Passive Transport Through Cell Membranes - Péter Hajdu 2023 - PDF

Passive transport through cell membranes Péter Hajdu, 2023 Based on the lecture by Prof. Gábor Szabó and Prof. György Vereb  This lecture: lecture slides marked by ! contain basic information absolutely...

Passive transport through cell membranes Péter Hajdu, 2023 Based on the lecture by Prof. Gábor Szabó and Prof. György Vereb  This lecture: lecture slides marked by ! contain basic information absolutely required for tests and exams! Outline of the lecture: Classification of membrane transports pH-dependent partitioning (HH eq. ) Passive transport though biological Facilitated diffusion (transporters) membranes Ion channels as tools of passive Diffusion transport Passive diffusion in the human body Gap Junction Transport of hydrophobic molecules Lipid-water partition coefficient Pharmacological concern (mechanism of action for local and general anesthetics, TDDS, liposomes)  Classification of membrane transport from various aspects ! 1. Membrane structure 3. Energetics – lipid bilayer – Passive – complete biological Simple diffusion membrane Facilitated – one or two bilayers transport – Active primary secondary 2. Number of different transported 4. Solubility of transported substances and direction of substance transport – hydrophylic – uniport – hydrophobic 𝑐𝑐𝑙𝑙 – co-transport: R= 𝑐𝑐𝑤𝑤 – symport – antiport Real (biological) membranes: passive and active transport ! Revision Electrochemical gradient * Simple Channel transporter diffusion / carrier Passive Active transport transport Along Against electrochemical electrochemical gradient gradient * For charged particle, make sure to use ”Electrochemical gradient”, concentration gradient is wrong in these cases! Simple passive diffusion through artificial lipid bilayers ! Partly revision (Biophysics) Rate of diffusion through the membrane determined by: Concentration gradient (chemical potential) Charge and membrane potential (electric potential) Together: electrochemical potential difference (use this term to cover charged particles!) Permeability constant (P) P determined by: 𝑅𝑅𝐷𝐷𝐿𝐿 𝑃𝑃 = 𝑑𝑑 R: lipid-water partition coefficient (hydrophobic vs. hydrophilic), see later DL: diffusion constant in lipid membrane d: thickness of the membrane Partly revision (Biophysics) info Simple passive diffusion through artificial lipid bilayers Fick’s 1st law in general (covering charged particles as well): ∆𝑐𝑐𝑋𝑋 𝐹𝐹𝑧𝑧𝑋𝑋 ∆𝜓𝜓 𝐽𝐽𝑋𝑋 = −𝐷𝐷 − 𝐷𝐷 𝑐𝑐𝑋𝑋 ∆𝑥𝑥 𝑅𝑅𝑅𝑅 ∆𝑥𝑥 Fick’s law for diffusion through a cell membrane: 𝑧𝑧𝑧𝑧 𝐽𝐽𝑋𝑋 = −𝑃𝑃𝑋𝑋 ∆𝑐𝑐𝑋𝑋 − 𝑃𝑃𝑋𝑋 𝑐𝑐𝑤𝑤 ∆𝜓𝜓 𝑘𝑘𝐵𝐵 𝑇𝑇 𝑅𝑅𝑋𝑋 𝐷𝐷𝐿𝐿 𝑃𝑃𝑋𝑋 = 𝑑𝑑 PX: permeability of the molecule X through the membrane RX: water-lipid partition coefficient for X (hydrophobic vs. hydrophilic) DL: diffusion constant for X in lipid membrane or through carriers d: thickness of the membrane Passive diffusion in the human body – examples ! Gas exchange in alveoli (lung) Passive diffusion in the human body – examples ! Reabsorption in renal tubules (kidney)  Transport of hydrophobic molecules ! Passive transport depends on: revision (Chemistry) R (lipid / water partition coefficient) 𝑐𝑐𝑙𝑙 Hydrophobic: 𝑅𝑅 = >1 𝑐𝑐𝑤𝑤 revision (Chemistry) pH, i.c. partition: Henderson-Hasselbalch eq. (pKa: the pH where the concentration of neutral and charged form is equal) [𝑀𝑀] [𝑀𝑀] 𝑝𝑝𝑝𝑝 = 𝑝𝑝𝐾𝐾𝑎𝑎 + 𝑙𝑙𝑙𝑙𝑙𝑙 + 𝑙𝑙𝑙𝑙𝑙𝑙 + = 𝑝𝑝𝑝𝑝 − 𝑝𝑝𝐾𝐾𝑎𝑎 [𝑀𝑀 ] [𝑀𝑀 ] If pH > pKa then [M] > [M+] i.e. more neutral form, if pH < pKa then [M] < [M+], i.e. more charged form, if pH = pKa then [M] = [M+], see above   Lipid-water partition coefficient (R) ! For small non-charged molecules, lipid-water partition coefficient alone determines accumulation in the membrane and in the cell. The higher the R,the more the concentration in the membrane and also in the cytosol. General anaesthetics are usually lipid soluble. Their efficacy is proportional to their R value, so effective concentration the higher their R, the smaller concentration is sufficient for reaching an anaesthetic effect. They affect receptors (e.g. GABA, glutamate receptor) and ion channels (e.g. K+ leak) in the cell membrane lipid-water partition coefficient  General anaesthetics may change the gating of ion 5* channels (effect of phosphatidic acid) through embedding into the membrane and as a result increasing lateral pressure within the membrane. This may also activate enzymes (PLD: phospholipase D) to generate phosphatidic acid. These both may activate K+ channels and thereby cause hyperpolarization. Hydro- static Effect reversed pressure Ether added to the fishtank This is an ether partitions into the cell membrane example, (also) of the fish’s neurons, and many other ion activates K+ channels. Increasing channels are hydrostatic pressure (e.g. filling modulated by up the tank with a high level of anaesthetics. water) exprimes the ether molecules from the membrane and the fish wakes up.  5*/info+ General anesthetics also affect various receptors in the cell membrane General anesthetics: Group 1: intravenous (iv) etomidate, propofol, barbiturates: GABAA (a chloride channel, opens for gamma-amino-butyric acid) receptor activation ⇒ hyperpolarization ⇒ suppression of neuronal excitability ⇒ unconsciousness Group 2: ketamine (iv), N2O, Xenon, cyclopropane (inhalational): NMDA receptor (glutamate receptor, Ca2+ selective) inhibition, leak K+ channels activation ⇒ hyperpolarization Group 3: halothane, flurane, isoflurane, sevoflurane, desflurane (all inhalational): activation: GABAA , leak K+ channels, inhibition: NMDAR, nAChR, mKATP, SR3 (serotonin receptor 3), Nav channels, HCN channels  TDDS (transdermal drug delivery system, topical and transdermal) 5*  transdermal exposure (ointment/cream/patch) Skin is mainly permeable to hydrophobic molecules Large total surface area, good blood and lymphatic supply The stratum corneum provides the most significant barrier to diffusion (~10 µm, keratin-filled dead cells) Significant transport of drugs having high lipid-water partition coefficient (R), can be increased by preventing the evaporation Transdermal patch: comfortable, well-controlled drug delivery, can be removed easily in case of overdose  Liposomes in medicine 5* How do liposomes „work”? Fusion with the cell membrane (non-specific uptake): e.g. cancer-therapy (doxorubicin), vaccination (Pfizer-Covid)  Transport of hydrophobic molecules ! Passive transport depends on: revision (Chemistry) R (lipid / water partition coefficient) 𝑐𝑐𝑙𝑙 Hydrophobic: 𝑅𝑅 = >1 𝑐𝑐𝑤𝑤 revision (Chemistry) pH, i.c. partition: Henderson-Hasselbalch eq. (pK: the pH where the concentration of neutral and charged form is equal) [𝑀𝑀] [𝑀𝑀] 𝑝𝑝𝑝𝑝 = 𝑝𝑝𝐾𝐾𝑎𝑎 + 𝑙𝑙𝑙𝑙𝑙𝑙 + 𝑙𝑙𝑙𝑙𝑙𝑙 + = 𝑝𝑝𝑝𝑝 − 𝑝𝑝𝐾𝐾𝑎𝑎 [𝑀𝑀 ] [𝑀𝑀 ] If pH > pKa then [M] > [M+] i.e. more neutral form, if pH < pKa then [M] < [M+], i.e. more charged form, if pH = pKa then [M] = [M+], see above   pH-dependent partitioning ! R-NH3+ R-NH3+ R-NH2 R-NH2 R-NH2 R-NH2 lysosome cytosol pH ≈ 5 pH ≈ 7 cytoplasm exctracellular R-NH3+ pH ≈ 7 R-NH3+ space pH ≈ 7.4 [𝑀𝑀] 𝑙𝑙𝑙𝑙𝑙𝑙 = 𝑝𝑝𝑝𝑝 − 𝑝𝑝𝐾𝐾𝑎𝑎 [𝑀𝑀+ ] Henderson-Hasselbalch eq.: determines intracellular distribution among subcellular compartments and penetration into the cell of amphiphilic materials ”Lysosomotropic amines” can accumulate in lysosomes. At physiological pH, these compounds are mostly unionized and passively diffuse across the lipid bilayers of organelles. Upon entering the acidic environment of the lysosome they become predominantly ionized and therefore less able to diffuse out, resulting in their accumulation. The high cc. (1) is harmful for the lysosomes and (2) sequesters much of the drug, decreasing its effect on other targets. Local anaesthetics (e.g. lidocaine) and some cytostatic drugs (e.g. daunorubicine) are examples.  pH, pKa, and local anaesthetics 5* Local anesthetics: bupivacaine, lidocaine, mepivacaine, procaine, ropivacaine, tetracaine % ionized - block sodium channel function ! - act from the intracellular side (need to get through the membrane first) ⇒ suppression of neuronal excitability [𝑀𝑀] 𝑙𝑙𝑙𝑙𝑙𝑙 = 𝑝𝑝𝑝𝑝 − 𝑝𝑝𝐾𝐾𝑎𝑎 [𝑀𝑀+ ] Lidocaine may penetrate better into cells when the extracellular space is acidic (below the usual pH=7.4), e.g. in inflammation, because it is less protonated than bupivacaine  Daunorubicin accumulation in lysosomes “low” pH 5* ! Daunorubicin, an amine that can be protonated, is a DNA-intercalating drug used in cancer treatment. It exerts its effect partly in the nucleus. Tumor cells with high lysosomal activity may defer some of its nuclear effect by sequestering it in acidic lysosomes. Blood,Vol 89, Nov 10 (May 15), 1997: pp 3745-3754 Facilitated diffusion through cell membranes ! 1. It happens along the electrochemical gradient (from high to low electrochemical potential site), no energy used. 2. A transporter molecule is needed, that specifically binds the molecule to be transported (e.g. GLUT1-5 uniporters binds D- glucose but not L isomeric variant, or ion channels are selective for certain type of ions)  Facilitated diffusion through cell membranes ! 3. Maximum rate of transport is limited as the # of transporting molecules and their transportation rate is finite. 4. Facilitated diffusion can be inhibited by specific antagonist, unlike simple diffusion (see TTX for Nav channels).  Facilitated diffusion (GLUTs) ! Glucose uniport into the cells (GLUTs) Types and tissue expression of GLUTs  Facilitated diffusion (antibiotics) ! A carrier type ionophore antibiotics Pore-forming type ionophore antibiotics Ion channels (selective holes) also allow facilitated ! diffusion 1. Selective pores for given type(s) of ions (electro-chemical gradient!!!) 2. Very high transport rate - effective in regulation of intracellular ion conc. and membrane potential 3. Regulated/controlled opening (gating): can serve as a switch in signalling processes (remember AP generation, i.c. Ca2+ level elevation)  Types of ion channels ! Voltage-gated Ligand-gated IC signal-gated Stretch/mechanically-gated G-protein (7-TM receptor) gated 7-TM receptor G-protein Leak/background channels (always open)  (Facilitated) diffusion between cells (gap junction): intercellular trafficking 5* info Diffusion of molecules between cells (fluorescent dye) EM* images on a gap junction Permeable to small molecules (below 2 kDa), both polar and ! non-polar. e.g.: ATP, ADP, cAMP, IP3, Ca2+, glutamate, glutathione, Na+, K+, Cl- *: electron microscope Henry J. Donahue, Roy W. Qu and Damian C. Genetos, 2018, Nature Review Rheumatology Morten Schak Nielsen, Lene Nygaard Axelsen, et al, 2012, Comprehensive Physiology, 2012  (Facilitated) diffusion between cells (gap junction): ! intercellular trafficking Structure of gap junction channels (connexin → connexon or connexin hemichannel → gap-junction channel) gap-junction channel (GJC) connexon connexin connexon or connexin hemichannel (half of the GJC)  Regulation of gap junction channels info Dopamine ⇒ [Ca ]i↑ ⇒ GJC closes 2+ [H+] i is high (low pH) ⇒ GJC closes ! (ball-and-chain model as in NaV) 5* Phosphorylation (reduced conductivity, [Ca2+]i is high ⇒ GJC pore closes Assembly/disassembly 5* ! (observe left and right images Voltage-gating (cardiac cells) of GJC) (pores)  Keywords ! apolar/nonpolar glucose uniport with example polar ion channel gating amphipatic molecule ion channel selectivity lipid-water partitioning coefficient gap junction (connexin, hemichannel/connexon, gap- facilitated diffusion junction channel (GJC)) passive transport voltage sensor