Structural and functional characterization of the human NBC3 sodium/bicarbonate co-transporter carboxyl-terminal cytoplasmic domain.
The sodium bicarbonate co-transporter, NBC3, is expressed in a range of tissues including heart, skeletal muscle and kidney, where it modulates intracellular pH and bicarbonate levels. NBC3 has a three-domain structure: 67 kDa N-terminal cytoplasmic domain, 57 kDa membrane domain and an 11 kDa C-terminal cytoplasmic domain (NBC3Ct). The role of C-terminal domains as important regulatory regions is an emerging theme in bicarbonate transporter physiology. This study determined the functional role of human NBC3Ct and characterized its structure using biochemical techniques. The NBC3 C-terminal domain deletion mutant (NBC3DeltaCt) had only 12 +/- 5% of wild-type transport activity. This low activity is attributable to low steady-state levels of NBC3DeltaCt and almost complete retention inside the cell, as assessed by immunoblots and confocal microscopy, suggesting a role of NBC3Ct in cell surface processing. To characterize the structure of NBC3Ct, amino acids 1127-1214 of NBC3 were expressed as a GST fusion protein (GST.NBC3Ct). GST.NBC3Ct was cleaved with PreScission Protease and native NBC3Ct could be purified to 94% homogeneity. Gel permeation chromatography and sedimentation velocity ultracentrifugation of NBC3Ct indicated a Stokes radius of 26 and 30 angstroms, respectively. Shape modelling revealed NBC3Ct as a prolate shape with long and short axes of 19 and 2 nm, respectively. The circular dichroism spectra of NBC3Ct did not change over the pH 6.2-7.8 range, which rules out a large change of secondary structure as a component of pH sensor function. Proteolysis with trypsin and chymotrypsin identified two proteolytically sensitive regions, R1129 and K1183-K1186, which could form protein interaction sites.
Loiselle
Frederick B
FB
CIHR Membrane Protien Research Group, Department of Physiology and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Jaschke
Paul
P
Casey
Joseph R
JR
D000595:Amino Acid Sequence; D001639:Bicarbonates / Q000378:metabolism; D001692:Biological Transport; D002460:Cell Line; D002942:Circular Dichroism; D003593:Cytoplasm / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion / Q000235:genetics; D027982:Sodium-Bicarbonate Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D001639:Bicarbonates; D010446:Peptide Fragments; C422022:SLC4A7 protein, human; D027982:Sodium-Bicarbonate Symporters