Localization of the nucleic acid channel regulatory subunit, cytosolic malate dehydrogenase.
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-PK(1) 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-PK(1) cells. LLC-PK(1) 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-PK(1) cells. This finding supports prior functional data that describe a role for cMDH as the regulatory subunit of NACh.
Hanss
Basil
B
Division of Nephrology, Department of Medicine, Mt Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029, USA. basil.hanss@mssm.edu
Leal-Pinto
Edgar
E
Teixeira
Avelino
A
Tran
Baohuong
B
Lee
Chun-Hui
CH
Henderson
Scott C
SC
Klotman
Paul E
PE
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure; D003600:Cytosol / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006497:Heparan Sulfate / Q000494:pharmacology; D007473:Ion Channels / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D018374:LLC-PK1 Cells; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008293:Malates / Q000494:pharmacology; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D013552:Swine
D049452:Green Fluorescent Proteins; D006497:Heparan Sulfate; D007473:Ion Channels; D008291:Malate Dehydrogenase; D008293:Malates; C030298:malic acid