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A TRPM4-dependent current in murine renal primary cilia
Richard J Flannery1, Nancy K Kleene2, Steven J Kleene1
1Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio.
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 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.
Insights
Researchers identified the transient receptor potential cation channel, subfamily M, member 4 (TRPM4) as a second Ca²⁺-gated channel in primary cilia. TRPM4 influences renal cell calcium dynamics and may play a role in kidney diseases.
Area of Science:
- Cell Biology
- Ion Channel Physiology
- Renal Physiology
Background:
- Defects in primary cilia are linked to human diseases, including polycystic kidney disease.
- Primary cilia contain Ca²⁺-gated ion channels, such as TRPP2, implicated in cystogenesis.
- Altered apical Ca²⁺ homeostasis is suspected in ciliary disease development.
Purpose of the Study:
- To identify and characterize Ca²⁺-gated ion channels in the primary cilia of renal epithelial cells.
- To investigate the functional properties and regulation of newly identified ciliary channels.
- To explore the potential role of these channels in renal primary cilia function and disease.
Main Methods:
- Direct electrophysiological recordings from native primary cilia of mIMCD-3 cells.
- Characterization of channel properties including Ca²⁺ sensitivity, ion permeability, and modulation by MgATP and PIP2.
- Gene silencing using short hairpin RNA (shRNA) to reduce TRPM4 expression and assess its impact on cilia length and channel activity.
Main Results:
- Identified transient receptor potential cation channel, subfamily M, member 4 (TRPM4) as a Ca²⁺-gated channel in primary cilia.
- TRPM4 exhibits low Ca²⁺ sensitivity (EC₅₀ = 646 μM), is inhibited by MgATP, and is permeable to K⁺ over Na⁺ (PK/PNa = 1.42).
- TRPM4 knockdown significantly reduced TRPM4 current and primary cilia length, and its activity is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2).
Conclusions:
- TRPM4 is a novel Ca²⁺-gated ion channel present in renal primary cilia.
- TRPM4 activity is regulated by intracellular Ca²⁺ levels and PIP2, with potential implications for apical Ca²⁺ dynamics.
- TRPM4 may contribute to renal primary cilia function, potentially in conjunction with TRPP2, and warrants further investigation in kidney diseases.
