that selectively bind to channels for a specific ion), genetic (e.g. mutations
of the Shaker protein), electrophysiological (examining changes in voltage across the cell
membrane), and x-ray crystallography methods can be used to identify specific amino acid
sequences that mediate ion channel selectivity.
Maintaining Ionic Concentration Gradients
The sodium-potassium pump
This symbol:
ADP ATP
Means breaks
down ATP for
energy
Breaks down ATP to pump three sodium ions out of the cell and two
potassium ions into the cell.
Kanai et al., Nature, 2013 Since the sodium-potassium pump uses ATP, this process requires energy.
Neuroscience 4th Edition, Bear, Connors, Paradiso
Maintaining Ionic Concentration Gradients
Higher concentration of sodium
outside the neuron The sodium-potassium pump maintains
sodium and potassium concentration gradients
across the membrane.
Equilibrium potentials can be different for
every neuron. In a “typical” neuron in which EK
= -84mV and ENa = +62mV, if the membrane
potential is -65mV:
1) The opening of potassium channels causes
K+ ions to exit the neuron via diffusion.
2) The opening of sodium channels allows Na+
ions to enter the neuron via diffusion.
Higher concentration of potassium
inside the neuron
Maintaining Ionic Concentration Gradients
The Ca2+ pump and the Na+/Ca2+ Exchanger
Plasma membrane CA2+ ATP-ase: breaks down ATP to remove calcium ions
from the neuron. It binds to Ca2+ with a high affinity even when
concentrations are very low, helping to keep Ca2+ levels low in the neuron.
Sodium-calcium exchanger: Uses the energy acquired from letting Na+ flow
down its gradient in exchange for the transport of one Ca2+ ion to the
outside of the neuron (reminder: the membrane can act like a capacitor and
store electrical energy)
Electrically charged plates:
This symbol: [Link]
ADP ATP
Means breaks
down ATP for
energy
It is estimated that the sodium-potassium pump expends as much as 70% of the
total ATP utilized by the brain (Neuroscience 4th Edition, Bear, Connors, Paradiso).
Why do you think this pump uses so much ATP?
Learning outcome: Describe mechanisms through which neurons generate ionic
concentration gradients across the neuronal membrane.
The distribution of ions across the neuronal membrane
Approximate ion concentrations on either side of a typical neuronal membrane
Neuroscience 4th Edition, Bear, Connors, Paradiso
Review of ion flow across a selectively permeable membrane
equilibrium potential (Eion): the electrical potential difference that exactly balances an ionic concentration gradient
no channels insertion of potassium channels causes movement ceases
K+ movement
Equilibrium results when electrical force pulling K+ ions inside exactly counterbalances the force of
diffusion pushing them out (equal and opposite).
Neuroscience 4th Edition, Bear, Connors, Paradiso
An excitable membrane
equilibrium potential (Eion): the electrical potential difference that exactly balances an ionic concentration gradient
no channels insertion of sodium channels causes movement ceases
Na+ movement
Equilibrium results when electrical force pulling Na+ ions outside exactly counterbalances the force of
diffusion pushing them in (equal and opposite).
Neuroscience 4th Edition, Bear, Connors, Paradiso
Relative Ion Permeabilities of the Membrane at Rest
The Nernst equation calculates the equilibrium potential given selective permeability to a single ion.
Scenario 1: The membrane is equally permeable to Na+ and K+
As an example, there are equal numbers of open Na+ and K+ channels.
K+ Channel
Na+ Channel
The membrane voltage would be an average of EK and ENa
Relative Ion Permeabilities of the Membrane at Rest
The Nernst equation calculates the equilibrium potential given selective permeability to a single ion.
Scenario 2: K+ permeability is 40 times greater than Na+ permeability
As an example, there are equal numbers of open Na+ and K+ channels.
10 K+ Channels
Na+ Channel
The membrane is 40x more permeable to potassium.
The membrane voltage would be much closer to EK.
Note: This is closer to what happens in “typical” neurons due
to the presence of potassium leak channels.
Goldman–Hodgkin–Katz equation
!" $' [&! ]"#$ ( $() [)*! ]"#$ ( $*+ [+,, ]%&
Vm = #
ln $ &! ( $ [)*! ] ( $ [+,, ] 𝑖𝑜𝑛 !"# : ionic concentration outside the cell
' %& () %& *+ "#$
𝑖𝑜𝑛 $% : ionic concentration inside the cell
At body temperature (37°C), this simplifies to (from textbook): R : gas constant
T : absolute temperature
$' [&! ]"#$ ( $() [)*! ]"#$ ( $*+ [+,, ]%&
F : Faraday’s constant
Vm = 61.54 log ln : natural logarithm
$' &! %& ( $() [)*! ]%& ( $*+ [+,, ]"#$
Intracellular sodium levels: 15 mM In a case where the membrane is only permeable
Serum sodium levels: 150 mM to potassium:
[' ! ]"#$
Vm = 61.54 mV log
Intracellular potassium levels: 100 mM ' ! %&
Serum potassium levels: 5 mM This is the equilibrium potential for potassium!
)
Intracellular chloride levels: 13 mM EK : 61.54mV x log
*++
Serum chloride levels: 150 mM
EK = -80 mV
Goldman–Hodgkin–Katz equation
!" $' [&! ]"#$ ( $() [)*! ]"#$ ( $*+ [+,, ]%&
Vm = #
ln $ &! ( $ [)*! ] ( $ [+,, ] 𝑖𝑜𝑛 !"# : ionic concentration outside the cell
' %& () %& *+ "#$
𝑖𝑜𝑛 $% : ionic concentration inside the cell
At body temperature (37°C), this simplifies to (from textbook): R : gas constant
T : absolute temperature
$' [&! ]"#$ ( $() [)*! ]"#$ ( $*+ [+,, ]%&
F : Faraday’s constant
Vm = 61.54 log ln : natural logarithm
$' &! %& ( $() [)*! ]%& ( $*+ [+,, ]"#$
Intracellular sodium levels: 15 mM In a case where the membrane permeability to K+
Serum sodium levels: 150 mM is 40 times greater than the permeability to Na+,
and the membrane is not permeable to Cl-:
Intracellular potassium levels: 100 mM
* *)+ ,-+())
Serum potassium levels: 5 mM Vm = 61.54 mV log
* *) ,-+(*++)
Intracellular chloride levels: 13 mM 0)+
Vm = 61.54 mV log -+*)
Serum chloride levels: 150 mM
Vm = -65mV
The equilibrium potential v. resting membrane potential
The equilibrium potential results from The resting membrane potential results
selective permeability of a single ion. from permeability to more than one ion, and
is determined by their relative permeability.
The equilibrium potential can be calculated The membrane potential can be calculated
using the Nernst equation. using the Goldman–Hodgkin–Katz equation.
Neurons are permeable to MORE THAN ONE ION.
Regulating extracellular potassium
Increasing extracellular potassium depolarizes neurons
Consequences:
depolarization: loss of the difference in
charge between the inside and outside
of the plasma membrane
∆ 48mV
Increasing extracellular K+ from 5mM to
50mM causes a 48mV depolarization
10x
Neuroscience 4th Edition, Bear, Connors, Paradiso
Regulating extracellular potassium
Increasing extracellular potassium depolarizes neurons
potassium spatial buffering: regulation of extracellular potassium concentration by astrocytes
This can be accomplished through a number of mechanisms,
including:
1) K+ uptake into astrocytes via K+ channels and
temporarily accumulates.
2) K+ uptake and release into other extracellular areas
containing less K+
Note: the blood-brain barrier also acts as a major defense against increases in serum potassium levels.
Why is it so important that the brain specializes in controlling extracellular
potassium? If astrocytes were unable to keep extracellular potassium in check,
what might be some of the consequences?
Learning outcome: Assess the importance of regulating extracellular potassium
levels and describe mechanisms through which extracellular potassium
regulation is achieved.
Summary
• Ionic concentration gradients are generated and maintained by ion pumps that use energy to
push ions inside or outside the neuron.
• Whereas the equilibrium potential accounts for the permeability of a single ion, the resting
membrane potential is the product of the relative permeabilities of multiple ions.
• The high permeability to K+ ions can have dramatic influences upon the resting membrane
potential and the brain has developed numerous mechanisms to help control extracellular
potassium levels.
