Bicarbonate transporters and intracellular pH regulation

Li-Ming Chen, Ph.D.Huazhong University of Science and Technology

Wuhan, China

7th Asia-Pacific Biotech Congress Beijing, China, July 2015

Physiological significance of pH regulation

Acid-base balance in vivo affects:(1) Cell survival(2) Cell migration and wound healing(3) Protein folding/assembly(4) Na+ homeostasis(5) Solute transport(6) Cell signaling(7) Bone remodeling…(8) Neuronal excitability….Ion channel gating, neurotransmitter releasing.(9) Muscle contraction(10) Fertility And many more others……

(Parker and Boron, 2013, Physiological Review)

Acid-Base Homeostasis

Ocular disorders

Short statue

Epilepsymigraine

Osteopenia

Mental retardation

Breast Cancer

Hypertension

Infertility

Renal tubule acidosis

For reviews, seeParker, Boron 2013Liu Y, et al 2012Guo YM, et al, 2014

HCO3–

CO2

HCO3–

H+

H2O

CO2

H2CO3

pHi

Gas channels

Proton transporters

Bicarbonate transporters

CA

CO2/HCO3- system contributes for about 70% of pH buffering

power in the body

Gas channel

Gas Channels across membrane

Conventional view (Overton rule, 1897)Gas molecules flux through membrane by simple diffusion.

Gas channel hypothesisMovement of gas molecules across plasma membrane can be mediated by protein channels.

Xenopus laevis Xenopus oocytes

Technique……

cRNA 4-5 days

Vm pHioocyte

Technique……

5% CO2

33 mM HCO3–

Xenopus oocyte:pH Changes Caused by CO2 Influx

HCO3–

H+

HCO3– pHS

[HCO3–]

pH … with 15-m tip

AQP1

H2O2 min

pHS

7.5

7.7

Bulk Extracellular Fluid

pHi

[CO2]S

CO2CO2

H2OCO2

H2OHCO3

–H+

0.5 mM

NH3 + NH4+

Xenopus oocyte:pH Changes Caused by NH3 Influx

pH … with 15-m tipNH4

+NH4+

pHipHS

[NH3]S

[NH4+]

2 minAQP1

H2OpHS 7.5

7.7

7.3

Bulk Extracellular Fluid

NH3NH3

H+

NH3 NH4+

H+

↑pHi

Relative permeability of channels to CO2 and NH3

AQP1

AQP4

AQP5

AmtB

(16)

RhAG

(16)(24)

(17)(10)

AQP0

0.00

0.05

0.10

(pHS*)CO2 (13)

(10)

(14)(24)

(17)

(13) (14)0.00

-0.10

-0.05(pHS*)NH3

AQP1

AQP4

AQP5

AmtB

RhAG

∞

AQP0

∞

0

3

2

1PC

O2*/

PN

H3*

∞

Relative CO2 permeability

Musa-Aziz, Chen et al, PNAS, 2009

Relative NH3 permeability

AQP1

AQP4

AQP5

AmtB

(16)

RhAG

(16)(24)

(17)(10)

AQP0

0.00

0.05

0.10

(pHS*)CO2 (13)

(10)

(14)(24)

(17)

(13) (14)0.00

-0.10

-0.05(pHS*)NH3

AQP1

AQP4

AQP5

AmtB

RhAG

∞

AQP0

∞

0

3

2

1PC

O2*/

PN

H3*

∞

Relative CO2 permeability

Musa-Aziz, Chen et al, PNAS, 2009

Relative NH3 permeability

First Example of Gas Selectivity by Channels

AQP1

AQP4

AQP5

AmtB

(16)

RhAG

(16)(24)

(17)(10)

AQP0

0.00

0.05

0.10

(pHS*)CO2 (13)

(10)

(14)(24)

(17)

(13) (14)0.00

-0.10

-0.05(pHS*)NH3

AQP1

AQP4

AQP5

AmtB

RhAG

∞

AQP0

∞

0

3

2

1PC

O2*/

PN

H3*

∞

Relative CO2 permeability

Musa-Aziz, Chen et al, PNAS, 2009

Relative NH3 permeability

First Example of Gas Selectivity by Channels

Walter F. Boron

Acid-Extruders↑pHi

Acid-Loaders↓pHi

Na-H exchangers(SLC9)

Na+

H+

Proton pump

Na+

Cl−

1 Na+

1 or 2 HCO3−

Anion exchangers(SLC4 & SLC26)

Cl−

HCO3−

1 Na+

3 HCO3−

Channels HCO3−

Na/HCO3– cotransporter

(SLC4)

Na-driven Cl-HCO3–

exchanger(SLC4)

Na/HCO3- cotransporter(SLC4)

2 HCO3−

ATP

ADPH+

Acid-base transporters in pH regulation

Na-Coupled Bicarbonate Transporters (NCBT)

SLC4 family HCO3- transporters

AE3AE1

AE2

NBCe1

NBCn1

NBCn2

NDCBE

AE4

NBCe2

BTR1

0.1

(SLC4A2 )

(SLC4A1 )(SLC4A3)

(SLC4A4)

(SLC4A5)

(SLC4A7)

(SLC4A10)

(SLC4A8)

(SLC4A9)

(SLC4A11)

Electrogenic

Electroneutral

Cl-HCO3Exchangers

Na-dependent

Na-independent

HCO3–

Cl –

1Na+

1HCO3–

1Na+ + 2HCO3–

1Cl –

NDCBE

1Na+

2HCO3–

NBCe1NBCe2

1Na+

3HCO3–

NBCe1NBCe2

Intracellular Extracellular

NBCn2

Electrogenic

Electroneutral

NBCn1

Ion Transport Modes of NCBTs

-120

-80

-40

0

Vm

(mV)7.17.27.37.47.5

pHi

7.05 min

Vm pHi

NBCe1

NBCe1

CO2/HCO3−HEPES

CO2

H2CO3

H+ CO2

H2O HCO3−

pH ↓

2 HCO3−

1 Na+

-120

-80

-40

0

Vm

(mV)7.17.27.37.47.5

pHi

7.05 min

2 HCO3−

1 Na+

Vm pHi

NBCe1

NBCe1

CO2/HCO3−HEPES

CO2

H2CO3

H+ CO2

H2O HCO3−

pH ↑

-120

-80

-40

0

Vm

(mV)7.17.27.37.47.5

pHi

7.05 min

2 HCO3−

Vm pHi

NBCe1

NBCe1

CO2/HCO3−

0 NaHEPES

CO2

H2CO3

H+ CO2

H2O HCO3−

1 Na+

-120

-80

-40

0

Vm

(mV)7.17.27.37.47.5

pHi

7.0

Vm pHi

CO2

H2CO3

H+ CO2

H2O5 min

NBCn1

NBCn1

CO2/HCO3−

0 NaHEPES

1 HCO3−

1 Na+

HCO3−

-120

-80

-40

0

Vm

(mV)7.17.27.37.47.5

pHi

7.0

▲

Control oocyte

Vm pHi

CO2

H2CO3

H+ CO2

H2O HCO3−

H2O injected

CO2/HCO3−

0 NaHEPES

Ct

EL4

Nt

7 82 9 13 1410 11431 65

EL3

12

Extracellular

A fundamental question:Why some transporters (NBCe1, NBCe2) are electrogenic, and some (e.g., NBCn1, NBCn2) are electroneutral? What is the structural basis to determine the electrogenicity vs electroneutrality of the transporter?

Ct

EL4

Nt

7 82 9 13 1410 11431 65

EL3

12

Extracellular

The fourth extracellular loop (EL4) is the key structural elements determining the electrogenicity versus electroneutrality of NCBTs

Manipulation of homologous replacement on EL4 can interconvert NCBTs between electrogenic and electroneutral modes. (Chen LM et al, J Physiol, 2011)

CO2/HCO3-

0 Na

(Chen LM et al, J Physiol, 2011)

▼

5 min

▼

-120-100-80-60-40-20

0

Vm

(mV)

7.1

7.2

7.3

7.4

7.5

pHi

7.0

-120-100-80-60-40-20

0

Vm

(mV)

7.1

7.2

7.3

7.4

7.5

pHi

7.0

NBCe1

NBCn1

NBCe1

CO2/HCO3-

0 Na

-120-100-80-60-40-20

0

Vm

(mV)

7.1

7.2

7.3

7.4

7.5

pHi

7.0

-120-100-80-60-40-20

0

Vm

(mV)

7.1

7.2

7.3

7.4

7.5

pHi

7.0NBCe1

NBCn1

NBCe1

(Chen LM et al, J Physiol, 2011)

NBCn1

Structural Diversity and Effect on the Function of NCBTs

SLC4 family HCO3- transporters

AE3AE1

AE2

NBCe1

NBCn1

NBCn2

NDCBE

AE4

NBCe2

BTR1

0.1

(SLC4A2 )

(SLC4A1 )(SLC4A3)

(SLC4A4)

(SLC4A5)

(SLC4A7)

(SLC4A10)

(SLC4A8)

(SLC4A9)

(SLC4A11)

Electrogenic

Electroneutral

Cl-HCO3Exchangers

Na-dependent

Na-independent

89kb

1 2 4 5 6 7 11 3028 298-10 12-27

P1 P2 P3MEIK MCDL MHAN

a a

3

# # # # ##

Structure of Slc4a10 gene

MEIK-NBCn2

MCDL-NBCn2

MHAN-NBCn2

Promoter P1

Promoter P3

Promoter P2

1 2 3 4 5 6 7 8

100 aa

TMD

NBCn2-ANBCn2-BNBCn2-CNBCn2-D

NBCn2-KNBCn2-LNBCn2-MNBCn2-N

NBCn2-ENBCn2-FNBCn2-GNBCn2-HNBCn2-INBCn2-J

Nt Ct

A C BCassette

'rb7NCBE'rb3NCBE

Expression of NBCn2 variants

MEIK

MCDL

MHAN

(Liu et al 2013, PLOS ONE; Wang DK et al, Scientific Reports, 2015)

** Identified by our group for the first time

*********

*

BrainEye Hea

rtLiv

erKidney

Pancre

as

SpleenMus

cle

BrainKidney

Duodenum

ColonTe

stisEpid

idymis

Vas de

feren

s

Uterus

a-MCDL-NBCn2

a-MEIK-NBCn2

Tissue specific expression of NBCn2 variants

(Liu et al 2013, PLOS ONE)

Promoter P1 is primarily expressed in the central nervous systemm, but is also expressed in much less extent in many other tissues.Promoter P2 is primarily expressed in the kidney.

Structure of Slc4a7 gene encoding NBCn1

MEAD MERF IIITruncationIVIII

1 2 4-7 17-27 298 9 10 11 12-15 16 28

MDEL

3

# # # # # #

P2P1

Wang DK et al, Scientific Reports, 2015

Nt TMD Ct

Cassettes I II IIIIV

100 aa

NBCn1-G

NBCn1-A

NBCn1-BNBCn1-CNBCn1-DNBCn1-E

NBCn1-F

NBCn1-HNBCn1-I

NBCn1-KNBCn1-J

MERF

MEAD

NBCn1-LNBCn1-M

NBCn1-ONBCn1-P

NBCn1-N

NBCn1-e/gNBCn1-c/h

*****

*******

* Identified by our group for the first time

Expression of NBCn1 variants

NBCn1-RMIPLNBCn1-QMDEL

Promoter P2

Promoter P1

-120

-80

-40

0

Vm

(mV)7.17.27.37.47.5

pHi

7.0

HCO3-

Na+

Vm pHi

CO2

H2CO3

H+ CO2

H2O

CO2/HCO3−

0 NaHEPES

5 minHCO3

-

Affect of structural variation on NBCn1 activity

Xenopus oocyte

mNBCn1 B C D E F G I J N O H2ONt MEAD MEAD MEAD MEAD MERF MEAD MEAD MERF MERF MEAD NACas. I + – + + + + – + + – NACas. II + + + – – – – – – – NACas. III – + + – – + + + + + NACas. IV – – – – – – – – + + NA

(6) (6)(7) (9)

(6)

(12)

(7)(8)

(11) (10) (11)

0

5

10

15

20

dpH

i/dt (

×10-5

)

* *

B C D E F G I J N O0

5

10

15

20

NBCn1-dependent

dpH i/dt ( × 10-5 )

(6) (6)(7) (9)

(6)

(7) (8)

(11) (10) (11)

Liu et al 2013, Journal of Physiology

Total NBCn1in cell lysate

268

171117

B C D E F G I J N O kDa460

Surface NBCn1(Biotinylated)

268

171

117

460

NBCn1 variants

Liu et al 2013, Journal of Physiology

RelativeSurface NBCn1

abundance

B C D E F G I J N O0

0.5

1.0

1.5

2.0 B C D E F G I J N O0

5

10

15

20

NBCn1-dependent

dpH i/dt ( × 10-5 )

(6) (6)(7) (9)

(6)

(7) (8)

(11) (10) (11)

dpHi/dt (×10-5)

Surface NBCn1(6) (6)

(7)

(9) (6)

(7)(8) (11)

(10) (11)

05

1015202530

B C D E F G I J N OIndices of single molecular activity. Liu et al 2013, Journal of Physiology

dpHi/dt (×10-5)

Surface NBCn1(6) (6)

(7)

(9) (6)

(7)(8) (11)

(10) (11)

05

1015202530

B C D E F G I J N OIndices of single molecular activity. Liu et al 2013, Journal of Physiology

A comprehensive analysis shows that cassettes II, III and IV have stimulatory effects on the intrinsic activity of NBCn1.

Cassettes I II IV III

Structural cassettes of NBCn1

Role of NCBTs in renal physiology

Role of kidney in acid-base balance

(Koeppen, Adv Physiol Edu, 2009) NH4+ excretion

HCO3- reabsorption

Proximal Tubule

Henle’s loop

mTAL Collecting duct

Renal Bicarbonate Reabsorption

Proximal Tubule

Henle’s loop

mTAL

100% offiltered HCO3

−

10%

6%

Virtually no HCO3−

is left in urine

80%reabsorbed

Renal Bicarbonate Reabsorption

Collecting duct

4%

H2O

H2CO3

HCO3–

CO2

H+ H+

H2CO3

CO2 H2O

Na+

NHE3

1 Na+

3 HCO3–

CAIV CAII

NBCe1

H-pump

3 Na+

2 K+

Lumen InterstitialSpace

50%

30%

JHCO3−other

20%

Molecular mechanism of HCO3− reabsorption by

proximal renal tubule epithelial cells

Na-K pump

(For review, see Guo et al, Acta Physiol Sinica, 2014)

H2O

H2CO3

HCO3–

CO2

H+ H+

H2CO3

CO2 H2O

Na+

NHE3

1 Na+

3 HCO3–

CAIV CAII

NBCe1

H-pump

3 Na+

2 K+

Na+

HCO3– HCO3–

NBCn2

Lumen InterstitialSpace

NBCn2 presumably contributes to HCO3

– reabsorption in proximal tubule epithelial cells. (Guo YM et al, unpublished)

Na-K pump

Molecular mechanism of HCO3− reabsorption by

proximal renal tubule epithelial cells

ExtracellularEL4

IntracellularNt

7 8 9 13 1410431 6

EL3

427 522

799

721

Q29X R298S S427L

T485S G486R R510H

W516X L522P 2311A

A799V R881C 65bp

BA

(2311A)592

617

2 11588112

485

486

516

CC

CC

29

Core Domain

Linker

Variable region

NN

N597

510

Ct (65bp)

982

298

(For review, see Guo et al, Acta Physiologica Sinica, 2015)

NBCe1 Mutations associated with proximal renal tubule acidosis

(Weiner & Hamm, Ann Rev Physiol, 2007)

Renal Ammonium Excretion

Renal Ammonium Excretion

(Weiner & Hamm, Ann Rev Physiol, 2007)

Na+

HCO3−HCO3 –

Na+

NH4+

2Cl−NH4

+ NH3

H+ CO2

H2O

Na+

ROMK

NH4+

NH4+

NH4+

NHE4

NBCn2

NKCC2

Interstitial SpaceLumen

Na+

HCO3−HCO3 –

NBCn1

Other have shown that NBCn1 mediate HCO3- uptake to titrate NH4+. (For review, see Parker & Boron, 2013)

Role of NBCn2 in ammonium excretion in mTAL

We found that NBCn2 is also involved in this process. (Guo YM et al, unpublished)

100 aa

TMD

NBCn2-ANBCn2-BNBCn2-CNBCn2-D

NBCn2-KNBCn2-LNBCn2-MNBCn2-N

NBCn2-ENBCn2-FNBCn2-GNBCn2-HNBCn2-INBCn2-J

Nt Ct

A C BCassette

'rb7NCBE'rb3NCBE

Expression of NBCn2 variants

MEIK

MCDL

MHAN

(Liu et al 2013, PLOS ONE; Wang DK et al, Scientific Reports, 2015)

NBCn2-Ct

MEIK-NBCn2

Kidney

Cortex

Cortex

OSOMISOM

IM

MCDL-NBCn2

NHE3

AQP1

Na-K

Actin

Different NBCn2 variants have distinct distribution in rat kidney

(Guo YM et al, unpublished)

S1

AQP1

G

MergeC

NBCn2E*

G

MergeF

50 μm

NBCn2B

D

50 μm

α1

ACortex Cortex

(Guo YM et al, unpublished)

Localization of NBCn2 in cortex of rat kidney

Anti-MEIK Anti-α1 Merge

Anti-NBCn2-Ct Anti-α1 Merge

Localization of NBCn2 in mTAL of rat kidney

Anti-NHE3 Anti-α1 Merge

A B C

D E F

G H I

20um

20um

20um

mTAL

mTAL

mTAL

(Guo YM et al, unpublished)

Actin

MCDL-NBCn2

p=0.5882.0

1.5

1.0

0.5

0Control NH4Cl

(9)(10)

NH4Cl

MCDL-NBCn2 Actin

(Guo YM et al, unpublished)

Effect of NH4Cl-induced metabolic acidosis on NBCn2 abundance in rat kidney

MEIK-NBCn2

Actin

2.0

0 Control NH4Cl

4.0

p=0.00011

6.0

8.0

(10)

(9)

NH4Cl

MEIK-NBCn2 Actin

NBCn1

Actin

8.0

Control NH4Cl

6.04.02.0

0

10.012.014.0

(9)

(10)

NH4Cl

Effect of NH4Cl-induced metabolic acidosis on NBCn2 abundance in rat kidney

(Guo YM et al, unpublished)

NBCn1Actin

p=0.00024

NHE3Actin

5.04.0

Control NH4Cl

3.02.01.0

0

NH4Cl

p=0.0059

(10)

(9)

NHE3Actin

1. The NCBTs of SLC4 family are a major group of pH regulators widely expressed in diverse tissues, playing vital physiological and pathological roles. They could be potential targets for drug development.

2. The five NCBT genes produce a great extend of diversities in their expression products by using alternative promoters and alternative splicing of pre-mRNAs.

3. The expression of different variants of a same NCBT is highly tissue and cell-type specific.

4. The optional structural elements of NCBTs could play important role in the functional regulation of the transporters.

Summary and conclusion

Acknowledgement

Chen Lab @HUST Case Western Reserve Univ

State Univ. New York @Buffalo

Dr. Mark D. ParkerEvan J. Myers

Dr. Walter F. BoronDr. Xue QIN

Dr. Ying LIUDeng-Keng WANG Yi-Min GUOJiu-Ying XUDe-Zhi JIANGZhang-Dong XIEMei LIUJin-Lin WANG Xiao-Yu WANG Pan SU

Dr. Nathan MorrisDr. Musa-Aziz Raif

MOE and MOF of PR ChinaCHUTIAN Scholar Project, HubeiStartup funding from HUST

Ion selective electrode

3 M KCl Standard pH solution

Liquid cocktail is permeable to proton only

Vm electrode pH electrode

Lee Boron Parker 2013Dimeski 2010

0.5 mM NH3/NH4

+5% CO2

33 mM/ HCO3

2 min

7.5

7.7

pHS

7.3

AQP1H2O

SGLT1

7.5

7.7

pHS

7.3AQP1H2O

NKCC2

7.5

7.7

pHS

7.3

AQP1H2O

Pept1

Negative Controls

[NH3]S

pHS

NH3

NH4+

H+

pHspHsHCO3– H+

H2O

pHS

[CO2]S

CO2

0.5 mM NH3 + NH4

+5%CO2

33 mM HCO3

D160A

7.5

7.5

AQP5

AmtB

2 min

7.5RhAG

D167N

AQP47.5

7.5

[NH3]S

pHS

NH3

NH4+

H+

pHspHsHCO3– H+

H2O

pHS

[CO2]S

CO2

Xenopus oocytes: Surface pH Changes Caused by Influx of CO2 or NH3

RBCs, “ubiquitous”

Astrocytic endfeet of BBB

Alveolar type I cells

Bacterial Rh homolog

RBC Rh complex

AQP0Eye (lens)

AQP1

H2O7.5

7.7

pHS

7.3

P=10-13

(13)

AQP5

H 2O

AQP4

H 2O

(11)

P=10-5 (17)

(14)

AQP1

(10)

H 2O

(9)

P=10-6

0.00

0.05

0.10

0.15

F G H

ΔpH S

for C

O2

I

ΔpH S

for C

O2

AmtB

0.00

0.05

0.10

0.15(16)

P=0.02

P<10-4

P=0.35

D160

A A

mtB

(10)

H 2O

(8)

J

D167

NRh

AG

RhAG

H 2O

(11)(24)

(26)

P=10-4

P<10-4

P=0.6

(13)(14) (11)

P=0.6P=10-4

(10)(9)

-0.15

-0.10

-0.05

0.00

P=0.8ΔpH S

for N

H 3

(17)

ΔpH S

for N

H 3

-0.15

-0.10

-0.05

0.00

(16)

(10) (8)

P<10-4

P<10-4

P=0.5

(26)(11)

P<10-4

P<10-4

P=0.3(24)

5%CO2/ 33mMHCO3

0.5 mM NH4Cl

7.5

7.5

7.7

pHS

A

B

C

D

E7.5

7.5

D160A

2 min

AQP5

AQP1

H2O

AmtB

RhAG

D167N

AQP47.5

7.3

Xenopus oocytes:Surface pH Summary

Musa-Aziz R, Chen LM, et al, PNAS, 2009

Xenopus oocytes:Subtract Day-Matched H2O Background

AQP1

AQP4

AQP5

AmtB

(16)

RhAG

(16) (24)

(17)(10)

AQP0

0.00

0.05

0.10

(pHS*)CO2 (13)

(10)(14) (24)

(17)

(13) (14)0.00

-0.10

-0.05(pHS*)NH3

Relative, channel-specific

NH3 permeability

AQP1

AQP4

AQP5

AmtB

RhAG

∞

AQP0

∞

0

3

2

1PCO

2*/PNH

3*

∞

Relative, channel-specific

CO2 permeability

Musa-Aziz, Chen et al, PNAS, 2009