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

NALCN is a neuronal cation channel carrying a small background leak Na⁺ current at the resting membrane potential¹⁰. When overexpressed in HEK293T fibroblast cells, it generates a Na⁺-permeable cation channel that is voltage-independent, non-inactivating, tetrodotoxin (TTX)-resistant and Gd³⁺-blockable¹⁰. It is not known whether, like background K⁺ channels, NALCN is also regulated by neuromodulators, but the biophysical and pharmacological properties of the NALCN currents (I_NALCN) closely resemble those of the SP-activated cation channel currents (I_SP) studied in several brain regions^11–14.

To test the possibility that I_SP requires NALCN, we recorded I_SP in wild-type and Nalcn knockout¹⁰ (Nalcn^−/−) neurons via patch clamp with measures taken to minimize K⁺ channel effects and to block voltage-gated Na⁺ channel and synaptic currents. In 16 of 34 wild-type hippocampal pyramidal neurons held at −67 mV, an inward current (> 50 pA) developed within 1.1 ± 0.2 min (range: 0.4 – 2.9 min) after a pulse of SP was delivered through a puffer pipette placed ~ 20 µm from the cell body (Fig. 1a, e). The time courses of I_SP vary between neurons, but are comparable to previous recordings^{11, 12, 15} and are in agreement with the “slow” nature of the signal transduction pathway (see below). I_SP was blocked by pre-incubating cells with a competitive peptide (n = 3, Fig. 1b) or an NK1R antagonist (L703606; n = 14; Fig. 1a), suggesting involvement of NK1R or another member of the tachykinin receptor family of GPCRs. Reducing bath concentration of Na⁺ from 155 mM to 5 mM largely abolished the I_SP (Fig. 1a), suggesting that Na⁺ was the major charge carrier of the current at this holding potential. I_SP has also been recorded in the rat ventral tegmental area (VTA)⁹. Of 43 putative dopaminergic (DA) neurons cultured from the VTA of wild-type mice (see Methods), 32 had an I_SP > 50 pA (Fig. 1d). In contrast to the wild-type, none of the 30 hippocampal (Fig. 1c, e) and 29 VTA (Fig. 1d) neurons from Nalcn^−/− mutant mice showed a significant I_SP (Fig. 1e).

Like I_NALCN¹⁰, the SP-activated currents did not inactivate within 300 ms at any voltage (Fig. 2a). The averaged current (I)-voltage (V) relationship was linear at negative membrane potentials, suggesting little voltage-dependence of conductance in this range (Fig. 2d). The overall properties of the I_SP in cultured mouse hippocampal neurons were similar to those of the I_SP recorded from numerous other neuronal preparations, primarily from brain slices^{12, 13, 16}. Due to low current amplitudes, potential space clamping problems in neuronal recordings, and possible contamination by voltage-gated currents at positive potentials, a detailed biophysical characterization could not be performed in the neurons.

I_SP could be restored in the Nalcn^−/− neurons by transfecting a NALCN cDNA (Fig. 2c, d). The restored I_SP (Supplementary Fig. 2b, d), like that from wild-type neurons (Supplementary Fig. 2a, d), was blocked by a trivalent NALCN blocker Gd³⁺ (10 µM). The current, however, became resistant to 10 µM Gd³⁺ (Supplementary Fig. 2c, d) when restored with a Gd³⁺-resistant NALCN pore mutant (EEKA) that has a single amino acid change (E to A) in the ion filter region (Supplementary Fig. 1). Taken together, the similarity of I_SP and I_NALCN, the absence of I_SP in Nalcn^−/− neurons and presence with NALCN cDNA transfection, and the alteration of I_SP pharmacology by the EEKA pore mutant strongly suggest that NALCN forms the pore conduit carrying the I_SP.

To determine the role of G-proteins (the immediate downstream effectors of GPCR NK1R) in the NALCN activation by SP, we “locked” G-proteins in active or inactive states with non-hydrolyzable analogs of GTP (GTP-γ-S) or GDP (GDP-β-S), respectively, applied via patch pipettes. Surprisingly, SP still induced inward currents of comparable sizes (Fig. 3a). Thus, the activation of NALCN by SP through GPCRs is likely via an unconventional mechanism that does not require G-protein stimulation.

Some GPCRs may also activate the Src family of tyrosine kinases (SFKs), which is a more recently discovered GPCR signaling cascade that regulates downstream factors such as mitogen-activated protein kinase and gene expression^{17, 18}. Bath application of genistein (a phosphotyrosine kinase inhibitor) or PP1 (an SFK inhibitor) abolished I_SP (Fig. 3b), suggesting that SFKs are required for the activation of NALCN by SP.

Similar to previous findings¹⁹, intracellular dialysis with an SFK activator via patch pipette induced a gradual increase of inward current (defined as I_Src; Fig. 3c, left panel). After the current plateaued, SP no longer activated an additional inward current (I_SP; Fig. 3c, d), suggesting that SP and SFKs activate a common channel. Similar to I_SP, the I_Src was blocked by Gd³⁺ (Fig. 3c, left). In contrast to wild-types, Nalcn^−/− neurons lacked I_Src (Fig. 3c,d); this current was largely restored by transfection with NALCN cDNA (Fig. 3d). These data suggest that SFK activation is both necessary and sufficient for I_SP. Given that many other ion channels can also be regulated by SFKs²⁰, the lack of I_Src in the Nalcn^−/− neurons was surprising, but seems to suggest that NALCN is a major cation channel target for SFKs near the resting membrane potential and may also be modulated by the diverse array of stimuli (e.g. neurotransmitters, growth factors, cytokines, cell stretch and adhesion molecules) that lead to SFK activation^{21, 22}.

Other neuropeptides such as NT also elicit slow depolarization of neurons and activate a cation current (I_NT) similar to I_SP⁹. In the wild-type VTA neurons, NT puffing (10 µM) elicited an inward current (−90.0 ± 25.3 pA at −67 mV; n = 29) that was blocked by an NT receptor antagonist SR48692 and the SFK inhibitor PP1 (not shown). In contrast, neurons from Nalcn^−/− mice lacked an I_NT (Supplementary Fig. 3), suggesting that the I_NT is also through NALCN.

To determine the role of the SP- and NT- activated NALCN currents in modulating neuronal excitability, we recorded action potentials in the VTA DA neurons. Firing frequencies were significantly increased by 1 µM SP (Supplementary Fig. 4a, c) or 1 µM NT (Supplementary Fig. 4b, c) in the wild-type, but not in the Nalcn^−/− neurons. The defect in the mutant was partially restored by transfecting with NALCN cDNA (Supplementary Fig. 4c). We conclude that the NALCN channel plays a major role in the potentiation of neuronal excitability by the neuropeptides in these neurons. Under other conditions or in other cells, the peptides may also excite neurons by suppressing K⁺ currents or by activating some of the TRP family members^{6, 8, 9, 23–26}.

Unlike in neurons, little robust I_SP was observed from HEK293T fibroblast cells co-transfected with NK1R and NALCN (see Fig. 4e), possibly because of a lack of key component for the channel activation. In Drosohpila melanogaster and C. elegans, Nalcn genetically interacts with others such as unc-79 and unc-80^27–29. To investigate whether UNC-80 forms a physical complex with NALCN in the brain, we cloned a mammalian UNC-80 homolog (hereafter called mUNC80) from mouse brain (Supplementary Fig. 5). The full-length mUNC80 is predicted to encode a 371 kDa protein (Fig. 4a) with no obvious domains of known function. It has high (96%) and moderate (~30%) identities with its human and invertebrate homologs, respectively. NALCN and mUNC80 form a complex in mouse brain (Fig. 4c), and in transfected HEK293T cells (Fig. 4b). In addition, both are tyrosine-phosphorylated and such phosphorylation can be inhibited by PP1 (Supplementary Fig. 6).

In HEK293T cells with moderate levels of co-expression of NK1R, mUNC80 and NALCN (see Methods), SP activated a current (−550.6 ± 102.9 pA at −100 mV; 0 to −4904 pA; Fig. 4d, e) with a linear I/V relationship (Fig. 4d, right, curve 2). Like I_SP in neurons, the current was blocked by the NALCN blocker Gd³⁺ (Fig. 4f), but became resistant to Gd³⁺ when NALCN was replaced with the EEKA pore mutant (Fig. 4g). Inclusion of GDP-β-S in the pipette did not prevent current activation (−914.0 ± 317.1 pA, n = 10), suggesting independence of G-protein activation.

Consistent with a requirement of SFKs, the current was suppressed by SFK inhibitors (Fig. 4g, h, Supplementary Fig. 7) and an anti-phospho-SFK antibody (Supplementary Fig. 8). Furthermore, when a constitutively active Src (with Y529 mutated to F) was co-transfected, a basal current was increased from −104.1 ± 22.1 pA (empty vector to replace Src Y529F in transfection; n = 22) to −434.3 ± 119.9 pA (n = 21), and application of SP no longer activated significant I_SP in the Src Y529F co-transfected cells (−64.6 ± 33.1 pA, n = 13; compared with −416.2 ± 89.6 pA in the empty vector cotransfection control, n = 13) (Supplementary Fig. 9). Finally, inclusion of a recombinant active Src protein in the pipette led to a gradual increase of inward current (Supplementary Fig. 10), suggesting that, like in neurons (Fig. 3c), SFK is sufficient to activate the current.

The simplest interpretation of our work is that the SP- and NT- activated cation current is through a channel complex consisting of NALCN and mUNC80. Our preliminary studies found that UNC-79 is also associated with mUNC80 and NALCN in the brain (not shown). Further studies need to investigate if UNC-79 affects I_SP’s properties such as the activation time courses. Similarly, the mechanisms underlying the involvement of SFKs in the coupling between receptor and channel remain to be uncovered. Due to the large driving force of Na⁺ at resting membrane potential, I_NALCN should be a potent excitatory current in neurons. The channel is voltage-independent and non-inactivating, and is thus ideal for regulating neuronal excitability by neuromodulators.