Search

ALTHOUGH the existence of ion conductance channels in excitable tissues is strongly suspected1, there has been no way of measuring the occurrence of such channels in extracts of nerve or brain until the advent of lipid bilayer techniques2, improved to the extent of allowing detection of a few channels3. The observation4 of ion channel formation in lipid bilayers by gramicidin A and its elucidation as a helical peptide structure5,6 has prompted the search for substances of similar properties in extracts of nervous tissue. This is made more plausible by the fact that the channel conductance Δ(Na+) for the alkyldiamide derivatives of gramicidin is close to the value of 2 × 10−10, which was estimated for nerve on pharmacological grounds7.

The catalytic Na,K-ATPase α-subunit is not able to exit the ER or catalyze ion transport unless assembled with the β-subunit. However, requirements for the ER exit of the Na,K-ATPase β-subunit that plays an additional, ion-transport-independent, role in intercellular adhesion are not clear. The Na,K-ATPase β1- or β2-subunits and their N-glycosylation-deficient mutants were expressed in renal MDCK cells. Confocal microscopy, immunohistochemistry, and immunoprecipitation were employed to evaluate the role of N-glycans of the β-subunit isoforms in the quality control of the Na,K-ATPase in the ER. Mutagenic removal of as few as two of the eight N-glycosylation sites from the β2-subunit precludes its assembly with the α1 subunit and results in full retention of the unassembled β2-subunit in the ER. However, removal of all three N-glycosylation sites from the β1-subunit only slightly affects its assembly with the α1-subunit and trafficking to the plasma membrane. The β2-subunit binds 4-5-fold more efficiently to the ER lectin chaperone, calnexin, and 2-3-fold less efficiently to the non-lectin ER chaperone, BiP, than the β1-subunit. These results indicate that folding of the β2- and β1-subunits is mediated by lectin and non-lectin chaperones, respectively, consistent with the essential role of N-glycosylation for folding and trafficking of the β2 but not of the β1-subunit. Disruption of the α1-β association by mutations in defined α1-interacting regions of either β1- or β2-subunits results in the ER retention of unassembled mutants, indicating that α-β assembly is essential for the ER export of either β-subunit isoform. In conclusion, the ER quality control system ensures that only properly folded β-subunits assemble with the α-subunits and only assembled α-β complexes are exported to the Golgi and delivered to the plasma membrane.Supported by NIH, DK077149 and DK58333.