Localization of the Nucleic Acid Channel Regulatory Subunit, Cytosolic Malate Dehydrogenase

• Published in: The Journal of membrane biology Vol. 226; no. 1-3; pp. 1 - 8
• Main Authors: Hanss, Basil, Leal-Pinto, Edgar, Teixeira, Avelino, Tran, Baohuong, Lee, Chun-Hui, Henderson, Scott C, Klotman, Paul E
• Format: Journal Article
• Language: English
• Published: New York New York : Springer-Verlag 01.12.2008; Springer-Verlag; Springer Nature B.V
• Subjects: Animals; Artifical planar membranes; Biochemistry; Biomedical and Life Sciences; Cell Membrane - metabolism; Cell Membrane - ultrastructure; Cell Nucleus - metabolism; Cell Nucleus - ultrastructure; Cytosol - enzymology; Dose-Response Relationship, Drug; electrophysiology; Epithelial ion transport; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Heparitin Sulfate - pharmacology; Human Physiology; ion channels; Ion Channels - antagonists & inhibitors; Ion Channels - metabolism; Ion Channels - physiology; Ion transport by epithelia; Ions; Kidneys; Life Sciences; LLC-PK1 Cells; Malate Dehydrogenase - metabolism; Malates - pharmacology; Membrane biology/immuno chemistry; Membrane transport; Membranes; Microscopy, Confocal; Microscopy, Immunoelectron; Planar bilayers; Proteins; Renal physiology; Rodents; Swine; Epithelial ion transport; Membrane transport; Electrophysiology; Ion channels; Membrane biology/immuno chemistry; Planar bilayers; Ion transport by epithelia; Artifical planar membranes; Renal physiology
• ISSN: 0022-2631; 1432-1424
• DOI: 10.1007/s00232-008-9133-5

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.