Peptide neurotransmitters activate a cation channel complex of NALCN and UNC-80

Abstract

Several neurotransmitters act through G-protein-coupled receptors to evoke a 'slow' excitation of neurons. These include peptides, such as substance P and neurotensin, as well as acetylcholine and noradrenaline. Unlike the fast (approximately millisecond) ionotropic actions of small-molecule neurotransmitters, the slow excitation is not well understood at the molecular level, but can be mainly attributed to suppressing K(+) currents and/or activating a non-selective cation channel. The molecular identity of this cation channel has yet to be determined; similarly, how the channel is activated and its relative contribution to neuronal excitability induced by the neuropeptides are unknown. Here we show that, in the mouse hippocampal and ventral tegmental area neurons, substance P and neurotensin activate a channel complex containing NALCN and a large previously unknown protein UNC-80. The activation by substance P through TACR1 (a G-protein-coupled receptor for substance P) occurs by means of a unique mechanism: it does not require G-protein activation but is dependent on Src family kinases. These findings identify NALCN as the cation channel activated by substance P receptor, and suggest that UNC-80 and Src family kinases, rather than a G protein, are involved in the coupling from receptor to channel.

Figure 1. NALCN is required for I_SP

Currents (at −67 mV) were recorded from wild-type (+/+) or Nalcn knockout (−/−) hippocampal (Hippo) and VTA neurons. Horizontal and vertical arrows indicate 0 current level and SP application (10 seconds of puffing), respectively. a, I_SP developed in bath containing 155 mM Na⁺, but not when Na⁺ was lowered to 5 mM (replaced with NMDG). Incubation with L703606 (10 µM, 6 min) blocked I_SP. b, Blockade by a peptide NK1R antagonist (10 µM, 6 min incubation). c, Nalcn^−/− hippocampal neuron. d, VTA neurons. e, Summary of the I_SP sizes. Numbers of cells are in parentheses. Error bars, mean ± s.e.m.

Figure 2. Characterization of the I_SP in hippocampal neurons

a–c, Net SP-activated currents at various voltages (right) were obtained by subtracting the currents before (left) from after (middle) SP bath application (1 µM) in wild-type (+/+) (a), mutant (−/−) (b), and mutant transfected with NALCN (−/−; NALCN) (c). Dotted lines indicate 0 current level. d, Averaged I_SP amplitudes. The lines from mutant (−/−) and mutant transfected with empty vector (−/−; mock) overlap. Right panel shows the voltage step protocols used. Error bars, mean ± s.e.m.

Figure 3. I_SP is G protein-independent but requires SFKs

Hippocampal neurons were used. a, I_SP (at −67 mV) from recordings with GTP-, GTP-γ-S - and GDP-β-S –containing pipette solutions. b, Inhibition of I_SP (at −67 mV) by genistein (30 µM; left) and PP1 (20 µM; middle). Right, summary. c, In wild-type neurons (left), a Gd³⁺-blockable current developed upon intracellular dialysis (start time indicated by arrow) with pipette solution containing an SFK activator (1 µM; a ramp from −67 to −47 mV in 1.4 s was given every 10 s to monitor input resistance). After the current reached a plateau (size defined as I_Src for d), SP did not induce an additional current (I_SP for d). Right, Nalcn^−/− neuron. d, Summary of I_Src (left) and additional currents activated by SP after the dialysis-induced currents plateaued (right). Error bars, mean ± s.e.m.

Figure 4. I_SP reconstituted in HEK293T cells

a, Immunoblot (IB) with lysates from transfected HEK293T cells and brain. b, c, Immunoprecipitation (IP) showing protein complex between mUNC80 and NALCN in HEK293T transfected as indicated (b) and in brain (c). Ctrl1 and ctrl2 were two unrelated proteins used as controls. Recordings in d–h were done using ramp protocols (Vh = −20 mV; −100 to +100 mV in 1 s, every 20 s). d, Recordings from a cell transfected with NK1R, mUNC80 and NALCN. Currents at three time points are expanded in the right panel (circled 1, before SP; 2, after SP; 3, after Na⁺ and K⁺ in the bath were replaced with NMDG). e, Summary of I_SP sizes (at −100 mV) from cells transfected with combinations as indicated. f, g, Recordings showing that I_SP was blocked by Gd³⁺ (f) but became resistant to Gd³⁺ when NALCN was replaced by the EEKA pore mutant (g). h, Inhibition of I_SP by PP1 (20 µM). Error bars, mean ± s.e.m.