Martin J. Cann

Signal transduction mediated by

inorganic ions.

The importance of the major biologically activeinorganic ions.

pH homeostasisVolume homeostasisSolute transportAction potentialsGas transportFluid secretion

The major biologically active inorganic ions

Although the physiology of the predominant inorganicions is well understood, sensor mechanisms remainelusive.

CationsNa+ ?K+ ?

AnionsCl- guanylyl cyclase-AHCO3

- sAC, Spirulina CyaC

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?1) What are the phenotypic consequences of the loss of inorganic

ion responsive genes?2) What insights can recombinant protein provide us regarding

the mechanism of enzyme activation by inorganic ions?

HCO3- responsive adenylyl cyclases

in prokaryotes and eukaryotes.

Biological functions for HCO3

Gas exchangepH homeostasisSperm maturationNucleotide synthesisCarbon fixation

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

GAF-A GAF-B ACPASN C

G595K465I394L385L237T207Q50 K859

CyaB1595-859

CyaB1

100

150

200

250

300

350

400

Bas

al

NaC

l

KC

l

NaH

CO

3

KH

CO

3

0

Spec

ific

Act

ivity

[nm

ol c

AM

P/m

g/m

in]

GAF-A GAF-B ACPASN C

CyaB1595-859

0 0.1 1.0 10.0 100.00

100

200

300

400

Salt [log mM]

Spec

ific

Act

ivity

[nm

ol c

AM

P/m

g/m

in]

GAF-A GAF-B ACPASN C

CyaB1595-859

NaHCO3

NaCl

1.11.1Hill coefficient

97.73.7

91.64.9Ea (kJ/mol)

3.5 x 103

3.7 x 103

7.02.6kcat (min-1)

238.036.3

93.58.2

Vmax(nmol/mg/min)

33.32.8

11.80.8KM (ATP) (M)

HCO3-Cl-

kcat/KM(M-1 sec-1)

0 2 4 6 8 100

100

200

300

Acc

umul

ated

cA

MP

[pm

ol/a

ssay

]

Time [mins]

GAF-A GAF-B ACPASN C

+

+

CyaB11-859

KHCO3

KCl

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

50

100

150

200

250

1 10 1000Salt [log mM]

Spec

ific

Act

ivity

[nm

ol c

AM

P/m

g/m

in]

01 10 100

0

2.5

5.0

7.5

10.0

Salt [log mM]

Spec

ific

Act

ivity

[nm

ol c

AM

P/m

g/m

in]

0

GAF-A GAF-B ACPASN C

CyaB1595-859

CyaB1595-859 CyaB1595-859 K646A

V722

NH2

N

N

O

1

O

O

NH3+K646

HO C

Anabaena CyaB1

I1019

NH2

N

N

O

1

O

O

NH3+

D1018

K938

VC1·IIC2

C

6

6

N

N

N

N

1 10 1000

5

10

15

20

Salt [log mM]

Spec

ific

Act

ivity

[nm

ol c

AM

P/m

g/m

in]

0 0Salt [log mM]

Spec

ific

Act

ivity

[nm

ol c

AM

P/m

g/m

in]

1 10 1000

2.5

5.0

7.5

10.0

12.5

15.0

Mycobacterium tuberculosisRV1319c Thr

Mycobacterium tuberculosisRV1264 Asp

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.

GAF domain mediatedNa sensing.

Biological functions for Na

pH homeostasisMaintenance of blood pressureAction potentialsSolute transportVolume homeostasis

0 2 4 6 8 100

100

200

300

Acc

umul

ated

cA

MP

[pm

ol/a

ssay

]

Time [mins]

GAF-A GAF-B ACPASN C

+

+

CyaB11-859

KHCO3

KCl

0

50

100

150

200

250

300pm

ol c

AM

P/as

say

Rb CsLi KNaNone

GAF-A GAF-B ACPASN C

CyaB11-859

0 1 10 1000

100

200

300

Salt [mM]

pmol

cA

MP/

assa

y

GAF-A GAF-B ACPASN C

CyaB11-859

0 1 2 3 4 5 6 7 8 9 10

0

1

250 mM NaCl0 mM NaCl

Time [mins]

nmol

cA

MP/

assa

y

GAF-A GAF-B ACPASN C

+

+

CyaB11-859

0

0

250

500

750

10000 mM NaCl

50 mM NaCl

-7 -6 -5cAMP log [M]

pmol

cA

MP/

assa

y

GAF-A GAF-B ACPASN C

+

+

CyaB11-859

0

50

100

150

200

250

1 2 3 4 5 6

pmol

cA

MP/

assa

y

+Na +Na +Na

CyaB1 1-859 CyaB1 1-859 D190A GAF-A

CyaB1 1-859 D360A GAF-B

GAF-A GAF-B ACPASN C

+

+

CyaB11-859

Anabaena sp. PCC7120

WT cyaB1

BG11

BG11/ 40 mM NaCl

3.01.00.80.60.40.2

Wild type

cyaB1

NaCl [mM]

0

0.01

0.02

0.03

0.04

Gro

wth

Rat

e[

/hr-1

]

Anabaena sp. PCC7120WT

Anabaena sp. PCC7120cyaB1

0 4 40NaCl [mM]

0.20.2 4.04.0

NaCl [mM]

pH

7.0

8.5

9.0

7.5

8.0

WT cyaB1

-50000

-40000

-30000

-20000

-10000

0

0 100 200 300 400 500 600

Time [secs]

WTcyaB1

Fl

uore

scen

ce In

tens

ity [A

rbitr

ary

Uni

ts] cells

Na

H2O + CO2 HCO3- + H+

H+

CA

Sym

HCO3- Na+

out

in

A

-9

-8

-7

-6

-5

-4

-3

-2

-1

0

1

2

0 100 200 300time [secs]

WTcyaB1

H

+ [pm

ol/

g ch

loro

phyl

l]

Light off

Light on

A

B

Ant

H+

Na+

out

in

B

4 mM Na+

CyaB1

out

in

CyaB1

cAMP

0.2 mM Na+

out

in

Na/HAnt

Na+

H+

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.

The major biologically active inorganic ionsCationsNa+ GAF domain of Anabaena CyaB1K+ ?

AnionsCl- guanylyl cyclase-AHCO3

- A defined subset of Class III adenylylcyclases

University of TübingenJoachim SchultzTobias KanacherJurgen Linder

University of DurhamMartin CannArne HammerJie Zhou

University of TokyoMasayuki Ohmori