Figure 7 a. Secondary transporters make up the largest subfamily of transport proteins. TAGI 2000. Nature 408, 796 1. Na+- or H+-coupled cotransporters - Secondary active transport 2/7-02 Energy released from downhill movement of Na+ (or H+) is enough to accumulate large conc gradient of S. Examples: a) Bacteria and yeast take up ions and metabolites by H+-coupled symport. b) Plant vacuoles accumulate S using H+-coupled antiporters c) Glucose is accumulated in polarized epithelial cell via Na/glu symport. 2. Methods to study cotransporter. Evidence for a H+-sucrose symporter in leaf PM. a) A ph gradient drives sucrose uptake. Or a Na gradient drives glucose uptake. b) A gene/protein is identified by functional expresssion in yeast mutant c) Transport assays verify protein activity d) Cotransporter has 12 TM domains 3. Regulation of cell volume: Water movement. 4. Channels: a. Measure channel activity in cells and patches with patch clamp method b. Study cloned genes by expression in frog oocyte c. Structure of channels d. Functional domains -regulation 15-25. Transport of glucose across epithelial cells: a. Gluc/Na cotransport; b. Gluc transporter 11-12 Alberts 11-10. Model of 2Na/ 1 Glu cotransporter Simultaneous binding model. Binding of both Na and Glu to sites causes conformational change. 15-18. Na-coupled Glucose transporter Lactose H+ Modelmechanism of bacteria lactose permease Most secondary transporters have 12 TM. 1
15-22. Plant vacuole takes up ions and metabolites actively with antiporters Vac ph 5 Problem: 1. How can you show whether glucose uptake into a cell is passive or dependent on a Na gradient? 2. How can you experimentally show whether active Ca uptake into a vacuole depends on a H+ gradient or on ATP? 1. Cells control and maintain their osmotic conc and their volume As cells increase in volume during growth, how do they maintain their osmotic conc? A. Take up more ions/solutes during growthusing pumps. Cotransporters and channels b. Take up water Some cells will lyse in dilute solution. E.g. red cells some cells do not. Why? Plant, yeast, bacteria have rigid walls, so they do not burst in hypotonic solutions. 2. Water moves from a region of high water potential to low water potential. I.e. water moves passively Cells control their osmotic conc and cell vol. 15-32. Evidence for a water channel protein (aquaporin) Microinject CHIP 28 mrna into oocyte Transfer cell from 0.1 M --> 0.035 M Why is control oocyte unchanged? control 15-33 4 subunits form a functional water channel 18-8. Stomata. Open and closed state 2
15-34. Opening and closing of stomates regulate CO 2 uptake for photosynthesis. Guard cell movement is regulated by pumps, cotransporters, and Why are ion channels important? Conduct ions extremely rapidly Can be opened, closed or inactivated Change membrane electric potential locally Change local ion conc [Ca] fast Therefore they provide a fast signal that cells can sense and respond. Channels are integral to many signal transduction pathways. Channels are also important for ion/nutrient distribution [uptake or efflux]. Channels : for nutrient uptake and for signaling 21-8. Channels are regulated by several mechanisms: 21-9 Origin of resting potential: K+ leak via open K channels generate a resting potential, - 70 mv inside. Conduct primarily ions: Na, K, Ca, or Cl Mechanically gated- e.g. osmotic Na+ influx will depolarize the cell Cl- influx will hyperpolarize the cell Methods 1. Intracellular electrode- measure cell potentials. Vary voltage and measure current: to show how membrane potential controls the opening or closing of ion Measures ion flow through the sum of many channels in the cell membrane. 2. Patch clamp method: detects ion flow through a single channel. Voltage clamp- to measure how membrane potential control opening or closing of one channel. +ligand or modulators in pipet or solution. 3. Heterologous expression of mrna in oocyte followed by patch clamping to test channel actviity. 4. Genetic approach: screen for functional rescue in yeast 21-19. Patch clamp method is used to measure current through a single channel. 3
21-20. Patch clamp method: Four configurations Single Voltage gated Na channel Shift in membrane potential Single Na channel opens Sum of 144 single channels 11-31 Alberts 21-21. Membrane patch Na (in) channel How do you identify a channel protein and determine ion specificity? 15-23. Test channel activity after expression in frog oocyte. K (out) channel: a voltage gated channel What does the result show? Test specificity by changing ion in patch pipet. Mutate residues to identify functional domains 21-26. K+ channel inactivation by N terminal region 21-24. Voltage-gated channels have similar structures. Functional regions of K+ channel Discovered in shaker mutant of fly Block pore 4
21-27. Two types of Shaker K+ channel is a tetramer 11-26. Neurons sense, conduct, and transmit signals as nerve impulses Target cell How? By an action potential Action potential arise when Na channel opens and closes; delayed K channel opens/closes 11-37. How a nerve impulse stimulates a muscle cell to contract Review: 1. You can measure glucose uptake into an animal or plant cell, but you do not know if this is active or passive, and the type of transport protein. How would you solve this? Working hypotheses? Approach and methods:? 2. A yeast mutant can grow on 100 mm K but not on 7 mm K+. You identify the mutated gene and obtain the wild type cdna. How would you proceed to identify the function of this gene X? Working hypothesis? Approach and methods? 5