Slide 1

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 Slide 2 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. Slide 3 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 Slide 4 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 Slide 5 Outline I Amino acids at the mouth of the central pore II Amino acids lining the central pore Slide 6 Outline I Amino acids at the mouth of the central pore II Amino acids lining the central pore Slide 7 water pore central pore Slide 8 Slide 9 3 Slide 10 D50 T41L43 V52 Protein sequence alignment Slide 11 bAQP1hAQP5 Slide 12 H2OH2O T41YT41WT41FT41G 0.000 0.001 0.002 0.003 0.004 P f (cm/s) Slide 13 0 50 100 150 200 hAQP5T41YhAQP5T41WhAQP5T41FhAQP5T41G Relative channel- dependent CO 2 permeability Slide 14 L43 T41 Slide 15 T41Y Slide 16 T41W Slide 17 T41F Slide 18 T41G Slide 19 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 Slide 20 Outline I Amino acids at the mouth of the central pore II Amino acids lining the central pore Slide 21 Amino acids lining the central pore T41 L43 L47 L51 L167 L163 G159 Slide 22 Sequence alignment of AQPs TM2 TM5 Slide 23 If mutate to Asp (D) T41 L43 L47 L51 L167 D D D D D Slide 24 Channel-dependent pH S for CO 2 0.00 0.03 0.06 0.09 hAQP5T41DhAQP5L43DhAQP5L47DhAQP5L51DhAQP5L167D LXXD pH S * Pf*Pf* Aspartic Acid (Asp) Slide 25 Channel-dependent P f (cm/s) 0.000 0.001 0.002 0.003 0.004 H2OH2O hAQP5T41DhAQP5L43DhAQP5L47DhAQP5L51DhAQP5L167D LXXD P f * Aspartic Acid (Asp) Slide 26 If mutate to Arg (R) T41 L43 L47 L51 L167 R R R R R Slide 27 -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12 hAQP5T41RhAQP5L43RhAQP5L47RhAQP5L51RhAQP5L167R Channel-dependent pH S for CO 2 LXXR pH S * Arginine (Arg) Pf*Pf* Slide 28 Channel-dependent P f (cm/s) 0.000 0.001 0.002 0.003 0.004 H2OH2O hAQP5T41RhAQP5L43RhAQP5L47RhAQP5L51RhAQP5L167R LXXR P f * Arginine (Arg) Slide 29 If mutate to Thr (T) T41 L43 L47 L51 L167 T T T T T Slide 30 Channel-dependent pH S for CO 2 0.00 0.03 0.06 0.09 hAQP5T41 wt AQP5 hAQP5L43ThAQP5L47ThAQP5L51ThAQP5L167T LXXT pH S * Threonine (Thr) Pf*Pf* Slide 31 Channel-dependent P f (cm/s) 0.000 0.001 0.002 0.003 0.004 H2OH2O hAQP5T41ThAQP5L43ThAQP5L47ThAQP5L51ThAQP5L167T LXXT P f * Threonine (Thr) L163T Slide 32 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. Slide 33 Acknowledgement Collaborator Emad Tajkhorshid Lab Walter F Boron Raif Musa-Aziz Mark D Parker