We then examined some of the ion permeation and gating properties of the acetylcholine-activated current in MIN6 cells. Substitution of sodium ions by N-methyl-D-glucamine or lithium in the extracellular solution had a robust inhibitory effect, suggesting that sodium is the main ion species contributing to the NALCN inward conductance (21.4±9.4% of control, N=5, P=0.001 and 53.2±9.7% of control, N=5, P=0.008, respectively; Fig 2A). The current was also insensitive to TTX (2 μM; 103.3±9.8% of control, N=7; Fig 2A), but was partly blocked by gadolinium (10 μM; 51.4±6.1% of control, N=9, P<0.0001; Fig 2A). Addition of heparin (1 mg/ml) to the patch pipette did not affect the current density, indicating that the current was independent of intracellular calcium stores mobilized through activation of IP3 receptors (97.1±22.2% of control, N=12, P=0.89; Fig 2A). Replacement of GTP with either GDP-β-S (1 mM) or GTP-γ-S (1 mM) in the pipette solution did not significantly change the current density (72.3±18.7% of control, N=9, P=0.178, and 86±23.8% of control, N=7, P=0.579, respectively; Fig 2A). Even the use of a pipette solution with no GTP did not have any effect on the current (data not shown). It has been shown that some G-protein-coupled receptors (GPCRs) can signal directly through SFKs (McGarrigle & Huang, 2007). Thus, we also examined the possibility that NALCN could be activated through an SFK-dependent pathway by using the SFK inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1) (Fig 2A,B). Bath application of PP1 (20 μM) almost fully and reversibly abolished the acetylcholine-activated current (6.1±2.5% of control, N=5, P<0.0001; Fig 2A,B). A strong inhibition was also observed with the SFK inhibitor SU6656 (20.1±11.9% of control, N=5, P=0.001; Fig 2A and supplementary Fig S3 online). The current–voltage (I–V) relationship of the acetylcholine-activated current was determined by using a voltage ramp protocol in the absence and presence of acetylcholine (N=8; Fig 2C). We found the relationship to be linear in the range of −110 to −20 mV, with a slope conductance of 76.8±2.5 pS/pF, and a reversal potential Erev of approximately −23 mV. As the activation of the current by acetylcholine was associated with an increase in membrane noise, we also carried out a noise analysis to estimate the single-channel amplitude and found a value of 1.6±0.1 pA at −80 mV with an estimated single-channel conductance of ∼27 pS (supplementary Fig S4 online). To determine further which MRs were involved in the modulation of NALCN, we first carried out an analysis by using RT–qPCR in MIN6 cells (Fig 2D). Our results indicated that M3R and M4R are the muscarinic receptor subtypes present in MIN6 cells (Fig 2D). We therefore used specific antagonists of M3R (7 μM 4-diphenylacetoxy-N-methylpiperidine methiodide) and M4R (10 μM tropicamide) to determine the MR subtype responsible for current activation (Fig 2E,F). Our results suggest that M3R was responsible for current activation (4-diphenylacetoxy-N-methylpiperidine methiodide: 20.9±2.6% of control, N=5, P=0.0002; tropicamide: 94.8±18.3% of control, N=9; Fig 2F). These data suggest that M3R activation induces an inward, primarily sodium, conductance through NALCN channels that is independent of intracellular calcium stores and G proteins, dependent on SFK activation, resistant to TTX and sensitive to gadolinium inhibition.
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.
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