MCB 110 - Spring 2008 - NogalesTransport 1V Membrane Potential and Nerve ImpulsesSuggested Reading: Lodish, Chapter 7Alberts, Chapter 11MCB 110 - Spring 2008 - NogalesTransport 2MCB 110 - Spring 2008 - NogalesTransport 3MCB 110 - Spring 2008 - NogalesTransport 4MCB 110 - Spring 2008 - NogalesTransport 5ENERGETICS OF SOLUTE MOVEMENT• Movement into the cellG = RT lnCi[]Co[]= 2.3RT log10Ci[]Co[]At room temperature, T = 25 ˚CG =1.4 kcal / mol log10Ci[]Co[]If Ci[]= Co[]G = 0Equilibrium!!If Ci[]< Co[]G < 0 Termodynamically favorableto move into the cell!Example:Ci[]=110Co[]G = 1.4kcal / molAs solute moves in Ci[] until Ci[]= Co[]G = 0 Equilibrium!!MCB 110 - Spring 2008 - NogalesTransport 6• If the solute has charge: Electrochemical GradientG = 2.3RT log10Ci[]Co[]+ zFEwhere: z is the charge number F is a constant, 23.06 kcal/mol.V E is the electric potential (V)The electric potential at the plasma membrane is about –70 mV+ + + + + + + + + + out+________________________ membrane- - - - - - - - - - - in -If Na+[]i=Na+[]o10 thenG = 1.4 kcal /mol + (1)(23.06 kcal/ mol.V)(0.07V)= 1.4 1.7 = 3.1kcal /molMCB 110 - Spring 2008 - NogalesTransport 7MCB 110 - Spring 2008 - NogalesTransport 8II C – Pores and Ion ChannelsSolutes diffuse through hydrophilic channels in integral membrane proteins• Aquaporin – Waterchannel that enhanceswater diffusion. Mostabundant in kidneywhere it is regulatedby the hormonevasopressin.Tetramer, with onewater channel permonomer. Theselectivity filter ishydrophobic exceptfor a few water-binding polar residues.MCB 110 - Spring 2008 - NogalesTransport 9• Ion Channels Essential for nerve impulse, secretion, muscle contraction, regulation ofcell volume, etc. Highly selective, bi-directional (down electrochemical gradient),regulated (voltage, phosphorylation, ligands) Recent technological development in the study of ion channels: Cloning: sequence, structure prediction, mutagenesis Incorporation into host cells (frog oocytes) Studies in liposomes (controlled environment) Conductance studies by patch clampingMCB 110 - Spring 2008 - NogalesTransport 10MCB 110 - Spring 2008 - NogalesTransport 11MCB 110 - Spring 2008 - NogalesTransport 12MCB 110 - Spring 2008 - NogalesTransport 13The channel is formed by a tetramer ofidentical monomers forming a pore in thecenter.Each monomer has 6 transmembrane helicalsegments (S1 to S6),Between the last two helices there is a highlyconserved region, the P segment, involved inthe formation of the ion selectivity channel inthe tetramer.Helix S4 has a high content of basic residuesthat is used as a voltage sensing mechanism.Upon depolarization of the cell membrane theS4 helix moves up toward the outside causinga conformational changes that opens thechannel.At the N-terminus a globular, cytosolicdomain is thought to plug the channel centralcavity during inactivation (“ball and chain”hypothesis).MCB 110 - Spring 2008 - NogalesTransport 14There are a number of different voltage-gated K+ channel that open atdifferent voltages. They are regulated by phosphorylation through theaction of hormones.Ball and Chain Model. Patch clamping experiments with mutants lacking the residuesforming the “ball” show permanent activation. This phenotype is rescued by adding asynthetic “ball” peptide.MCB 110 - Spring 2008 - NogalesTransport 15MCB 110 - Spring 2008 - NogalesTransport 16MCB 110 - Spring 2008 - NogalesTransport 17MCB 110 - Spring 2008 - NogalesTransport 18MCB 110 - Spring 2008 - NogalesTransport 19Voltage-gated Ion Channels – Most or all of voltage-gated channels have a conservedarchitecture and mode of action that indicate they may have evolved from a commonancestor.Voltage-Gated Na+ Channel MechanismMCB 110 - Spring 2008 - NogalesTransport 20MCB 110 - Spring 2008 - NogalesTransport 21MCB 110 - Spring 2008 - NogalesTransport 22MCB 110 - Spring 2008 - NogalesTransport 23In humans there are 5 different isotypes ofthe glucose transporter.Insulin is a hormone involved in the control ofsugar levels in the blood. Insulin-responsivecells (muscle, adipocytes) contain the isoformGLUT4 in vesicles that are exocytosed asinsulin concentration goes up.Type I diabetes is caused by low insulinproduction, while Type II diabetes is causedby failure of the insulin receptor or theGLUT4 transporter.MCB 110 - Spring 2008 - NogalesTransport 24IV Active TransportMCB 110 - Spring 2008 - NogalesTransport 25MCB 110 - Spring 2008 - NogalesTransport 26MCB 110 - Spring 2008 - NogalesTransport 27MCB 110 - Spring 2008 - NogalesTransport 28MCB 110 - Spring 2008 - NogalesTransport 29Ca2+-ATPase - Present in the plasma membrane and ER. Pumps Ca2+ out of the cytoplasm.H+/K+- APTase - Responsible for secretion of acid in the stomach.MCB 110 - Spring 2008 - NogalesTransport 30MCB 110 - Spring 2008 - NogalesTransport 31MCB 110 - Spring 2008 - NogalesTransport 32MCB 110 - Spring 2008 - NogalesTransport 33MCB 110 - Spring 2008 - NogalesTransport 34MCB 110 - Spring 2008 - NogalesTransport 35IV B – CotransportCoupling of transport to existing ion gradientsNa+/Glucose cotransporter – Activates transport of glucose in the intestine.• Binding of 2 Na+ and 1 glucose on the outside• Na+ release on the inside (lower concentration)• Conformational change• Loss of affinity for glucose• This symporter can pump glucose against a concentration gradient greater than 20,000 foldMCB 110 - Spring 2008 - NogalesTransport 36MCB 110 - Spring 2008 - NogalesTransport 37MCB 110 - Spring 2008 - NogalesTransport 38V Membrane Potential and Nerve ImpulsesIon concentrations gradients and selective movement of ions through channels createsa difference in voltage across the plasma membrane of ~ -70 mV, with the inside ofthe cell being always negative with respect to the exterior. In nerve and muscle cellsthis potential is subject to large changes during impulse transmission that are due tothe movement of a small percentage of the ions in and out of the cell.V A – Resting PotentialThe Na+/K+ A TPase generates large concentrationgradients for these ions. In the resting state only K+leaking channels a re open (most Na+, C l- and Ca2+ areclose). The movement of K+ down theirelectrochemical gradients, thus, out of the cell,creates a n et negative charge inside and positivecharge outside that results in t he resting electricpotential of –70
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