Channels, Carriers and Pumps

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Channels, Carriers and Pumps
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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.

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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.

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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

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Ion channels Gramicidin A

• Let us determine permeation of Na through a
  Gramicidin A channel
• We take Ficks law to calculate the number of Na
  ions crossing the channel.

• 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

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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

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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.

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A weakly specific cation channel

• The nicotinic acetylcholine receptor an example
  of an ionotropic receptor
• Hardly discriminates between Na and K.
• Heteropentamer a2ß?d
• Each subunit has 4 transmembrane helices (M1-M4)

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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

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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!

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Sodium channels II

• Epithelial Na channels (not voltage-dependent)
• a2ß?, with each subunit having 2 transmembrane
  helices
• 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!

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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

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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

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All these channels? How can they be distinguished?

• Ion selectivity
• Conductance
• Pharmacology (Activators/Inhibitors)
• Localization
• Molecular structure

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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 junction
Unspecific cation channel
Voltage-dependent cation channels
Ø 1.5-2.0 nm Ø 0.65 nm Ø 0.3-0.5 nm

• But there is still a problem!

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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.

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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.

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Distinguishing carriers and channels
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Carrier molecules must interact specifically with
each molecule transported
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Carrier saturation

• Passive transport by simple diffusion is
  described by Ficks law

• 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.

• 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.

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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

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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)

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Na/K- ATPase

• Two subunits
• Operates in membranes as dimer (a2ß2)
• 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.

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The cycle of the Na/K ATPase or Na/K pump
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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, blood-brain
  barrier)

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The Na/glucose cotransporter
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Examples of countertransporters

• Na/Ca exchanger (keeps 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 keeps intracellular pH and
  HCO3- above their equilibrium values