Membranes 2: Transportation Steven E. Massey, Ph.D. Associate Professor Bioinformatics Department of Biology University of Puerto Rico Río Piedras Office & Lab: NCN#343B Tel: 787-764-0000 ext. 7798 E-mail: stevenemassey@gmail.com
Membranes are impermeable to polar solutes Thus, a transport system is required to import and export polar molecules : organisms need to acquire compounds from their environment and to excrete side-products of metabolism or secrete functional molecules In addition, membranes can be used to form a gradient of ions Ion gradients have many functions Eg. in neurons, electrochemical potential for the synthesis of ATP, using a proton gradient for facilitating transport of other molecules
Movement of solutes across a permeable membrane Uncharged molecules are affected by chemistry only Charged molecules are affected by charge and chemistry. This is called an electrochemical gradient. The membrane potential = 0
Different types of protein membrane transporters
Energy changes (ΔG) accompany passage of a charged molecule through an impermeable membrane (lipid bilayer) a) the energy change is high due to removal of water molecules b) the energy change is lower using a transporter because the transporter forms bonds with the ion Transporters promote the process of facilitated diffusion or passive transport
Transporters are of two types: channels and carriers Channels are usually not highly specific, much faster than carriers and are non-saturatable; they are effectively a hole in the membrane Carriers are saturatable, which means that they have a maximum velocity. This implies that they have a specific interaction with the substrate Pumps are a type of carrier that requires energy and are also called active transporters This is necessary when transporting a molecule against a concentration gradient Passive transport occurs along a gradient Eg. the glucose transporter of erythrocytes GLUT1
Different types of transport Ionophores are a third category of transporter that are mobile and lipid soluble
The structure of GLUT1, a passive transporter, facilitates the transport of glucose, which is negatively charged, along a concentration gradient The transmembrane regions are hydrophobic, but contain charged residues
The polar residues (ser, asn, thr) mean that one face of the helix is polar. This is called an amphiphilic helix When they are grouped together, they may form a hole (pore) that forms a polar environment; this has not been experimentally determined yet
Kinetics of glucose transport into erythrocytes Vo = Vmax [S]out Kt + [S]out Vo is the initial velocity of glucose accumulation in the cell [S]out is the concentration of substrate outside the cell Kt is analogous to the Michaelis constant (Km) However, many transporters are not enzymes...
Double reciprocal plot (1/Vo vs 1/[S]out) allows the calculation of Vmax and Kt This derived from an experiment where [S]out is varied and the change in Vo recorded
The process of transporting glucose involves conformational changes in the GLUT1 transporter There are two conformational states; T1 and T2 Glucose binds to the T1 conformation and then converts into T2
There are different isoforms of the GLUT transporter in human tissue. Isoforms are different forms of the same protein; they have different kinetic properties GLUT3 (brain) has a high affinity, which allows glucose to be transported when glucose is low, GLUT2 (liver) has a low affinity, which allows it to be transported when glucose is high
In type I diabetes, insulin in not released in sufficient levels from the pancreas Insulin acts to increase the numbers of GLUT4 glucose transporters at the cell surface - this enhances glucose uptake
Three classes of transport system Transporters differ in the direction and number of molecules transported: Uniporter one direction Symporter two molecules (ore more) in the same direction Antiporter two molecules (or more) in opposite directions
Two types of active transport, which is transport against a concentration gradient a) primary involves the hydrolysis of ATP b) secondary involves the creation of an ion gradient using ATP that drives transport
Na+K+ ATPase is responsible for producing a membrane potential in all animal cells The antiporter transports 3 Na+ ions out and 2 K+ ions in
The transporter results in a membrane potential (net negative charge inside the cell), facilitates transport and regulates cell volume Na+K+ ATPase is a P-type ATPase These are cation transporters that are reversibly phosphorylated
Glucose transport in intestinal epithelial cells utilizes secondary active transport to enter the cell using a gradient created by Na+K+ ATPase and a Na+-glucose symporter Then the GLUT2 uniporter is utilized
F0F1 ATPase is responsible for pumping protons across a membrane, creating a proton gradient. Fo spins in relation to the rest of the transporter It is an F type ATPase, which hydrolyze ATP in order to transport protons
Proton gradients can be used to generate ATP in oxidative phosphorylation and photophosphorylation The enzyme is called ATP synthase and the reaction is opposite to the previous slide
Aquaporins are channels that allow the movement of water This is important for the regulation of cell volume They facilitate changes in volume, which is regulated by changes in osmolarity Ion channels allow the rapid movement of ions across membranes They differ from ion transporters in the following way: 1) movement is more rapid 2) they are not saturatable 3) they are gated ie. opened or closed in response to an event Ligand gated channels open in response to binding of a ligand eg. a neurotransmitter such as serotonin Voltage gated channels open in response to a change in Vm
An example of a ligand gated channel is the nicotinic acetylcholine receptor This facilitates an electrical signal from a neuron to muscle via the neuromuscular junction On binding to acetylcholine it allows the entry of Na+, K+ and Ca2+ into the cell An example of a voltage-gated channel is the voltage gated Na+ channel of neurons Membrane depolarization causes the channel to open, allowing Na+ to enter the neuron promoting the action potential to move along the neuron
Defects in membrane transporters can cause disease Cystic fibrosis is due to a mutation in a gene called cystic fibrosis transmembrane conductance regulator (CFTR) This is a Cl- channel A mutation leads to a reduced export of Cl-, this leads to diminished export of water from the cell and a sticky membrane surface Mucus accumulates in the lungs as a consequence, which can lead to bacterial infections
The three states of CFTR There are two segments, each with six transmembrane helices When the R domain is phosphorylated and no ATP is bound to the Nucleotide Binding Domain (NBD), the channel is closed When ATP binds to the NBD, the channel opens When the R domain is unphosphorylated, it prevents binding of ATP and so remains closed Cystic fibrosis usually results from a mutation in F508, which leads to misfolding and reduced transport of Cl-