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.
Li
Qiang
Q
Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Liu
Yan
Y
Shen
Patrick Y
PY
Dai
Xiao-Qing
XQ
Wang
Shaohua
S
Smillie
Lawrence B
LB
Sandford
Richard
R
Chen
Xing-Zhen
XZ
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D015220:Calcium Channels; D004347:Drug Interactions; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007473:Ion Channels / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000737:chemistry; D009206:Myocardium / Q000737:chemistry; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques / Q000379:methods; D010750:Phosphoproteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface; D017386:Sequence Homology, Amino Acid; D016324:Sequence Tagged Sites; D019210:Troponin I / Q000378:metabolism* / Q000494:pharmacology*; D020798:Two-Hybrid System Techniques; D014981:Xenopus
D015220:Calcium Channels; D007473:Ion Channels; D008562:Membrane Glycoproteins; C117185:PKD2L1 protein, human; D010750:Phosphoproteins; C486487:Pkd2l1 protein, mouse; D020033:Protein Isoforms; D011956:Receptors, Cell Surface; D019210:Troponin I; D005982:Glutathione Transferase; D002118:Calcium