NBCe1-A Transmembrane Segment 1 Lines the Ion Translocation Pathway

Area of Science:

• Biochemistry
• Molecular Biology
• Physiology

Background:

• The electrogenic Na(+)/HCO(3)(-) cotransporter (NBCe1-A) is crucial for sodium and bicarbonate transport across the basolateral membrane of the renal proximal tubule.
• Understanding the structural requirements of NBCe1-A is essential for elucidating its transport mechanism.

Purpose of the Study:

• To investigate the structural role of transmembrane segment 1 (TM1) residues in mediating NBCe1-A transport.
• To identify specific residues within TM1 that are critical for cotransporter function.

Main Methods:

• Site-directed mutagenesis was used to introduce 25 cysteine mutants in TM1 (Gln-424 to Gly-448).
• Mutants were assessed for sensitivity to methanethiosulfonate (MTS) reagents (MTSES, MTSET, MTSEA).
• Topology analysis using biotin maleimide and MTS-TAMRA, along with helical wheel plots, were employed.

Main Results:

• Mutants T442C, A435C, and A428C showed significant sensitivity to MTS reagents, indicating their proximity to the pore.
• Thr-442 was identified as marking the C-terminal end of TM1 and residing in a narrow region of the ion translocation pore.
• A delta(-) helical dipole at the C-terminal end of TM1 involving Thr-442 was suggested to be critical for NBCe1-A function.

Conclusions:

• Residues Thr-442, Ala-435, and Ala-428 in TM1 line the ion translocation pore of NBCe1-A.
• Thr-442 plays a critical role in NBCe1-A function, potentially through a delta(-) helical dipole influence.
• The orientation of TM1 in NBCe1-A differs from that in AE1, as suggested by functional insensitivity of AE1-Thr-422 to MTS reagents.