Transcript Channels, Carriers and Pumps - Washington State University

Channels, Carriers and
Pumps
Characteristics of Membrane Channels
• Nonenergetic - protein-lined membrane openings that mediate
downhill flow of ions or molecules (almost) as if they were diffusing
in free solution.
• Selective – most channels prefer one ion species or one family of
molecules, but selectivity varies. This means that some part of the
channel interior must serve as a selectivity filter.
• Usually can open and close (channels that are always open are
called pores) – channels typically open and close spontaneously,
but may also be voltage-gated or chemically gated. This means that
some part or parts of the channel structure must serve as a gate or
gates.
• Some channels serve as receptors for extrinsic chemical messages
– hormones or neurochemical transmitters – these are termed
ionotropic receptors.
Ion channels are electrical conductors
• The current that flows through a single
channel is the product of the
electrochemical driving force (V) and the
single-channel conductance (G).
• Classically, an individual channel was
regarded as having a characteristic
conductance, but a number of channels
are now known that have multiple open
states with different conductances.
Ion channels: ionophores

Gramicidin is an antibiotic obtained from the bacterial species Bacillus brevis

Gramicidin is a peptide of 15 amino acids

Its sequence contains alternatively D- and L-amino acids and the molecule builds a
helix with an inner pore of 0.4 nm in diameter.

Two molecules build a transmembrane, unspecific cation channel through which K+
and Na+ can permeate. The channel is open whenever the two molecules are in
position with each other
Ion channels: Gramicidin A

Let us determine permeation of Na+ through a Gramicidin A channel

We take Fick’s law to calculate the number of Na+ ions crossing the channel.
J  D  A

(c1  c2 )
x1  x2
Suppose DNa is 1.33 cm2 s-1, c1-c2 is 100 mmol/l and that x1-x2 is the thickness of a
membrane (5 nm). After converting all terms to cm, we obtain
3,14  0,2  10 7 cm   1,33  10 5 cm 2  s 1   100  10  6 mol  cm 3 

5  10 7 cm 
2
J Na




J Na  3.31018 mol s 1
J Na  2.0 106 íons s 1
What is measured?

Below models and channel current traces of voltage-dependent K+ and Na+ channels
in an axon.

Na+ channel has two gates and four states, K+ channel has one gate and two states
Gap junction channels

Very unspecific!

Connecting different cells.

Each channel consists of 2 “connexons”. Each connexon consists of 6 connexins.
Each connexin is a polypeptide that crosses 4 times the membrane.

The pore has a diameter of 1.5-2.0 nm

Inorganic ions, water, and many small organic molecules (like amino acids) up to
about 1200 D can pass the gap junction channel.
A weakly specific cation channel

The nicotinic acetylcholine receptor – an example of an ionotropic receptor

Hardly discriminates between Na+ and K+.

Heteropentamer α2βγδ
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Each subunit has 4 transmembrane helices (M1-M4)
There are lots of potassium channels

Many different families of K+ channels with very different structure and function:
•
Delayed rectifier K+-channels
•
Inward rectifier K+-channels
•
Ca++-sensitive K+-channels
•
ATP-sensitive K+-channels
•
Na+-activated K+-channels
•
Cell volume sensitive K+-channels
•
Type A K+-channels
•
Receptor-coupled K+-channels
Sodium channels I

Voltage-dependent Na+ channels:
•
Similar structure to voltage-dependent K+ channels, but here the channel is formed
by one huge protein sequence with 4x6 membrane spanning helices
•
Action potential!
Sodium channels II

Epithelial Na+ channels (not voltage-dependent)
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α2βγ, with each subunit having 2 transmembrane helices
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Important for transepithelial Na+ absorption in tight epithelia (distal nephron, distal
colon, amphibian skin and bladder, freshwater fish gill, other freshwater animals

Important for sensing salt!
Calcium channels


Voltage-dependent Ca++-entry channels
•
L-type (long lasting) Ca++ channel: a1C, a1D, a1F, or a1S, a2d, b3a
•
N-type Ca++ channel: a1A, a2d, b4a
•
P-type Ca++ channel: a1B, a2d, b1b
•
Q-type Ca++ channel: a1A, a2d, b4a
•
R-type Ca++ channel: a1G, a1H, a1I
•
T-type Ca++ channel: a1G, a1H, a1I
Ligand-gated Ca++ channels
Homotetramer complex, 6 transmembrane helices

•
Ca++ release channels: Ryanodine receptors
•
Calcium channel and Inositol-1,4,5-triphosphate (IP3) receptor in ER
•
Calcium channel and receptor of nicotinic acid-ADP (NAADP)
•
Calcium channel and receptor of sphingolipíds
Functions
•
In general cause an increase in cellular Ca++ which is a messenger for many
processes
Anion channels


Different types:
•
Extracellular ligand-gated Cl- channels
•
Cystic fibrosis transmembrane conductance regulator (CFTR)
•
Voltage-gated Cl- channels
•
Nucleotide sensitive Cl- channel
•
Intracellular Cl- channel
•
Calcium-activated Cl- channel
Functions:
 involved in NaCl absorption and secretion across epithelia
 HCl secretion in mammalian stomach
 Cell volume regulation
 Postsynaptic, inhibitory GABA and Glycin receptors
All these channels? How can they be distinguished?

Ion selectivity

Conductance

Pharmacology (Activators/Inhibitors)
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Localization
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Molecular structure

Ion channels: How do they distinguish between
ions?
Selectivity for charge:
•

Negatively charged groups at the “mouth” of the channel can attract cations
and push away anions. Positively charged groups at the “mouth” of the
channel can attract anions and push away cations.
Selectivity for size:
•
The diameter of the pore could determine the size of the particles that can
pass.
•
Interestingly, channels with 6, 5 and 4 transmembrane domains were found:
Gap
junctio
n
Ø = 1.5-2.0 nm
Unspecific
cation channel
nm
 But there is still a problem!
Ø = 0.65 nm
Voltagedependent cation
channels
Ø = 0.3-0.5
Ion channels: How do selectivity filters work?

Why do Na+ ions (rNa = 0.095 nm) not permeate through K+ channels (rK = 0.133 nm)?
Na+
K+

K+ ions permeate through K+ channels without their hydrated shell (“naked”). Amino
acid side groups of the channel protein mimic the presence of the water molecules in
a way that K+ ions can easily give up their hydrated shell and pass through the
channel.

Na+ ions are smaller and their “naked” form is not stabilized by K+ channels.
Together with their hydrated shell Na+ ions are too big to pass K+ channels.
Ion channels: How do they distinguish between ions?

K+ ions travel naked through their channels. Na+ ions travel together with a water
molecule.

Naked Na+ ions are not stabilized in K+ channels. They cannot strip off their hydrate
shell. K+ ions with a water molecule are too big to pass Na+ channels. Their naked
form is not stabilized either.
Distinguishing carriers and
channels
Carrier molecules must interact specifically with
each molecule transported
Carrier saturation

Passive transport by simple diffusion
is described by Ficks law
J  D  K dist 


c1  c 2
xm
Here, the rate is determined by the
gradient
Facilitated diffusion through carriers
does not only depend on the
concentration
gradient
of
the
substrate, but also on the number of
carriers, on their turnover (which
determines Vmax) and on their affinity to
the substrate.
Vmax  (c1  c2 )
J
K m  (c1  c2 )



Carriers show saturation!
Channels show saturation only at very
high concentrations.
Free diffusion across the membrane
saturates only when the membrane
area becomes rate limiting.
Active Transport
• Metabolic energy is spent to drive solutes
against their chemical or electrochemical
gradients
• The driving force may be
– reducing power (H+ transport by ETC)
– ATP (Na+/K+ pump, V-type H+ pump) – we call these
primary active transport
– Transmembrane gradient of some other substance
(frequently Na+), which is the result of primary active
transport – we call these secondary active processes
P-type ATPases

P-ATPases form an intermediate during their reaction cycle in which phosphate is
covalently bound to the ATPase.

P-ATPases are much smaller proteins (less subunits) than V- and F-ATPases and they
have a different mechanism.

P-ATPases make a flip-flop conformational change that exposes ion binding sites to
different sides of the membrane.

They generate transmembrane ion gradients and transmembrane voltages.

In this way they can energize other transporters and, thus, many transport processes.

There are several families of P-ATPases:
•
Na+/K+-ATPase (in almost all animal cells)
•
K+/H+-ATPase (stomach acidification in mammals)
•
Ca2+-ATPase (in plasma membranes and in endomembranes, e.g. ER)
•
K+-ATPase (in plant plasma membranes)
Na+/K+- ATPase

Two subunits

Operates in membranes as dimer (α2β2)


Translocates 3 Na+ ions out of the cell in exchange for 2 K+ ions.
Na+ and K+ distributions across the plasma membrane are kept away from diffusional
equilibrium by the Na+/K+ pump. The energy is provided by hydrolysis of ATP.

Is electrogenic and contributes to membrane voltage (only slightly though: 6-15 mV).

It is a major part of the energy budget of excitable cells, especially small ones.

It is inhibited by specific drugs: ouabain, digitalis and other cardiac glycosides
derived from plants.
The cycle of the Na+/K+ ATPase or Na+/K+ pump
Cotransport and exchange:
gradient-mediated active transport
• Examples of cotransporters:
• NK2C cotransporter (renal tubule) Na+, K+,
2 Cl-, inhibited by furosemide-type
diuretics
• NCC Na+-amino acid cotransporter (most
cells, inc. intestinal cells)
• SGLT Na+ -coupled glucose transporter 2
Na+/glucose (intestine, renal tubule, bloodbrain barrier)
SGLTs (sodium glucose linked transporters) are multifunctional proteins
The SGLT cotransporter
The SGLT Na+/glucose cotransporter has negative ionic binding sites within its
central pore. 2 Na+ from the extracellular fluid first occupy these sites – this
allows glucose to bind to its high affinity site within the pore. When the carrier is
loaded, the pore undergoes a conformational change that both lowers the
affinity of the binding sites for the substrates and presents the substrates to the
intracellular side. This cycle can be repeated perhaps 1000 times/sec at 38oC.
Functional domains within the SGLT1 transporter can be identified
The SFF (sodium-solute symporter) gene family has over 450
members. Of the 11 human genes in this family, 9 have known
functions. Six of them are glucose transporters.
The Na+/glucose cotransporter is energized
by the Na+/K+ pump
Examples of some countertransporters
• Na+/Ca++ exchanger (helps keep intracellular
Ca++ four orders of magnitude lower than
extracellular Ca++)
• Cl-/HCO3- exchanger – transports Cl- into
cytoplasm in exchange for metabolic HCO3• Na+/H+ exchanger – drives exit of metabolic
protons in exchange for Na+; keeps intracellular
pH and HCO3- above their equilibrium values