Membrane Transport 1.The question: How does a cell Membrane serves as both “barrier” and “gate” for communication between the outside and inside of the cell (or among organelles. Lipid bilayer—barrier, transport proteins—gates. 2. Permeability of “lipid bilayer” and a “cell membrane” Biologically important molecules: bilayer membrane Polar small: water, glycerol, oxygen Polar large: amino acids, sugars, nucleotides Ions: K+, Na+, Ca2+, Cl-, NO3- etc Non-polar small/large: phenolics, lipids, steroids + +++

3. Membrane Transport 1)Carriers and channels: carriers function like enzymes that bind small molecules and release to the other side of the membrane (“mechanical hands”); channel is a aqueous pore formed by membrane proteins (“tunnel”). 2) Passive and active transport: transport down the electrochemical gradient across the membrane –without the need of immediate energy requirement (ATP) (passive); or requires ATP and transport against he electrochemical gradient (active). For uncharged molecules just mean concentration gradient. Out in

4. ATP-driven carriers or pumps The carrier protein is an enzyme called ATPase that hydrolyzes ATP to get needed energy for transport. 1) Example/model: 3 Na+ ATP ADP P 2 K+ P

Summary: ATP-dependent; conformational change powered by reversible phosphorylation (at aspartate residue forming a high-energy intermediate); conformational changes generate binding sites for Na/K and “movement” associated with the translocation of the ions. This example Na/K pump is only found in animals but not in fungi and plants! 2) Plant pumps a.H-ATPase: the most important “active” transporter that produce a proton gradient and maintain the membrane potential for other secondary transport (across PM). pH gradient between inside and outside of the cell. Membrane potential: charge Difference across the membrane For plant cells: typically around –120 mV Meaning more negative inside the cell Amino acids Other organic acids

A lot of secondary transport is dependent on H+-gradient Amino acids(symport) And Anions Cations (channels)

b. Vacuolar H+-ATPase: A complex molecular Machine that consists of 13 distinct subunits. It pumps H+ into the Vacuole from cytoplasm. The two H-ATPases in PM or tonoplast take Care of the H+ in the Cytoplasm and pump them into the “inactive” Space and keep the pH Neutral in the cytosol.

c. Other pumps: Ca2+-ATPases in the PM, ER, vacuole These are the pumps that like the H-ATPases keep the Ca2+ concentration in the cytoplasm low by pumping Ca2+ into the cell wall, ER, or vacuole. Very important because Ca2+ serves as a signal for many processes—discuss in later sessions. 5. Ion channels: structure and function Ion channels conduct ion flows down the electrochemical gradient and is considered as “passive” transport. 1)General properties: a) selectivity b) gating: open / close (like a door) 2) Voltage- or ligand-gated channels: the gate/door is operated by voltage or ligand (chemical binding to the channel protein) 3) Voltage-gated channels from plants: K+ channels a) Identification of the first plant ion channel KAT1 and AKT1—the yeast and oocyte model system

Yeast mutant that cannot survive on low [K+] medium---transform this mutant with a cDNA library that represents all possible genes----select the mutant cells that can survive the low [K+] medium---isolate the plant cDNA inside the yeast cells---likely represent the gene coding for K+ transporter. One of such clones was KAT1—standing for K+ transporter of Arabidopsis thaliana. KAT1 expression in the yeast rescued the mutant on low [K+] medium. Interestingly, it turns out to be a voltage-gated K+ channel like those found in our nerves!

b) Characteristics of voltage-gated K+ channels Tetrameric complex and each subunit has: ---6 transmembrane domains (one subunit) ---voltage sensor (voltage sensing) ---pore domain (for K selectivity) Originally discovered in Animals And KAT1 has all these Elements! The structure is solved at atomic level

c) Functional Analysis Oocyte expression And patch-clamp Involves: --microinjection of mRNA --recording of electrical Current across the membrane ---analysis of the current

This shows that the channel conduct both inward and outward current—the ions can flow both ways depending on the membrane potential (voltage) given by the machine.

Results: The K+ current conducted by KAT1: *rectifying inward current---unidirectional influx of K+ when the membrane voltage is negative enough (more negative than – 100 mV)—this is consistent with the voltage gating theory. **K-selective: not permeable to other monovalent cations such as Na+. This is consistent with the Selectivity property. D) Functions Transport and signaling

The journey of K+: Soil---root epidermis (inward channels)---root cortex---endodermis--- xylem cells (outward channels)---xylem vessels --transpiration stream/mass flow---leaf xylem---mesophyll cells (inward/outward channels)---back to phloem and recycling

6. Water channels---new concept on water permeability Earlier idea: lipid bilayer is somewhat permeable to water New idea: water transport across Membrane is facilitated by channels Discovery of water channels In plants: The most abundant protein in vacuole membrane Turns out to be an ion-channel -like molecule that conduct water And glycerol in oocyte system. Make the oocytes burst due to Excessive water uptake! Not only vacuole but plasma membrane also has them./

Atomic structure of a water channel from red blood cells It is formed by 4 subunits (like the K-channel)