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. 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

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 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) α2βγ, 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!

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) Localization 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 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!

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 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.

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, blood-brain 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