A TRPM4-dependent current in murine renal primary cilia.
Defects in primary cilia lead to a variety of human diseases. One of these, polycystic kidney disease, can be caused by defects in a Ca²⁺-gated ion channel (TRPP2) found on the cilium. Other ciliary functions also contribute to cystogenesis, and defects in apical Ca²⁺ homeostasis have been implicated. By recording directly from the native cilia of mIMCD-3 cells, a murine cell line of renal epithelial origin, we have identified a second Ca²⁺-gated channel in the ciliary membrane: the transient receptor potential cation channel, subfamily M, member 4 (TRPM4). In excised primary cilia, TRPM4 was found to have a low sensitivity to Ca²⁺, with an EC₅₀ of 646 μM at +100 mV. It was inhibited by MgATP and by 9-phenanthrol. The channel was not permeable to Ca²⁺ or Cl⁻ and had a permeability ratio PK/PNa of 1.42. Reducing the expression of Trpm4 mRNA with short hairpin (sh) RNA reduced the TRPM4 current by 87% and shortened primary cilia by 43%. When phospholipase C was inhibited, the sensitivity to cytoplasmic Ca²⁺ greatly increased (EC₅₀ = 26 μM at +100 mV), which is consistent with previous reports that phosphatidylinositol 4,5-bisphosphate (PIP2) modulates the channel. MgATP did not restore the channel to a preinactivation state, suggesting that the enzyme or substrate necessary for making PIP2 is not abundant in primary cilia of mIMCD-3 cells. The function of TRPM4 in renal primary cilia is not yet known, but it is likely to influence the apical Ca²⁺ dynamics of the cell, perhaps in tandem with TRPP2.
Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio.
Richard J
RJ
Flannery
Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio steve@syrano.acb.uc.edu.
Nancy K
NK
Kleene
Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio.
Steven J
http://orcid.org/0000-0001-7663-3590
SJ
Kleene
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002923:Cilia / Q000187:drug effects / Q000378:metabolism; D055724:Electrophysiological Phenomena / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects; D055785:Gene Knockdown Techniques; D015640:Ion Channel Gating / Q000187:drug effects; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D054801:Phosphoinositide Phospholipase C / Q000494:pharmacology; D050053:TRPM Cation Channels / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D050396:TRPP Cation Channels / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
D000255:Adenosine Triphosphate; D002118:Calcium; D054801:Phosphoinositide Phospholipase C; D050053:TRPM Cation Channels; D050396:TRPP Cation Channels; D010738:Type C Phospholipases; C476766:TRPM4 protein, mouse
TRPM4; TRPP2; polycystic kidney disease; primary cilium