martin j. cann
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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 - PowerPoint PPT PresentationTRANSCRIPT
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
Ba
sa
l
Na
Cl
KC
l
Na
HC
O3
KH
CO
3
0
Sp
ec
ific
Ac
tiv
ity
[nm
ol
cA
MP
/mg
/min
]
GAF-A GAF-B ACPASN C
CyaB1595-859
0 0.1 1.0 10.0 100.0
0
100
200
300
400
Salt [log mM]
Sp
ec
ific
Ac
tiv
ity
[nm
ol
cA
MP
/mg
/min
]
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
11.80.8
KM (ATP) (M)
HCO3-Cl-
kcat/KM
(M-1 sec-1)
0 2 4 6 8 100
100
200
300
Ac
cu
mu
late
d c
AM
P[p
mo
l/a
ss
ay
]
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 1000
Salt [log mM]
Sp
ec
ific
Ac
tiv
ity
[nm
ol
cA
MP
/mg
/min
]
01 10 100
0
2.5
5.0
7.5
10.0
Salt [log mM]
Sp
ec
ific
Ac
tiv
ity
[nm
ol
cA
MP
/mg
/min
]
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]
Sp
ec
ific
Ac
tiv
ity
[nm
ol
cA
MP
/mg
/min
]
0 0
Salt [log mM]
Sp
ec
ific
Ac
tiv
ity
[nm
ol
cA
MP
/mg
/min
]
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
Ac
cu
mu
late
d c
AM
P[p
mo
l/a
ss
ay
]
Time [mins]
GAF-A GAF-B ACPASN C
+
+
CyaB11-859
KHCO3
KCl
0
50
100
150
200
250
300p
mo
l c
AM
P/a
ss
ay
Rb CsLi KNaNone
GAF-A GAF-B ACPASN C
CyaB11-859
0 1 10 1000
100
200
300
Salt [mM]
pm
ol c
AM
P/a
ssay
GAF-A GAF-B ACPASN C
CyaB11-859
0 1 2 3 4 5 6 7 8 9 10
0
1
250 mM NaCl
0 mM NaCl
Time [mins]
nm
ol c
AM
P/a
ssay
GAF-A GAF-B ACPASN C
+
+
CyaB11-859
0
0
250
500
750
10000 mM NaCl
50 mM NaCl
-7 -6 -5cAMP log [M]
pm
ol
cA
MP
/as
sa
y
GAF-A GAF-B ACPASN C
+
+
CyaB11-859
0
50
100
150
200
250
1 2 3 4 5 6
pm
ol
cAM
P/a
ssay
+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
Ra
te[
/hr-1
]
Anabaena sp. PCC7120WT
Anabaena sp. PCC7120cyaB1
0 4 40
NaCl [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]
WT
cyaB1
F
luo
resc
enc
e In
ten
sity
[A
rbit
rary
Un
its] 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
ph
yll
]
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 ions
CationsNa+ 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