Troponin I binds polycystin-L and inhibits its calcium-induced channel activation.
Polycystin-L (PCL) is an isoform of polycystin-2, the product of the second gene associated with autosomal dominant polycystic kidney disease, and functions as a Ca(2+)-regulated nonselective cation channel. We recently demonstrated that polycystin-2 interacts with troponin I, an important regulatory component of the actin microfilament complex in striated muscle cells ... and an angiogenesis inhibitor. In this study, using the two-microelectrode voltage-clamp technique and Xenopus oocyte expression system, we showed that the calcium-induced PCL channel activation is substantially inhibited by the skeletal and cardiac troponin I (60% and 31% reduction, respectively). Reciprocal co-immunoprecipitation experiments demonstrated that PCL physically associates with the skeletal and cardiac troponin I isoforms in overexpressed Xenopus oocytes and mouse fibroblast NIH 3T3 cells. Furthermore, both native PCL and cardiac troponin I were present in human heart tissues where they indeed associate with each other. GST pull-down and microtiter binding assays showed that the C-terminus of PCL interacts with the troponin I proteins. The yeast two-hybrid assay further verified this interaction and defined the corresponding interacting domains of the PCL C-terminus and troponin I. Taken together, this study suggests that troponin I acts as a regulatory subunit of the PCL channel complex and provides the first direct evidence that PCL is associated with the actin cytoskeleton through troponin I.
Mesh Terms:
3T3 Cells, Amino Acid Sequence, Animals, Calcium, Calcium Channels, Drug Interactions, Glutathione Transferase, Humans, Ion Channels, Membrane Glycoproteins, Mice, Molecular Sequence Data, Muscle, Skeletal, Myocardium, Oocytes, Patch-Clamp Techniques, Phosphoproteins, Protein Isoforms, Receptors, Cell Surface, Sequence Homology, Amino Acid, Sequence Tagged Sites, Troponin I, Two-Hybrid System Techniques, Xenopus
3T3 Cells, Amino Acid Sequence, Animals, Calcium, Calcium Channels, Drug Interactions, Glutathione Transferase, Humans, Ion Channels, Membrane Glycoproteins, Mice, Molecular Sequence Data, Muscle, Skeletal, Myocardium, Oocytes, Patch-Clamp Techniques, Phosphoproteins, Protein Isoforms, Receptors, Cell Surface, Sequence Homology, Amino Acid, Sequence Tagged Sites, Troponin I, Two-Hybrid System Techniques, Xenopus
Biochemistry
Date: Jun. 24, 2003
PubMed ID: 12809519
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