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Martin J. Cann

Signal transduction mediated by inorganic ions. Martin J. Cann. The importance of the major biologically active inorganic ions. pH homeostasis Volume homeostasis Solute transport Action potentials Gas transport Fluid secretion. The major biologically active inorganic ions

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Martin J. Cann

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  1. Signal transduction mediated by inorganic ions. Martin J. Cann

  2. The importance of the major biologically active inorganic ions. pH homeostasis Volume homeostasis Solute transport Action potentials Gas transport Fluid secretion

  3. The major biologically active inorganic ions Although the physiology of the predominant inorganic ions is well understood, sensor mechanisms remain elusive. Cations Na+ ? K+ ? Anions Cl- guanylyl cyclase-A HCO3- sAC, Spirulina CyaC

  4. Anabaena strain PCC7120 Why Anabaena strain PCC7120? • Clear role for inorganic ions in organismal biology • Genome is sequenced • Wild type and mutant strains are available What do we hope to learn? What are the phenotypic consequences of the loss of inorganic ion responsive genes? What insights can recombinant protein provide us regarding the mechanism of enzyme activation by inorganic ions?

  5. HCO3- responsive adenylyl cyclases in prokaryotes and eukaryotes.

  6. Biological functions for HCO3 Gas exchange pH homeostasis Sperm maturation Nucleotide synthesis Carbon fixation

  7. Class I - Enterobacteriacae Class II - Toxin producing eubacteria Class III - ‘Universal’ Class, mammals and some prokaryotes Class IV - Aeromonas hydrophila Class V - Prevotella ruminicola Class VI - Genomes of Rhizobiaceae HCO3- responsive e.g. mammalian sAC, Spirulina CyaC HCO3- non-responsive e.g. mammalian tmAC

  8. CyaB1595-859 GAF-A GAF-B PAS AC CyaB1 N C Q50 T207 L237 L385 I394 K465 G595 K859

  9. N C 400 350 CyaB1595-859 Specific Activity [nmol cAMP/mg/min] 300 GAF-A GAF-B PAS AC 250 200 150 100 0 KCl NaCl Basal KHCO3 NaHCO3

  10. N C 400 CyaB1595-859 300 NaHCO3 NaCl GAF-A GAF-B PAS AC Specific Activity [nmol cAMP/mg/min] 200 100 0 0 0.1 1.0 10.0 100.0 Salt [log mM]

  11. Cl HCO - - 3 11.8 33.3 ± K ( M) m M (ATP) 2.8 0.8 ± V 93.5 238.0 max 8.2 36.3 (nmol/mg/min) ± ± 3.7 x 103 3.5 x 103 kcat/KM (M-1 sec-1) kcat (min-1) 2.6 7.0 Hill coefficient 1.1 1.1 91.6 97.7 E (kJ/mol) 4.9 3.7 a ± ±

  12. CyaB11-859 N C + + GAF-A GAF-B PAS AC 300 200 Accumulated cAMP [pmol/assay] 100 0 0 2 4 6 8 10 Time [mins] KHCO3 KCl

  13. Anabaena CyaB1 638 FNYEGTLDKFIGDALM (59) GSHKRMDYTVIGDGVN---LSSRLETV 736 Rattus sAC C1 87 LIFGGDILKFAGDALL (55) GDETRNYFLVIGQAVDDVRLAQNMAQM 184 Rattus sAC C2 336 FMFD------KGCSFL (51) GHTVRHEYTVIGQKVN---IAARMMMY 420 Spirulina CyaC 1049 FENQGTVDKFVGDAIM (66) GSQERSDFTAIGPSVN---IAARLQEA 1154 Stigmatella CyaB8 203 LTCGGTLDKFLGDGLM (66) GGSMRTEYTCIGDAVN---VAARLCAL 308 Mycobacterium Rv1319 399 DRHHGLINKFAGDAAL (50) GAKQRFEYTVVGKPVN---QAARLCEL 488 Mycobacterium Rv1264 253 TAPPVWFIKTIGDAVM (40) -----RAGDWFGSPVN---VASRVTGV 327 Bos AC1 C1 345 HCR---RIKILGDCYY (54) GLR-KWQYDVWSNDVT---LANVMEAA 434 Bos AC1 C2 915 FYKDLEKIKTIGSTYM (62) GAR-RPQYDIWGNTVN---VASRMDST 1015 Rattus AC3 C1 359 HQL---RIKILGDCYY (54) GQK-RWQYDVWSTDVT---VANKMEAG 448 Rattus AC3 C2 967 KFRVITKIKTIGSTYM (72) GAR-KPHYDIWGNTVN---VASRMEST 1077 Mus AC9 C1 434 KCE---KISTLGDCYY (54) GMR-RFKFDVWSNDVN---LANLMEQL 519 Mus AC9 C2 1096 DYNSIEKIKTIGATYM (62) GTT-KLLYDIWGDTVN---IASRMDTT 1196 Rattus GCA 912 DVY---KVETIGDAYM (57) GLK-MPRYCLFGDTVN---TASRMESN 1004

  14. N C 250 10.0 200 CyaB1595-859 7.5 GAF-A GAF-B PAS AC Specific Activity [nmol cAMP/mg/min] Specific Activity [nmol cAMP/mg/min] 150 5.0 100 2.5 50 0 0 1 10 100 0 Salt [log mM] 0 1 10 100 Salt [log mM] CyaB1595-859 CyaB1595-859 K646A

  15. 15.0 20 12.5 15 10.0 Specific Activity [nmol cAMP/mg/min] Specific Activity [nmol cAMP/mg/min] 7.5 10 0 5.0 5 2.5 1 10 100 0 0 1 10 100 Salt [log mM] 0 Salt [log mM] Mycobacterium tuberculosis RV1319c Thr Mycobacterium tuberculosis RV1264 Asp

  16. Mechanism of HCO3- activation of AC • HCO3- increases rate of substrate turnover. • HCO3- is hypothesized to be co-ordinated in • the active site by an essential lysine residue. • HCO3- is hypothesized to mimic a carboxy group. • A Thr/Asp polymorphism can be used as a • predictor of HCO3- responsiveness. • HCO3- responsive ACs can be detected in the • genomes of many prokaryotes and several eukaryotes.

  17. GAF domain mediated Na sensing.

  18. Biological functions for Na pH homeostasis Maintenance of blood pressure Action potentials Solute transport Volume homeostasis

  19. CyaB11-859 N C + + GAF-A GAF-B PAS AC 300 200 Accumulated cAMP [pmol/assay] 100 0 0 2 4 6 8 10 Time [mins] KHCO3 KCl

  20. N C 300 250 200 pmol cAMP/assay GAF-A GAF-B PAS AC 150 100 50 0 None Li Na K Rb Cs CyaB11-859

  21. N C 300 200 GAF-A GAF-B PAS AC pmol cAMP/assay 100 0 0 1 10 100 Salt [mM] CyaB11-859

  22. CyaB11-859 2 N C 50 mM NaCl 0 mM NaCl + + nmol cAMP/assay 1 GAF-A GAF-B PAS AC 0 0 1 2 3 4 5 6 7 8 9 10 Time [mins]

  23. CyaB11-859 N C 1000 + + 750 0 mM NaCl 50 mM NaCl GAF-A GAF-B PAS AC 500 pmol cAMP/assay 250 0 0 -7 -6 -5 cAMP log [M]

  24. CyaB11-859 N C 250 + + 200 GAF-A GAF-B PAS AC 150 pmol cAMP/assay 100 50 0 +Na +Na +Na 1 2 3 4 5 6 CyaB1 1-859 CyaB1 1-859 D190A  GAF-A CyaB1 1-859 D360A  GAF-B

  25. Anabaena sp. PCC7120 WT cyaB1 BG11 BG11/ 40 mM NaCl

  26. NaCl [mM] 0.2 0.4 0.6 0.8 1.0 3.0 Wild type cyaB1

  27. 0.04 0.03 Growth Rate [/hr-1] Anabaena sp. PCC7120WT 0.02 Anabaena sp. PCC7120cyaB1 0.01 0 0 4 40 NaCl [mM]

  28. NaCl [mM] 0.2 4.0 0.2 4.0 WT cyaB1 7.0 7.5 8.0 pH 8.5 9.0

  29. WT cyaB1 cells 0 -10000 Na -20000 Fluorescence Intensity [Arbitrary Units] -30000 -40000 -50000 0 100 200 300 400 500 600 Time [secs]

  30. A H+ CA H2O + CO2 HCO3- + H+ out Sym in HCO3- Na+ 2 A 1 0 -1 B -2 Light on H+ [pmol/g chlorophyll] -3 Light off WT cyaB1 -4 -5 -6 -7 -8 -9 0 100 200 300 time [secs] B Na+ out Ant in H+

  31. H+ 4 mM Na+ 0.2 mM Na+ out out in in Na+ CyaB1 CyaB1 cAMP Na/H Ant

  32. GAF domain mediated Na sensing • CyaB1 is the first identified Na sensor. • GAF domains are found throughout the animal, plant, • and microbial kingdoms. • GAF domains may mediate at least some aspects of • Na detection in diverse organisms.

  33. The major biologically active inorganic ions Cations Na+ GAF domain of Anabaena CyaB1 K+ ? Anions Cl- guanylyl cyclase-A HCO3- A defined subset of Class III adenylyl cyclases

  34. University of Durham Martin Cann Arne Hammer Jie Zhou University of Tübingen Joachim Schultz Tobias Kanacher Jurgen Linder University of Tokyo Masayuki Ohmori

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