Abstract
NACh is a nucleic acid–conducting channel found in apical membrane of rat kidney proximal tubules. It is a heteromultimeric complex consisting of at least two proteins: a 45-kDa pore-forming subunit and a 36-kDa regulatory subunit. The regulatory subunit confers ion selectivity and influences gating kinetics. The regulatory subunit has been identified as cytosolic malate dehydrogenase (cMDH). cMDH is described in the literature as a soluble protein that is not associated with plasma membrane. Yet a role for cMDH as the regulatory subunit of NACh requires that it be present at the plasma membrane. To resolve this conflict, studies were initiated to determine whether cMDH could be found at the plasma membrane. Before performing localization studies, a suitable model system that expressed NACh was identified. A channel was identified in LLC-PK1 cells, a line derived from pig proximal tubule, that is selective for nucleic acid and has a conductance of approximately 10 pS. It exhibits dose-dependent blockade by heparan sulfate or l-malate. These characteristics are similar to what has been reported for NACh from rat kidney and indicate that NACh is present in LLC-PK1 cells. LLC-PK1 cells were therefore used as a model system for immunolocalization of cMDH. Both immunofluorescence and immunoelectron microscopy demonstrated cMDH at the plasma membrane of LLC-PK1 cells. This finding supports prior functional data that describe a role for cMDH as the regulatory subunit of NACh.
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Acknowledgments
We thank Valerie Williams for electron microscopy and immunogold labeling. Microscopy was performed at the MSSM–Microscopy Shared Resource Facility, supported with funding from NIH-NCI shared resources grant (5R24 CA095823), NSF Major Research Instrumentation grant (DBI-9724504), and NIH shared instrumentation grant (1S10 RR0 9145). These studies were supported by grants from the NIDDK/NIH to B. G. (DK065838) and P. E. K. (DK63610). Portions of this work were presented at the annual meetings of FASEB (2004) and ASN (2005).
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Hanss, B., Leal-Pinto, E., Teixeira, A. et al. Localization of the Nucleic Acid Channel Regulatory Subunit, Cytosolic Malate Dehydrogenase. J Membrane Biol 226, 1–8 (2008). https://doi.org/10.1007/s00232-008-9133-5
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DOI: https://doi.org/10.1007/s00232-008-9133-5