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A TRPM4-dependent current in murine renal primary cilia.

ABSTRACT: 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 EC?? of 646 ?M 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 (EC?? = 26 ?M 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.

SUBMITTER: Flannery RJ

PROVIDER: S-EPMC4609916 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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A TRPM4-dependent current in murine renal primary cilia.

Flannery Richard J RJ Kleene Nancy K NK Kleene Steven J SJ

American journal of physiology. Renal physiology 20150819 8

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 re ...[more]

PMID: 26290373

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Project description:UnlabelledBackgroundThe central tenet of cilia function is sensing and transmitting information. The capacity to directly contact extracellular surfaces would empower primary cilia to probe the environment for information about the nature and location of nearby surfaces. It has been well established that flagella and other motile cilia perform diverse cellular functions through adhesion. We hypothesized that mammalian primary cilia also interact with the extracellular environment through direct physical contact.MethodsWe identified cilia in rod photoreceptors and cholangiocytes in fixed mouse tissues and examined the structures that these cilia contact in vivo. We then utilized an MDCK cell culture model to characterize the nature of the contacts we observed.ResultsIn retina and liver tissue, we observed that cilia from nearby cells touch one another. Using MDCK cells, we found compelling evidence that these contacts are stable adhesions that form bridges between two cells, or networks between many cells. We examined the nature and duration of the cilia-cilia contacts and discovered primary cilia movements that facilitate cilia-cilia encounters. Stable adhesions form as the area of contact expands from a single point to a stretch of tightly bound, adjacent cilia membranes. The cilia-cilia contacts persisted for hours and were resistant to several harsh treatments such as proteases and DTT. Unlike many other cell adhesion mechanisms, calcium was not required for the formation or maintenance of cilia adhesion. However, swainsonine, which blocks maturation of N-linked glycoproteins, reduced contact formation. We propose that cellular control of adhesion maintenance is active because cilia adhesion did not prevent cell division; rather, contacts dissolved during mitosis as cilia were resorbed.ConclusionsThe demonstration that mammalian primary cilia formed prolonged, direct, physical contacts supports a novel paradigm: that mammalian primary cilia detect features of the extracellular space, not just as passive antennae, but also through direct physical contact. We present a model for the cycle of glycoprotein-dependent contact formation, maintenance, and termination, and discuss the implications for potential physiological functions of cilia-cilia contacts.

Dataset Information

A TRPM4-dependent current in murine renal primary cilia.

Publications

A TRPM4-dependent current in murine renal primary cilia.

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