BISC 421 1st Edition Lecture 6 Outline of Current LectureI. Ion Channels ContinuedCurrent LectureIon Channels (continued) ‐Transporters Another mechanism that allows the movement of other ions across the membrane (usually larger) •These aren’t channels but they are proteins that need to use energy to transport a molecule across the membrane-‐ against concentration gradient •Ex. For potassium these transporters move potassium from out to in (against gradient) •Use energy to “pump” the molecule acrossTransport Proteins•Two main categories•First: ATPase or a “pump”o Hydrolyzes ATP to gain energyo Na+/K+ ATPase moves Na+ and K+ against concentration gradients (Na+ in to out and K+ out to in)o Ca2+ pumpo Need a molecule of ATP•Second: Ion exchanges which use the energy from moving one molecule to drive the movement of another molecule (use concentration gradients)•Concentration of Na+ is 10-20X greater out than in cells. Reverse for K+. Gradients are maintained by the plasma membrane Na+/K+-ATPase.Each cycle moves 3 Na+ out and 2 K+ in. Electrogenic: a net + charge across membrane. Cell negative inside.Look above-‐ use ATP to move these against concentration gradients•These are crucial to maintain gradient•3 Na+ out for every 2 K+ in-‐ cause an electrical potential across the membrane-‐ charge formed by this•The protein has the binding sites for Na+ and K+ in the protein-‐ special conformations•Becomes selective because it can change its conformation•Has ATP binding site (where hydrolysis of ATP occurs) and the phosphorylation site(transfer of a phosphate group-‐ negative charge changes the shape and conformation)Steps in ion movement c y cle.1. Na+ and ATP binds to the intracellular side.1. Increases ATPase activity.2. Hydrolyses ATP to ADP.2. The hydrolysis leads to phosphorylation.1. Binding of thenegatively chargedphosphate causesa conformationchange in theprotein3. Now Na+-binding site is extracellular1. Na+ ions are now released.2. K+ ions can now bind.4. The ATPase is then dephosphorylated.1. Dephosphorylation dependent upon K+.2. The conformation reverts to original state.3. Now K+ -binding siteis intracellular and the ions are released.•4 Basic Phases:•1. Inverted horseshoe-‐ conformation is such that the pockets are pointed inside the cell.Na+ ions will bind to the protein (the affinity for Na+ at this stage is very high and affinity for K+ at this point is very low). .•2. ATP will also bind to the intracellular sites and then the ATPase pump becomes active which will hydrolyze ATP to ADP prhosphorylating the protein and changing the conformation•3. The protein is now flipped to the outside and the affinity for Na+ is reduced and Na+is released. Affinity for K+ now becomes high. 2 K+ molecules bind•4. ATPase becomes dephosphorylated and then the conformation is switched back again making the affinity for K+ to decrease and K+ is released into the cell. The cyclecontinues from here. Net difference is the cell becomes just a little more negativeSlide 14-‐Na+/K+ pump can influence membrane •hyperpolarization occurs due to this pump-‐ making the cell more negative•Ca2+ has an important role in synaptic activity •Ca2+ ATPase helps get rid of Ca2+•Binds Ca2+ binds ATP phosphorylationchange confomation release Ca2+ to diffuse out into the ECF change conformationcycle continuesThree types of transporters.Ń Uniporters transport single molecules down a gradient.x Glucose, amino acids.Ń Symporters and antiporters couple twomoleculesx One against its concentration gradient, one down its gradient.x Also called cotransporters.y Neurotransmitters and their pre- cursors are taken into the nerve or to a vesicle via a transporter.Ń Exciatory amino acid transporter (EAAT)Ń Vesicular Glutamate transporter (VGLUT)Ń Vesicular inhibitory amino acid transporter(VIATT).Ń Glycine transporterAlso maintain ion gradients in vesicles.(VIATT).Ń Glycine transportery Also maintain ion gradients in vesicles•Do NOT use ATP•Energy is from coupling gradients of one molecule versus the gradient of another molecule•Uniport: move one molecule-‐ glucose transporter. Binds glucose at a high concentration and will move the molecule down concentration gradient•Symport/antiport: use the energy of one gradient to move another moelecule againstconcentration gradient. One molecule is moving down its concentration gradient and the other is moving against•Na+/neurotransmitter transport uses the concentration gradient of Na+ to move NTagainst concentration gradient‐Topology of principal subunits of voltage gated Na+ and K+ channels•Selective for an ion-‐ does size make a difference? Na+ is a little smaller than K+ so why can’t it go through K+ channels?•How do they sense voltage? And how do they inactivate?•K+ channel: Beta subunits are accessory subunits so ignore-‐ focus on the Alpha subunits- ‐the parts that span the membrane. The yellow one is the part of the protein that has all of the positive charge in it. The positive charge senses the membrane potential changeo An intact protein has four monomers coming together to form a functionalchannel•Na+ channel: more complex-‐ as opposed to being four monomers it is actually one protein with four domains that resemble just one of the monomers of the K+ channelOther channel types•There are Ca2+ voltage gated channels-‐ similar structures as Na+•The others may not have voltage sensing domains•There are also channels that are non gated‐Topology of Voltage-‐gated Na+ and K+ channelsK+-channels are tetrameric proteins. 6 transmembrane (S) domains and 1 P-segmentS4 contains many (+)-charges residues.x Each is structurally like the K+- channel subunits.Only one inactivating domain.Between the III & IVtransmembrane domains.The analogous S4 domains are the voltage sensors.•The Na+ ones are linked into one protein•Within each of these though they have similar regions-‐ 6 transmembrane domains, in the 4th one there is the positively charged residues•In the green area this is the part of the protein called the pore domain-‐ where the selectivity for the ion occurs-‐ between 5 and 6. Only have one