BIOL 408 Ch. 11 Plasma Membrane Permeability PDF

Chapter 11 -​ Plasma membrane permeability -​ The concentration of NaCl in the extracellular fluid of animals is >150 mM -​ Na+ concentration in cytosol is tenfold lower -​ In contrast, K+ concentration is higher in cytosol -​ Proton concentration in the lysosom...

Chapter 11 -​ Plasma membrane permeability -​ The concentration of NaCl in the extracellular fluid of animals is >150 mM -​ Na+ concentration in cytosol is tenfold lower -​ In contrast, K+ concentration is higher in cytosol -​ Proton concentration in the lysosome interior is about 100-fold greater -​ Only a few gases and uncharged, small, water-soluble molecules can readily diffuse across a pure phospholipid bilayer -​ CO2, N2, O2 -​ Ethanol -​ Water, urea (Slightly permeable) -​ Membrane transport proteins -​ ATP-powered pumps -​ 100-103 ions/s -​ Ion Channels -​ 107-108 ions/s -​ Ex. Aquaporins -​ 3 Groups of transporters (102-104 mol/s) -​ Uniporters -​ moves one substance at a time -​ Facilitated diffusion -​ Ex. GLUT1 -​ Symporters -​ transports two different substances in the same direction -​ Ex. Na+/glucose symporter -​ Antiporters -​ moves two different substances in different directions across the membrane -​ Ex. Na+/proton antiporter -​ GLUT1 transport -​ Binding of glucose to the outward-facing site triggers a conformational change in the transporter. -​ As a result of the conformational change, the binding site now faces toward the cytosol. -​ Glucose is then released to the inside of the cell. -​ Finally, the transporter regenerates the outward-facing binding site. -​ Osmotic pressure -​ Osmosisis one of the four main types of passive transport. (Other three: simple diffusion, facilitated diffusion and filtration.)  -​ Spontaneous movement of molecules through a selectively-permeable membrane from a region of high water potential to a region of low water potential -​ Hydrostatic pressure required to prevent net water flow -​ Aquaporin -​ Expression of aquaporin by frog oocytes increases their permeability to water -​ Aquaporins are water-selective channels that increase water permeability in cell membranes, driven by osmotic gradients. -​ Frog oocytes microinjected with aquaporin mRNA swelled and burst in a hypotonic solution, indicating that aquaporins are water-channel proteins. -​ AQP1 transports 3x109 water molecules per subunit per second -​ Impermeable to ions -​ Can transport water, glycerol, and urea -​ Four classes of ATP-powered transport proteins -​ P-class pumps: ​ Transport ions across membranes (e.g., Na⁺/K⁺ ATPase, Ca²⁺ ATPase). ​ Involved in active transport, using ATP to move ions against concentration gradients. ​ Key feature: Catalyze autophosphorylation of a key conserved Aspartate (Asp) residue within the pump during the transport cycle. -​ V-class pumps: ​ Primarily found in vacuolar membranes (e.g., in lysosomes and endosomes). ​ Pump protons (H⁺) into vacuoles or organelles, establishing acidic environments. ​ Key feature: Use ATP to pump protons, not involved in phosphorylation. -​ F-class pumps: ​ Found in mitochondria, chloroplasts, and bacterial membranes (e.g., ATP synthase). ​ Function in both proton pumping (via the proton gradient) and ATP synthesis. ​ Key feature: Can synthesize ATP using the proton gradient (reversible). -​ ABC superfamily: ​ Transport a wide range of molecules, including ions, lipids, and drugs. ​ Includes multidrug resistance proteins (MDRs) and other transporters. ​ Key feature: Utilize ATP binding and hydrolysis for molecule transport, without forming a phosphorylated intermediate. -​ Ca²⁺ in Skeletal Muscle Cells ​ Storage: Ca²⁺ ions are concentrated in the sarcoplasmic reticulum (SR).  ​ Concentration in cytosol: ○​ Resting cells: 10⁻⁷ M (100 nM). ○​ Contracting cells: 10⁻⁶ M (1 µM). ​ Concentration in SR lumen: 10⁻² M (10 mM). -​ Muscle Contraction & Relaxation ​ Contraction: Release of stored Ca²⁺ from the SR into the cytosol causes contraction. ​ Relaxation: The Ca²⁺-ATPase in the SR membrane pumps Ca²⁺ from the cytosol back into the SR, promoting relaxation. -​ Ca²⁺-ATPase Mechanism ​ P-Class Ca²⁺-ATPase: ○​ Located in the SR membrane of skeletal muscle cells. ○​ Two Ca²⁺-binding sites in the membrane-spanning domain. ○​ E1 conformation: Ca²⁺-binding sites face the cytosolic side and have a high affinity for Ca²⁺. ○​ ATP binding and hydrolysis: ATP binds at the cytosolic side, and hydrolysis results in phosphorylation of an Aspartate (Asp) residue. ○​ Asp~P bond: The phosphorylated intermediate (E1~P) is a high-energy acyl phosphate. ○​ Conformational change: Phosphorylation causes a shift to the E2 conformation, enabling the transport of Ca²⁺ back into the SR. ○​ Dephosphorylation: Dephosphorylation of Pi causes conformational change back to E1 -​ Na+/K+ ATPase -​ Steps in the Na⁺/K⁺-ATPase Pump Mechanism: ​ Step 1: ○​ Na⁺ binding: Three Na⁺ ions bind to the α-subunits of the enzyme, which contains six binding sites for Na⁺. ​ Step 2: ○​ ATP binding and hydrolysis: The binding of Na⁺ changes the local polarity, enabling ATP to bind to the α-subunit. ○​ ATP is then hydrolyzed to ADP, and a phosphate (P) is transferred to a conserved Asp residue on the α-subunit. ○​ The phosphorylation causes a conformational change in the α-subunit: the inside cavity closes, and the outside cavity opens. ​ Step 3: ○​ The three Na⁺ ions are released from the inside of the cell to the outside, as they are weakly bound. ​ Step 4 & Step 5:  ○​ K⁺ binding: Two K⁺ ions from the outside bind to the open cell cavity. ○​ Dephosphorylation: The phosphate group is removed (dephosphorylation). ○​ This results in another conformational change, allowing the two K⁺ ions to move inside the cell with the help of the β-subunit. ​ Step 6: ○​ The two K⁺ ions are released inside the cell, and the enzyme returns to its initial configuration. -​ Additional Key Points: ​ Ion exchange: The pump transports three Na⁺ ions out of the cell and two K⁺ ions into the cell. ​ Electrogenic effect: This charge difference creates an electrical potential across the cell membrane, contributing to the membrane potential. -​ V-Class ATPase Mechanism: ​ Transport of H⁺ ions: V-class ATPases transport only H⁺ ions (protons). ​ Function: These pumps acidify the lumen of lysosomes, endosomes, and plant vacuoles. -​ Key Features of V-Class ATPases: ​ Proton Gradient Maintenance: ○​ They maintain a 100-fold or more proton gradient between the lumen and cytosol: ​ Lysosomal lumen: pH 4.5-5.0. ​ Cytosol: pH 7.0. ​ ATP-powered: V-class H⁺ pumps are powered by ATP to pump protons across membranes. ​ Structural similarity to F-Class Pumps: ○​ V-class pumps are structurally similar to F-class proton pumps, but they operate in the reverse direction. ○​ F-class pumps generate ATP by pumping protons, while V-class pumps use ATP to pump protons. -​ Effect on Membrane Potential and pH: ​ Electrical Potential: ○​ For each H⁺ ion pumped across the membrane, a negatively charged ion (e.g., OH⁻ or Cl⁻) is left behind on the cytosolic side. ○​ This causes the cytosolic side to become negative and the luminal side to become positive, creating an electrical potential. ​ Effect on pH: ○​ If the organelle only has V-class pumps, H⁺ pumping generates an electrical potential without significant change in intraluminal pH.  ○​ If the organelle also contains Cl⁻ channels, Cl⁻ ions passively follow the pumped H⁺, resulting in a low luminal pH and no significant electric potential across the membrane. -​ ABC Transporters ​ Function: Use ATP to transport substances across membranes. ​ Structure: Consist of two transmembrane domains (T) and two ATP-binding domains (A). Each T domain is built of 10 membrane-spanning a-helices ​ Bacterial Permeases: Import various nutrients from the environment. ​ Multidrug Resistance: ABCB1 (MDR1) is involved in drug resistance by expelling toxins. ​ CFTR: An ABC transporter-class ion channel that conducts chloride ions and consumes ATP during operation. -​ CFTR (Cystic Fibrosis Transmembrane Regulator) Overview: ​ Class: CFTR is an ABC transporter ion channel or ATP-gated chloride channel (not a pump). ​ Structure: ○​ Composed of two transmembrane (T) domains and two ATP-binding (A) domains (like other ABC proteins). ○​ Contains an additional R domain on the cytosolic face, which links the two homologous halves of the protein. ​ Function: ○​ CFTR is crucial for the reuptake of chloride ions lost during sweating. ​ Channel Regulation: ○​ The CFTR chloride channel is normally closed. ○​ Opening is triggered by phosphorylation of the R domain by a protein kinase (PKA). ○​ ATP binding: Requires the binding of two ATP molecules to the A domains for the channel to open. -​ Ion Selectivity -​ P segment of pore forms ion-selectivity filter ​ K+ Ions: ○​ Lose their bound water molecules. ○​ Become coordinated with 8 backbone carbonyl oxygens from conserved amino acids in the P segment. ○​ The positively charged S4 serves as the primary sensor. ​ Na+ Ions: ○​ Have a tighter water shell that cannot coordinate perfectly with the channel's oxygen atoms. ○​ Pass through the channel rarely. ​ Ion Selectivity:  ○​ The ability of the K+ channel to select K+ ions is due to the backbone carbonyl oxygens from the Gly-Tyr-Gly sequence in the P segment. -​ Patch clamping ​ Purpose: Allows measurement of opening, closing, regulation, and ion conductance of a single ion channel. ​ Method: ○​ A patch electrode filled with a current-conducting solution is applied to the plasma membrane (PM). ○​ Slight suction is applied to form a seal with the membrane for accurate measurements. -​ Two-Na+/one-glucose symporter ​ Function: Used by cells (e.g., small intestine and kidney tubules) to import glucose against a concentration gradient by coupling it with the import of two Na+ ions. ​ Steps: 1.​ Binding: Na+ and glucose simultaneously bind to the outward-facing conformation of the protein. 2.​ Conformational Change: Binding causes a conformational change, temporarily occluding the substrates (unable to dissociate). 3.​ Inward-facing Conformation: The protein assumes an inward-facing state. 4.​ Dissociation: Na+ and glucose dissociate into the cytosol. 5.​ Reversion: The protein reverts to its outward-facing conformation to repeat the cycle. -​ Transcellular transport of glucose from the intestinal lumen into the blood -​ Dissolution of Bone by Osteoclasts: ​ Function: Osteoclasts dissolve bone for bone remodeling, which helps in repairing damaged bones. ​ Process: ○​ Polarized osteoclasts form tight seals with the bone, creating an enclosed extracellular space. ○​ Carbonic anhydrase catalyzes the conversion of CO2 and H2O into bicarbonate and H+. ​ Key Components: ○​ V-class Proton Pump: Pumps H+ into the extracellular space, acidifying the enclosed area. ○​ ClC-7 Chloride Channel: Facilitates Cl- diffusion to maintain electroneutrality in the space. ○​ Cl-/HCO3- Antiporter: On the opposing membrane, this maintains cytosolic pH by exchanging HCO3- for Cl-. ​ Outcome: The combined operation of these proteins and carbonic anhydrase acidifies the space and aids in bone dissolution.