Structure determinants for CO 2 transport of human aquaporin5 Xue Qin PI: Walter F Boron Department of Physiology and Biophysics Case Western Reserve University Gas Channel Workshop

Background Aquaporin 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.

Background H 2 O permeability 2 filter regions is important for H 2 O transport Selectivity filter: ar/R region NPA region CO 2 permeability Not well defined Horsefield et al, 2008 PNAS n n

H 2 O permeability--P f CO 2 permeability--  pH S Volumetric assay to measure osmotic water permeability How fast the volume of oocytes change with time Microelectrode to measure pH on the surface of the oocytes Methods

Outline I Amino acids at the mouth of the central pore II Amino acids lining the central pore

Outline I Amino acids at the mouth of the central pore II Amino acids lining the central pore

water pore central pore

3

D50 T41L43 V52 Protein sequence alignment

bAQP1hAQP5

H2OH2O T41YT41WT41FT41G P f (cm/s)

hAQP5T41YhAQP5T41WhAQP5T41FhAQP5T41G Relative channel- dependent CO 2 permeability

L43 T41

T41Y

T41W

T41F

T41G

Conclusion I I Amino acids at the mouth of the central pore Changes of  pH S (CO 2 permeability) is more sensitive than P f (H 2 O permeability) T41 is more important than L43. II Amino acids lining the central pore

Outline I Amino acids at the mouth of the central pore II Amino acids lining the central pore

Amino acids lining the central pore T41 L43 L47 L51 L167 L163 G159

Sequence alignment of AQPs TM2 TM5

If mutate to Asp (D) T41 L43 L47 L51 L167 D D D D D

Channel-dependent  pH S for CO hAQP5T41DhAQP5L43DhAQP5L47DhAQP5L51DhAQP5L167D LXXD −  pH S * Pf*Pf* Aspartic Acid (Asp)

Channel-dependent P f (cm/s) H2OH2O hAQP5T41DhAQP5L43DhAQP5L47DhAQP5L51DhAQP5L167D LXXD − P f * Aspartic Acid (Asp)

If mutate to Arg (R) T41 L43 L47 L51 L167 R R R R R

hAQP5T41RhAQP5L43RhAQP5L47RhAQP5L51RhAQP5L167R Channel-dependent  pH S for CO 2 LXXR −  pH S * Arginine (Arg) Pf*Pf*

Channel-dependent P f (cm/s) H2OH2O hAQP5T41RhAQP5L43RhAQP5L47RhAQP5L51RhAQP5L167R LXXR − P f * Arginine (Arg)

If mutate to Thr (T) T41 L43 L47 L51 L167 T T T T T

Channel-dependent  pH S for CO hAQP5T41 wt AQP5 hAQP5L43ThAQP5L47ThAQP5L51ThAQP5L167T LXXT −  pH S * Threonine (Thr) Pf*Pf*

Channel-dependent P f (cm/s) H2OH2O hAQP5T41ThAQP5L43ThAQP5L47ThAQP5L51ThAQP5L167T LXXT − P f * Threonine (Thr) L163T

Conclusion II I Amino acids at the mouth of the central pore Changes of  pH S (CO 2 permeability) is more sensitive than P f (H 2 O permeability) T41 is more important than L43. II Amino acids lining the central pore Changes of P f (H 2 O permeability) is more sensitive than  pH S (CO 2 permeability) Of all the amino acids lining the central pore, L51 is most sensitive to determine P f and  pH S.

Acknowledgement Collaborator Emad Tajkhorshid Lab Walter F Boron Raif Musa-Aziz Mark D Parker