Structure determinants for CO2 transport of AQP5

Structure determinants for CO2 transport of human aquaporin5

Xue Qin PI: Walter F Boron

Department of Physiology and BiophysicsCase Western Reserve UniversityGas Channel Workshop

BackgroundAquaporin 5 is a water channel highly expressed in salivary glands, eye, lung and trachea.Aquaporins are composed of 6 transmembrane domains, with N- and C- terminus on the cytoplasmic side of the membrane.

Fig 5Topology of AQP1H2N124563COOHRepeated NPA motifsAEBCD

BackgroundH2O permeability2 filter regions is important for H2O transportSelectivity filter: ar/R regionNPA regionCO2 permeabilityNot well defined

Horsefield et al, 2008 PNASnn

H2O permeability--PfCO2 permeability--pHSVolumetric assay to measure osmotic water permeabilityHow fast the volume of oocytes change with timeMicroelectrode to measure pH on the surface of the oocytesMethods

OutlineI Amino acids at the mouth of the central poreII Amino acids lining the central pore

OutlineI Amino acids at the mouth of the central poreII Amino acids lining the central pore

water porecentral pore

3

D50T41L43V52Protein sequence alignment

bAQP1hAQP5H2OhAQP5T41YT41WT41FT41G0.0000.0010.0020.0030.004Pf (cm/s)050100150200hAQP5T41YhAQP5T41WhAQP5T41FhAQP5T41GRelative channel-dependent CO2 permeability

L43T41

T41YT41Y

T41WT41W

T41F

T41GConclusion II Amino acids at the mouth of the central poreChanges of pHS (CO2 permeability) is more sensitive than Pf (H2O permeability)T41 is more important than L43.

II Amino acids lining the central pore

OutlineI Amino acids at the mouth of the central poreII Amino acids lining the central pore

Amino acids lining the central pore

T41L43L47L51L167L163G15921Sequence alignment of AQPs

TM2TM5If mutate to Asp (D)

T41L43L47L51L167DDDDDChannel-dependent pHS for CO20.000.030.060.09hAQP5 T41DhAQP5 L43DhAQP5 L47DhAQP5 L51DhAQP5 L167DLXXD pHS*Pf*

Aspartic Acid (Asp)

Channel-dependent Pf (cm/s)0.0000.0010.0020.0030.004H2OhAQP5 T41DhAQP5 L43DhAQP5 L47DhAQP5 L51DhAQP5 L167DLXXD Pf*

Aspartic Acid (Asp)If mutate to Arg (R)

T41L43L47L51L167RRRRR-0.020.000.020.040.060.080.100.12hAQP5 T41RhAQP5 L43RhAQP5 L47RhAQP5 L51RhAQP5 L167RChannel-dependent pHS for CO2LXXR pHS*Arginine (Arg)

Pf*

Channel-dependent Pf (cm/s)0.0000.0010.0020.0030.004H2OhAQP5 T41RhAQP5 L43RhAQP5 L47RhAQP5 L51RhAQP5 L167RLXXR Pf*

Arginine (Arg)

If mutate to Thr (T)

T41L43L47L51L167TTTTTChannel-dependent pHS for CO20.000.030.060.09hAQP5 T41wt AQP5hAQP5 L43ThAQP5 L47ThAQP5 L51ThAQP5 L167TLXXT pHS*Threonine (Thr)

Pf*

Channel-dependent Pf (cm/s)0.0000.0010.0020.0030.004H2OhAQP5 T41ThAQP5 L43ThAQP5 L47ThAQP5 L51ThAQP5 L167TLXXT Pf*

Threonine (Thr)

L163TConclusion III Amino acids at the mouth of the central poreChanges of pHS (CO2 permeability) is more sensitive than Pf (H2O permeability)T41 is more important than L43.II Amino acids lining the central poreChanges of Pf (H2O permeability) is more sensitive than pHS (CO2 permeability) Of all the amino acids lining the central pore, L51 is most sensitive to determine Pf and pHS.

AcknowledgementCollaboratorEmad Tajkhorshid

LabWalter F BoronRaif Musa-AzizMark D Parker