The NALCN ion channel is activated by M3 muscarinic receptors in a pancreatic beta-cell line

Abstract

A previously uncharacterized putative ion channel, NALCN (sodium leak channel, non-selective), has been recently shown to be responsible for the tetrodotoxin (TTX)-resistant sodium leak current implicated in the regulation of neuronal excitability. Here, we show that NALCN encodes a current that is activated by M3 muscarinic receptors (M3R) in a pancreatic beta-cell line. This current is primarily permeant to sodium ions, independent of intracellular calcium stores and G proteins but dependent on Src activation, and resistant to TTX. The current is recapitulated by co-expression of NALCN and M3R in human embryonic kidney-293 cells and in Xenopus oocytes. We also show that NALCN and M3R belong to the same protein complex, involving the intracellular I-II loop of NALCN and the intracellular i3 loop of M3R. Taken together, our data show the molecular basis of a muscarinic-activated inward sodium current that is independent of G-protein activation, and provide new insights into the properties of NALCN channels.

Figure 1

Knockdown and overexpression of NALCN in MIN6 cells alter an atropine-sensitive acetylcholine-activated current. (A) Endogenous NALCN expression in MIN6 cells assessed by RT–qPCR (data are presented as a percentage of expression compared with the β2M level) and (B) western blot analysis in the absence and presence of shRNAs. (C) Representative sample of acetylcholine (ACh) activated current on a MIN6 pancreatic β-cell held at −80 mV in a standard whole-cell voltage-clamp configuration. (D) This current is substantially reduced with shRNA targeted against NALCN and increased with NALCN overexpression. (E) Summary of the effects of NALCN shRNA knockdown and overexpression on acetylcholine current density, expressed as a percentage of mean control-cell current. NALCN, sodium leak channel, non-selective; RT–qPCR, reverse transcription quantitative PCR; shRNA, short hairpin RNA.

Figure 2

Permeation and gating properties of the acetylcholine-activated current in MIN6 cells. (A) Replacement of sodium ions by NMDG or lithium in the extracellular solution greatly diminishes the acetylcholine (ACh)-induced inward current. The current is also resistant to 2 μM TTX and was partly blocked by 10 μM Gd³⁺. Inclusion of heparin (1 mg/ml), GTP-γ-S (1 mM) or GDP-β-S (1 mM) in the pipette solution does not significantly affect the current, whereas the SFK inhibitors PP1 (20 μM) and SU6656 (5 μM) have a strong inhibitory effect. (B) Representative traces showing the effects of an SFK inhibitor (PP1, 20 μM) on the acetylcholine-activated inward current in MIN6 cells. (C) A voltage-ramp protocol (−100 to 100 mV over 0.2 s) was used to determine the current–voltage relationship of the acetylcholine-activated current. Values obtained in the absence of acetylcholine were subtracted from values obtained in the presence of acetylcholine to determine the I–V curve attributable to acetylcholine. Currents obtained at voltages greater than −20 mV were too variable for meaningful analysis and were therefore excluded. (D) RT–qPCR analysis shows that only mRNA from M3R and M4R can be detected in MIN6 cells. (E) Representative traces showing the effects of an M3R-specific antagonist (4-DAMP, 7 μM) and an M4R-specific antagonist (tropicamide, 10 μM) on the acetylcholine-activated inward current in MIN6 cells. (F) Summary of the effects of 4-DAMP and tropicamide on the acetylcholine-activated current, expressed as a percentage of control current. 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine methiodide; Gd³⁺, gadolinium; MR, muscarinic receptor; NMDG, N-methyl-D-glucamine; mRNA, messenger RNA; PP1, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine; RT–qPCR, reverse transcription quantitative PCR; SFK, Src family of tyrosine kinase; TTX, tetrodotoxin.

Figure 3

Co-expression of NALCN and M3R in the HEK-293 cell line and in Xenopus oocytes results in the expression of an M3R agonist-activated current. (A) Co-expression of NALCN and M3R cDNAs in HEK-293 cells results in the expression of an acetylcholine (ACh)-activated inward current with the same profile as in the MIN6 cells. (B) Mean acetylcholine-activated current density in HEK-293 cells expressing NALCN and M3R. (C) Co-expression of NALCN and an M3R deletion mutant (M3R^*) that is deficient in the activation of G proteins in Xenopus oocytes results in the expression of a carbachol (CCh)-activated inward cationic current. (D) Mean carbachol-activated current density in Xenopus oocytes, mock-injected or expressing M3R, NALCN, M3R^* or NALCN+M3R^*. (E) Leak activity in HEK-293 cells and (F) in Xenopus oocytes is not significantly altered by NALCN overexpression. cDNA, complementary DNA; HEK, human embryonic kidney; M3R, M3 muscarinic receptor; NALCN, sodium leak channel, non-selective.

Figure 4

NALCN and M3R belong to the same protein complex in HEK-293 cells. (A) NALCN and M3R co-immunoprecipitate (IP) when co-expressed in the HEK-293 cell line. (B) M3R co-immunoprecipitates with the NALCN's GFP-tagged intracellular I–II loop. (C) NALCN co-immunoprecipitates with the M3R's GFP-tagged intracellular segment i3 and the carboxy-terminus (Cter) part. (D) Overexpression of the NALCN's GFP-tagged intracellular loop I–II and (E) M3R's GFP-tagged intracellular segment i3 in the MIN6 cell line results in a strong inhibition of the acetylcholine (ACh)-activated inward current. GFP, green fluorescent protein; HA, haemagglutinin; HEK, human embryonic kidney; M3R, M3 muscarinic receptors; NALCN, sodium leak channel, non-selective; WB, western blot.