Transporters in cell physiology

1. Transporters in cell physiology Hana Sychrová Department of Membrane Transport Institute of Physiology, AS CR, v.v.i. Videnska 1083, 142 20 Prague 4 Tel: (420) 241 062 667 E-mail: sychrova@biomed.cas.cz

2. Membranes Integrity of cells, organelles Flow • material - transport of substances • energy - radiation - oxidation - electrochemical potential • information - physical signals - light - heat - electrical potential - magnetic field - mechanical pressure - chemical signals - attractants and repellents - „sense“ signals

3. Transport mechanisms 1. Nonspecific permeation (diffusion down the concentration gradient) 2. Specific transport (membrane protein) Transport systems: Pore -(oligomer) permitting nonspecific passage of solutes of different size ranges Transporter - relatively high specificity - binding site opens alternately to the one and to the other membrane side - antiporter/symporter - uniporter - exporter/importer - old names: permease (symporter) carrier (transporter) Channel - one protein or an oligomeric cluster - when open, the specific site in the channel can transiently bind solutes from both sides of the membrane

4. Transporter • membrane protein showing a relatively high specificity • - binding site opens alternately to the one and to the other membrane side • - antiporter/symporter (at least one ionic) • - uniporter • - exporter/importer Facilitated (mediated) diffusionwithout input of energy (glc in yeast) Active transport – against solutes’ chemical or electrochemical potential gradient – primary driven by exergonic chemical and photochemical reactions (ATP hydrolysis, mainly ions) - secondary driven by electrochemical potential gradients of H+ and Na+ (exceptionally of K+)

5. Channel • membrane protein (or an oligomeric cluster) • specific transport of ions or uncharged molecules • down their chemical/electrochem. potential gradient • two conformational states, open and closed • opening by • (a) spreading electric field (potential-gated) • (b) binding a specific ligand (chemically gated) • (c) mechanical stress (mechanically gated) • when open, the specific site in the channel can • transiently bind solutes from both sides of the membrane

6. Transport mechanisms 1. Nonspecific permeation (diffusion down the concentration gradient) 2. Specific transport (membrane protein) Transport systems Pore-(oligomer) permitting nonspecific passage of solutes of different size ranges Transporter- relatively high specificity - binding site opens alternately to the one and to the other membrane side - antiporter/symporter - uniporter - exporter/importer - old names: permease (symporter) carrier (transporter) Channel - one protein or an oligomeric cluster - when open, the specific site in the channel can transiently bind solutes from both sides of the membrane

7. Nonspecific permeation • diffusion down the concentration gradient • 1.1 hydrophobic domains of membranes    (various small, medium-sized and large lipophilic molecules) • 1.2 water-filled pores • (polyene antibiotics) • some opening only after a stimulus (connexons between adjacent cells -     all solutes up to a certain size) • 1.3 true pores in the lipid bilayertransiently formed at higher • temperature and with applied transmembrane electric potential    (all solutes, including macromolecules ("electroporation")) • 1.4via non-receptor endocytosis in membrane vesicles    (all solutes present in extracellular aqueous medium)

8. 2. Specific transport 2.1 Selective channels 2.2 Specific transporters 2.3 Group translocation Gram-positive and Gram-negative bacteria, brain tissue (mono- and disaccharides; amino acids) 2.4 Receptor-mediated pinocytosis 2.4.1 endocytosis, mainly in animal cells    (ferritransferrin) 2.4.2 exocytosis, mainly in fungal and animal cells    (hormones)

9. 2. Specific transport 2.1 Selective channels 2.1.1 nongated (porins in bacterial, outer mitochondrial and chloroplast membranes, aquaporins)     (cations, anions, water; also nonelectrolytes - glycerol) 2.1.2 electrically or potential-gated (nerve and muscle-cell) (generation and propagation of action potential, Na+, K+, Ca2+) 2.1.3 chemically or ligand-gated (hormone receptor-channels, acetylcholine receptor)    (cations) 2.1.4 mechanically or stress-gated (channels in blood capillary walls, inner ear hair cells)    (cations, especially K+) 2.2 Specific transporters 2.3 Group translocation 2.4 Receptor-mediated pinocytosis

10. 2. Specific transport 2.1 Selective channels 2.2 Specific transporters 2.2.1 mediated (or facilitated) diffusion    (monosaccharides in animal, noninsulin-dependent tissues, yeast cells) 2.2.2 primary active transport 2.2.3 secondary active transport 2.3 Group translocation 2.4 Receptor-mediated pinocytosis

11. 2.2.2 primary active transport • 2.2.2.1 driven by ATP or diphosphate hydrolysis • P-type, F-type and ABC-type ATPases in all cells    (cations, anions, amino acids, sugars, xenobiotics) • 2.2.2.2 driven by oxidation reactions, • in bacterial, inner mitochondrial and chloroplast membranes    (H+, Na+) • 2.2.2.3 driven by light absorption, • in halobacteria (bacteriorhodopsin)   •  (H+, Cl-) • 2.2.2.4 driven by decarboxylation, • in alkalifilic bacteria    (Na+) • 2.2.2.5 driven by methyl transfer, • some methanobacteria    (Na+)

12. 2.2.3. secondary active transport 2.2.3.1 symport type, using H+, Na+ and, exceptionally, K+ as driving ion, bacterial, fungal, plant and animal cells, net charge transporting    (various nonelectrolytes, mainly nutrients) with H+ - lactose in Gram- bacteria with Na+ - glucose in kidney tubules with K+ - lysine in insect intestines 2.2.3.2 antiport type, electrically silent, in a variety of cells    (cations) 2.2.3.3 antiport type, net charge transporting, in outer mitochondrial membranes    (ADP/ATP)

13. Transport mechanisms

14. [Na+ or K+]:H+ antiporter Nha1 2.A.36.4.1 • 2. Electrochemical-potential-driven transporters • 2.A. Transporters or carriers (uniporters, symporters and antiporters) • 2.A.36 Univalent cation:H+ antiporter-1

15. Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) Membrane Transport Proteins http://www.chem.qmul.ac.uk/iubmb/mtp/ • Introduction • Glossary of terms used in membrane transport work • List of families and subfamilies of the TC system • 1. Pores and channels • 1.A a-Helical channels • 1.B b-Strand porins • 1.C Pore-forming toxins • 1.D Non-ribosomally synthesized channels • 1.E Holins • 2. Electrochemical-potential-driven transporters • 2.A Transporters or carriers (uniporters, symporters and antiporters) • 2.B Non-ribosomally synthesized transporters • 3. Primary active transporters

16. Characterization of transport systems • Kinetic parameters - KT , Jmax , ( sin/sout ), substrate specificity • pH optimum, inhibitors, source of energy (active transport) • Isolation of the geneand sequencingprimary structure and properties of the protein • Protein purification reconstitution, crystalization • existence of similar systems (overlap of substrate specificity) • small amount of protein (low expression, short half-life) • denaturation of protein during membrane solubilization = loss of characteristic properties  expression of the gene in host organisms (yeast Saccharomyces cerevisiae)

17. Transport mechanisms in S. cerevisiae • 400 transport systems  23 families Passive transport simple diffusion - O2, CO2, glycerol facilitated diffusion- channels– metal cations, Cl- transporters– monosaccharides Active transport primary89 H+-ATPasa (gen PMA1) secondary 201 symport with H+- aa, K+, Pi antiport withH+- Na+, K+ André B., Yeast 11, 1575-1611 (1995) Nelissen B., FEMS Microbiol. Rev. 21, 113-134 (1997)

18. sugars- facilitated diffusion (glc, gal), 17 regulated systems - active symport with H+ (maltose) glycerol - simple diffusion - facilitated diffusion - channel (FPS1) – only outward - active symport with H+ - inward amino acids (nitrogen source) - active symport with H+ - accumulation 102 - 105 - 24 genes - 20 transporters (permeases) characterized kinetically - substrate specificity overlap - 16 mutants isolated (resistance to toxic aa analogs) - 14 gene isolated, products characterized (homology 20 - 60 %)

19. Transport of K+ and Na+ in S. cerevisiae K+ Na+ Ena1-5 Trk1-2 Mkh1 Na+ K+ H+ Nha1 ? K+ K+ H+ H+ Nhx1 ATP ADP+Pi H+ Tok1 Na+ Vnx1 Na+ K+ K+ H+ K+ Kha1 ADP+Pi ATP NSC1 ? Pma1 H+ Growth: 1 M – 2.5 M K+ 0 – 1.8 M Na+ Intracellular concentration: 200 - 300 mM K+ < 100 mM Na+ • 10 transport systems Channels (1 -2) ATPases (1) Symporters (2 – 4) Antiporters (5) K+ influx: Trk1,2p si/so~3 x 105 Sequestration in organellles: antiporters Nhx1pKha1p Vnx1p, Mkh1p(?) Alkali-metal-cation efflux: (Na+, Li+, K+, Rb+): Nha1p: Na+/H+ antiporter(acidic pHout) Ena1/Pmr2p: Na+-ATPase(neutral pHout) Tight regulation: expression, biogenesis (targeting, post-translational modif.), degradation (post-translational modif., delocalization)

20. K+ Ena1-5 Na+ Trk1-2 Mkh1 Na+ K+ H+ Nha1 ? K+ K+ H+ H+ Nhx1 ATP ADP+Pi H+ Tok1 Na+ Vnx1 Na+ K+ K+ H+ K+ Kha1 ADP+Pi ATP NSC1 ? Pma1 H+ Transport of K+ and Na+ in S. cerevisiae X X Growth: 1 M – 2.5 M K+ 0 – 1.8 M Na+ Intracellular concentration: 200 - 300 mM K+ < 100 mM Na+ X X Gene deletion: Phenotype: growth on YNB (pH 5.0) + salt [mM] Strain NaCl LiClKCl