September 6, 2012Cleveland, Ohio

Department of Physiology & BiophysicsCase Western Reserve University School of Medicine

10900 Euclid AvenueCleveland, OH 44106-4906

Walter F. Boron, M.D., Ph.D.

Gas Channels

Gas Channels Workshop

Department of Physiology & BiophysicsOffice of Naval Research &

Outline• Background• Computer simulations• Gas selectivity by channels• Physiological significance

Introduction of the “CO2 Pulse”(squid giant axon)

pH i

6.8

7.0

7.2

7.4

Vm-58

-56

5% CO2 / 50 mM HCO3–

10 minHCO3

–

CO2

H+

H2O+

+

CO2 H2O

HCO3–H+ +

+

pHi

Boron & De Weer, J Gen Physiol 67, 1976 Paul De Weer

Introduction of the “CO2 Pulse”(squid giant axon)

pH i

6.8

7.0

7.2

7.4

Vm-58

-56

10 min

Boron & De Weer, J Gen Physiol 67, 1976

5% CO2 / 50 mM HCO3–

HCO3–

CO2

H+

H2O+

+ HCO3–H+ +

CO2 H2O+pHi

Energy

(squid giant axon)

pH i

6.8

7.0

7.2

7.4

Vm-58

-56

10 min

Boron & De Weer, J Gen Physiol 67, 1976

5% CO2 / 50 mM HCO3–

First Example of Active Regulation of pHi

(squid giant axon)

pH i

6.8

7.0

7.2

7.4

Vm-58

-56

10 min

Boron & De Weer, J Gen Physiol 67, 1976

5% CO2 / 50 mM HCO3–

First Example of Active Regulation of pHipHi

Na+

CO3=

Cl–CO2 pHi

NDCBE

#1 #2

Na-Driven Cl-HCO3 Exchanger

#1

#2

Roger C. Thomas John M. Russell

7.0

7.2

7.4

7.6

7.8

Vm -62

-58

Boron & De Weer, J Gen Physiol 67, 1976

pHi

15 min NH4+

NH3H+ +

pHi

+

NH4+

NH3 H+

NH4+

pHi

10 mM NH4Cl

The Ammonium Prepulse(squid giant axon)

The Ammonium Prepulse(squid giant axon)

7.0

7.2

7.4

7.6

7.8

-62

-58

Boron & De Weer, J Gen Physiol 67, 1976

pHi

Vm

15 min

NH4+

H+ + +NH3 H+

NH4+

pHi

NH3

10 mM NH4Cl 10 mM NH4Cl

… inspired by the work of Overton

The Dogma…

… more than a century ago …

All gases move through all membranes simply by dissolving in the membrane lipid.

Gas diffusion through a membrane#1

Access

[X]W = sW pX [X]L = sL pX

#2Solubility

#3Diffusion

D

Henry’s Law

This how gases cross artificial membranes and some biological membranes …

… but not all

#4Egress Solubility theory

P sL/sW“Overton’s rule”Overton (1897)

JK Mitchell (1831)Solubility-

Diffusion theoryP (sL/sW) D

T Graham (1866)

Access-Solubility-Diffusion-Egress theory

P (A/E)(sL/sW) DBoron (2010)

When would a gas channelmake physiological sense?

D

JX = PX([X]o – [X]i)

#2 Gradient is low

#3 Physiological demand is high

Fick’s Law:

[X]i

#2#1#3

A gas channel could:

[X]o

(3) be under physiological regulation

(1) enhance flux if PX is low, (2) display selectivity for a particular gas, or

(4) Be amenable to pharmacological intervention

#1 Background permeability is low

(unstirred layers cannot overwhelm membrane )

*An absolute sine qua non

*

*

*In mammals, ULs are tiny in high-flux systems*Includes access, s, D, egress

Takamori … Jahn, Cell 127, 2006

Molecular Anatomy of a Trafficking Organelle

“Note that the model … accounts for approximately 2/3 of the protein mass of [synaptic vesicles].

It can be envisioned that, viewed from the outside, the lipidic surface is hardly visible when all [integral membrane] proteins are present …”

WFB: This model does not include the soluble proteins that bind to the vesicle …

… further limiting access of dissolved gases such as CO2.

Gas Channels Workshop

We will hear more about permeability barriers from Volker Endeward …

… and this will be a subject of discussion tomorrow

Gas Channels Workshop

We will hear more about the regulation of permeability to water and gases from Bhanu Jena

pHi

6.8

7.0

7.2

7.4

5 min

The First Gas-Impermeable Membrane

Luminal Change

100% CO2 pH 6.1

1% CO2 pH 7.4

5% CO2 pH 7.4

Basolateral Changes

Perfusion SideCollection SideCO2

Parietal CellEndocrine CellChief Cell

Waisbren et al,Nature 368, 1994

Parietal Cell

cRNA

3 days

cRNA

ExpressedAQP1

pH Vm

CO2 CO2

Xenopus-Oocyte Expression System

?

7.3

7.2

7.1

pHi

10 sec

CO2 / HCO3-

“Ooze”Time(s)

DpHi/Dtx10-4 pH/s

180 -9.6

82 -25.1

50 -35.8

Cooper & Boron, AJP Cell 275, 1998

Effect of AQP1 Expression on CO2 Permeability The First Gas Channel

Nakhoul et al, AJP Cell 274, 1998

Outline• Background• Computer simulations• Gas selectivity by channels• Physiological significance

Technical Approach

Molecular Dynamics (MD) simulations

Start with crystal structure and interatomic forces

Calculate vibrational movements of atoms, every 1 fs in real time … for a total of ~10 ns

Sui et al, Nature 414, 2001

Central pore:Mainly hydrophobic~3 A at narrowestGated by

hydrophobic residues

Aquapore:Hydrophilic &

hydrophobicLength: 18 – 20 ADiameter: 2.8 – 4 A at

narrowest (near bilayer center)

AQP1 Structure (top view)

Wang et al, J Struct Biol 157, 2007

Molecular Dynamics Simulation:CO2 through the Central Pore of AQP1

CO2

Emad Tajkhorshid

Running Conclusions

O2 and CO2 movement through AQP1 is feasible …

… both via the aquapores and the central pore

The central pore (a ~vacuum) may be the perfect channel for nonpolar gases

Gas Channels Workshop

We will hear more about Molecular Dynamics modeling from Emad Tajkhorshid

Gas Channels Workshop

We will hear more about the structural biology of proteins that act as gas channels from Bob Stroud

Outline• Background• Computer simulations• Gas selectivity by channels• Physiological significance

Technical Approach

Express mammalian channels in Xenopus (frog) oocytes.

Study dissolved gases that change pH

Measure pH on the surface of the oocyte using pH-sensitive microelectrodes

Xenopus oocyte:pH Changes Caused by CO2 Influx

CO2CO2

HCO3–

H+

H2O

HCO3–

CO2

H2OHCO3

–H+

pHi

pHS

[CO2]S

[HCO3–]

pH … with 15-m tip

5% CO2

33 mM HCO3–

AQP1

H2O2 min

pHS

7.5

7.7

Musa-Aziz et al, PNAS, 2009

Bulk Extracellular Fluid

Xenopus oocyte:pH Changes Caused by NH3 Influx

NH3

pH … with 15-m tip

NH3

NH4+

H+

NH4+

NH3 NH4+

H+

pHipHS

[NH3]S

[NH4+]

0.5 mM

NH3 + NH4+

2 minAQP1

H2OpHS 7.5

7.7

7.3

Musa-Aziz et al, PNAS, 2009

Bulk Extracellular Fluid

Intracellular Fluid(ICF)

HCO3-

+

A

HA

H+

+

CO2

H2OH2O

1k1k

2k 2k

2k 2k

H2CO3

HCO3-

+

A

HA

+

CO2

H2OH2O

1k1k

2k 2k

2k 2k

H2CO3

H+

HCO3-

+

A

HA

H+

+

CO2

H2OH2O

1k1k

2k 2k

2k 2k

H2CO3

HCO3-

+

A

HA

+

CO2

H2OH2O

1k1k

2k 2k

2k 2k

H2CO3

H+

Extracellular Unconvected Fluid (EUF)

Free Diffusion

Bul

k Ex

trac

ellu

lar F

luid

(BE

CF)

d

Somersalo, Occhipinti, Boron, Calvetti, J Theor Biol, 2012

0 200 400 600 800 1000 12007.500

7.502

7.504

7.506

7.508

Time (sec)

pHS

2M,CO 34.2 cm/secP =2

1M,CO /10P

2

2M,CO /10P

2

3M,CO /10P

2

4M,CO /10P

2

4M,CO / 2.5 10P ×

2

4M,CO / 5.0 10P ×

2

4M,CO / 7.5 10P ×

2

5M,CO /10P

(A)

0 200 400 600 800 1000 12007.00

7.05

7.10

7.15

7.20

Time (sec)

pHi

(C)

10-4

10-2

100

1020

2

4

6

8

(DpHS)max

PM,CO 2 (cm/sec)

x 10-3 (B) (D)

0

x 10-3

10-4

10-2

100

102

1

2

3

-(dpHi/dt )max

PM,CO 2 (cm/sec)Rossana Occhipinti

Implications

The background permeability of the membrane (i.e., in the absence of gas channels) must be very low.

With additional refinements to the model, we ought to be able to be able to estimate absolute permeabilities.

Gas Channels Workshop

We will hear more about the macroscopic modeling of CO2 influx into oocytes from Rossana Occhipinti, tomorrow morning

Pf* (cm/s)

0.000

0.001

0.002

0.003

0.004

(14)AQ

P1

AQP2

AQP4

M1

AQP0

AQP3

AQP4

M23

(7)(6)

(7) (7)

(5)

rAQ

P7

(4)

hAQ

P8

(5)

hAQ

P9

(4)

Channel-specific H2O permeability

More Aquaporins

Musa-Aziz, Geyer, Boron

R. Ryan Geyer Raif Musa-Aziz

More Aquaporins

0.00

0.02

0.04

0.06

0.08

(5)

(9) (12)

-0.08

-0.06

-0.04

-0.02

0.00

hAQ

P2

rAQ

P3

hAQ

P1

Relative, channel-specificCO2 permeability

Relative, channel-specificNH3 permeability

(11)

rAQ

P4

M1

(6)

rAQ

P4

M23

(5)

(9)

(12)

(11)(6)

NS from zero

NS from zero

AQP0

NS from zero

(13)

(13)(3)

rAQ

P7

(3)

(5)

hAQ

P8

(5)

(4)

hAQ

P9

(4)

NS from zero

NS from zero

Musa-Aziz, Geyer, Boron

(DpHS*)CO2

(DpHS*)NH3

0102030405060

Xenopus oocytes:CO2 over Pf or NH3 over Pf

0102030405060

AQP1

AQP4

M23

AQP5

AQP6

N60G

AQP0

AQP9

AQP3

AQP7

AQP8

AQP6

wt

AQP2

AQP4

M1

(17)

CO2

(13) (5)(6)

(17)NH3

(10)

(5)(13)

(10)

(9) (12)(17)

∞

(13)∞(13)

(13) (12)(12) (13) (6)(12)(12) (17) (9) (12)

(DpHS*)CO2

Pf*

(DpHS*)NH3

Pf*

Xenopus oocytes:CO2 over NH3

Musa-Aziz … Boron, unpublished

AQP1

AQP4

M23

AQP5

AQP6

wt

AQP9

∞ ∞ ∞

AQP0

0123

(13) (6) (17)(13)

(17)

(5)

AQP6

N60G

(10)

AQP2

(12)

AQP4

M1

(11)AQ

P3‡ ‡

AQP7

AQP8

(9) (12)

(11)

(DpHS*)CO2

(DpHS*)NH3

‡ Undefined (0/0)

More Rhesus Proteins: RhBG & RhCG

Geyer, Toye, Boron, Musa-Aziz

0.00

0.02

0.04

0.06

0.08

(8)

(12)(14)

-0.08

-0.06

-0.04

-0.02

0.00

(8)

RhBG

RhCG

RhAG

(12)

(14)

0.00

0.02

0.04

0.06

0.08

(8)

(12)(14)

-0.08

-0.06

-0.04

-0.02

0.00

(8)

(12)

(14)

0.0

0.2

0.4

0.6

0.8

(8)

(12)

(14)

1.0

RhBG RhCGRhAG

(DpHS*)CO2

(DpHS*)NH3

Relative index of CO2/NH3 permeability

0.0

0.2

0.4

0.6

0.8

(8)

(12)

(14)

1.0

(DpHS*)CO2

(DpHS*)NH3

Relative index of CO2/NH3 permeability(DpHS

*)CO2

(DpHS*)NH3

Relative, channel-specificCO2 permeability

Relative, channel-specificNH3 permeability

Gas Channels Workshop

We will hear more about the role of Rh proteins as NH3 channels from David Weiner

What is the molecular basis of gas selectivity?

Question

Sui et al, Nature 414, 2001

Central pore:Hypothesis …

blocked by DIDS

Aquapore:Blocked by HgCl2 and

pCMBS

AQP1 Structure (top view)

H2O & NH3 Pathways through hAQP1

0% H2O and NH3 (DIDS has no effect)

25% H2O & NH3 (blocked by pCMBS) = 100%

CO2 Pathways through hAQP1

60% CO2 (blocked by DIDS)

10% CO2 (blocked by pCMBS) = 40%

DIDS + pCMBS blocks ~100%?

Gas Channels Workshop

We will hear more about the use of surface-pH measurements to assess CO2 influx into oocytes from Xue Qin, tomorrow morning

Are there other families of gas channels?

Questions

Might oligomers with ‘central pores’ be a place to look?

These will be subjects of discussion tomorrow

Running Conclusions

Like ion channels, gas channels can exhibit selectivity

It appears that it is possible to block the alternate pathway (but not the aquapore) with DIDS

It appears that, in the case of AQP1, it is possible to block CO2 transport through the aquapore (but not the alternate pathway) with pCMBS

Outline• Background• Computer simulations• Gas selectivity by channels• Physiological significance:

The renal proximal tubule

Running Conclusions

Knocking out AQP1 reduces HCO3 reabsorption by 40%–60%

About 60% of the CO2 permeability of the proximal-tubule epithelium requires AQP1

AQP1-null mice exhibit a major defect in defending arterial pH in the face of metabolic acidosis

Gas Channels Workshop

We will discuss the physiological role of gas channels tomorrow

Outline• Background• Computer simulations• Gas selectivity by channels• Physiological significance:

The renal proximal tubuleRed Blood Cells

Technical Approach

Mass Spec of 18O-labeled CO2

18O label is lost to H2O as CO2 enters RBCs and becomes exposed to CA II

HCO3–H+

CO2H2O+

+

CO2 H2O

HCO3–H+ +

Human Red Blood Cells:CO2 Permeability of AQP1

+

Rh complex(~35% of total)

AQP1(~60% of total)

Lipid(at most ~5%)

Endeward et al, FASEB J 20, 2006

½ blocked by DIDS All blocked by DIDS

Conclusions

In human RBCs, ~60% of the CO2 moves via AQP1 … most of the remainder probably moves via RhThe lipid of the plasma membrane can account for,

at most, 5% of the CO2 permeability.The tightness of the RBC membrane lipids may

reflect an adaptation that allows the RBC to resist physical abuse

Gas Channels Workshop

We will hear more about the CO2 permeability of RBCs—and the use of 18O exchange—from Gerolf Gros

Next Steps

Extend experiments to mouse RBCs, using RhAG-null and AQP1/RhAG double knockouts

Use stopped-flow device to measure O2 fluxes across RBC membranes (exploiting the absorbance spectrum of Hb) …

… and …

Effect of knocking out AQP1 or RhAG on Oxygen efflux from mouse Red Blood Cells

(7)

Control

(7)

AQP1-KO

(1)

RhAG-KO

0.00

0.25

0.50

0.75

1.00Re

lativ

e O

2 effl

ux

Geyer RR & Boron WF.

9% 15%

Although AQP1 and Rh complex account for 90-95% of CO2 permeability in human RBCs …… might contribute a maximum of only ~25% to O2 permeability.

Are AQP1 and Rh primarily CO2 channels with an O2 “leak”?Do RBCs have “dedicated” O2 channels?

Effect of Inhibitors onOxygen efflux from mouse Wild-Type Red Blood Cells

(10)

Control

(10)

+pCMBS

(7)

DIDS +

(3)+

pCMBS + Phloretin

(8)

+Phloretin

0.00

0.25

0.50

0.75

1.00

Rela

tive

O2 e

fflux

Geyer RR & Boron WF.

50% 40%20%

75%

All P 10-4 vs Control

Conclusions

In mouse RBCs, ~10% of the O2 may move via AQP1 (we still need WBs for major RBC proteins)

The Rh complex may mediate a similar fraction (we need to increase ‘N’)

About 70% of O2 permeability is blocked by the combination of pCMBS + phloretin …

… suggesting to us that RBS have at least two O2 channels (perhaps with CO2 leak) with partially overlapping sensitivities to pCMBS and phloretin

Gas Channels Workshop

We will hear more about the O2 permeability of oocytes from Ryan Geyer, tomorrow morning

Gas Channels Workshop

We will hear about channel-mediated fluxes of NO from Jeff Garvin

Outline• Background• Computer simulations• Gas selectivity by channels• Physiological significance:

The renal proximal tubuleRed Blood Cells: AQP1 (60%) & Rh (35%) of CO2

Voluntary Exercise: AQP1 KOs vs WT

Overall Conclusions• AQPs, Rhs, UTs conduct dissolved gas• Computer simulations: reasonable for AQP1

• Gas selectivity of AQP/Rh : AQPs > Rh

• Can block CO2 vs. NH3 transport selectively• Physiology: Movement of dissolved gas through

channels appears to be significant for proximal tubules, RBCs, and exercise.

Department of Physiology & BiophysicsCase Western Reserve University School of Medicine

10900 Euclid AvenueCleveland, OH 44106-4906

Walter F. Boron, M.D., Ph.D.Phone: 216-368-3400

charleen.bertolini@case.eduwalter.boron@case.edu

Contact Information

DIDS appears to block the Central Pore here, too

AmtB0% CO2

100% CO2

DIDSblocks

RhAG0% CO2

100% CO2

DIDS blocks

33% NH3

0% NH3

DIDS blocks none

33% NH3

0% NH3

DIDS blocks none

Takamori … Jahn. Molecular anatomy of a trafficking organelle. Cell 127: 831-846, 2006.

“Note that the model … accounts for approximately 2/3 of the protein mass of [synaptic vesicles].

It can be envisioned that, viewed from the outside, the lipidic surface is hardly visible when all [integral membrane] proteins are present …”

WFB: This model does not include the soluble proteins that bind to the vesicle …

… further limiting access of dissolved gases such as CO2.

Rossana Occhipinti