Excitable tissues are strongly regulated by extracellular [Ca²⁺] ([Ca²⁺]_o) (Neher and Sakaba, 2008; Ma et al., 2014; Jackman and Regehr, 2017). Movement of extracellular Ca²⁺, through voltage-activated Ca²⁺ channels (VACC), to the intracellular space is central to many of these processes (Ma et al., 2012a; Nanou and Catterall, 2018). However, a distinct, extracellular mechanism that is independent of synaptic transmission also contributes to [Ca²⁺]_o-dependent regulation of nerve and muscle function (Adrian and Gelfan, 1933; Weidmann, 1955; Frankenhaeuser, 1957; Frankenhaeuser and Hodgkin, 1957). Decreases in [Ca²⁺]_o and [Mg²⁺]_o substantially facilitate spontaneous and evoked action potential generation which represents increased intrinsic excitability (Weidmann, 1955; Frankenhaeuser, 1957; Frankenhaeuser and Hodgkin, 1957). In the brain, physiological neuronal activity decreases [Ca²⁺]_o (Nicholson et al., 1978) leading to further increases in action potential firing in neighboring neurons (Anderson et al., 2013). The firing patterns and computational properties of local circuits are impacted substantially by this positive feedback leading to changes in brain behaviors (Titley et al., 2019). Furthermore, under pathological conditions, larger decreases in [Ca²⁺]_o occur, resulting in even greater changes in circuit activity, and implicating [Ca²⁺]_o-dependent excitability in the pathogenesis of brain injury (Ayata and Lauritzen, 2015).

Classical studies proposed that the mechanism underlying [Ca²⁺]_o-dependent excitability centers on voltage-gated sodium channel (VGSC) sensitivity to extracellular Ca²⁺. Reduced [Ca²⁺]_o was proposed to shift the effective voltage-dependent gating of the sodium conductance in the hyperpolarizing direction by reducing the screening of local negative charges on the extracellular face of the membrane or channel by external Ca²⁺ (Frankenhaeuser and Hodgkin, 1957; Hille, 1968). This surface potential screening model accounted for [Ca²⁺]_o-dependent excitability in nerves and muscle without a need for additional molecular players and was widely accepted (Hille, 2001), although direct binding of Ca²⁺ to the VGSC was also proposed as contributing (Armstrong and Cota, 1991). However, this theory was challenged by new data demonstrating that activation of the sodium leak channel (NALCN), a non-selective cation channel, by the intracellular proteins, UNC79 and UNC80 (Lu et al., 2009; Lu et al., 2010) was necessary for [Ca²⁺]_o-dependent excitability to occur in hippocampal neurons. Following the deletion of NALCN or UNC79, [Ca²⁺]_o-dependent excitability was completely lost suggesting the increased excitability resulted from the activation of the non-rectifying NALCN which depolarized neurons and increased the likelihood of action potential generation independent of changes in VGSC function (Lu et al., 2010). The calcium-sensing receptor (CaSR), a G-protein-coupled receptor (GPCR), was hypothesized to detect and transduce the [Ca²⁺]_o changes and signal to the downstream multistep pathway (Lu et al., 2010). CaSR is well-positioned as a candidate [Ca²⁺]_o detector because at nerve terminals it detects [Ca²⁺]_o and regulates a non-selective cation channel (Smith et al., 2004; Chen et al., 2010) and because it transduces changes in [Ca²⁺]_o into NALCN activity following heterologous co-expression of CaSR, NALCN, UNC79, and UNC80 (Lu et al., 2010). Interest in the UNC79-UNC80-NALCN pathway has also risen, due to its essential role in the maintenance of respiration (Lu et al., 2007), the regulation of circadian rhythms (Lear et al., 2013; Flourakis et al., 2015), and because mutations of UNC80 and NALCN cause neurodevelopmental disorders, characterized by development delay and hypotonia (Al-Sayed et al., 2013; Perez et al., 2016).

Here, we address the question of whether the G-protein mediated NALCN pathway or VGSCs transduce the [Ca²⁺]_o-dependent effects on excitability. We test if CaSR is a modulator of neuronal excitability via its action on a nonselective cation channel, determine the impact of CaSR expression on factors of intrinsic neuronal excitability, and examine the relative contributions of [Ca²⁺]_o-regulated changes on VGSC and NALCN gating. In recordings from neocortical neurons, isolated by pharmacological block of excitatory and inhibitory inputs, we determine that neuronal firing is increased by decreasing external divalent concentrations and that this is almost entirely attributable to [Ca²⁺]_o-dependent shifts in VGSC gating. Surprisingly, CaSR deletion substantially shifted VGSC gating, but had no effect on NALCN sensitivity to [Ca²⁺]_o. Taken together our experiments indicate that acute [Ca²⁺]_o-dependent increases in neuronal excitability result from changes in VGSC and NALCN gating and that CaSR contributes by an, as yet, uncharacterized action on VGSCs.

Extracellular calcium concentration regulates both synaptic transmission and intrinsic neuronal excitability, thereby strongly affecting the probability of action potential generation. Consequently, physiological and pathological changes in [Ca²⁺]_o will impact neuronal computation in a complex manner. We have investigated the mechanisms underlying divalent-dependent changes in intrinsic neuronal excitability and tested if CaSR is transducing decreases in [Ca²⁺]_o into NALCN-mediated depolarizations to trigger action potentials (Lu et al., 2010). We found no evidence that this specific mechanism was active in neocortical neurons (Figure 2). Instead, we determined that the vast majority of divalent-dependent neuronal excitability was mediated via VGSCs in three ways. Decreasing the concentration of external divalents activated VGSCs at the resting membrane potential and depolarized the membrane toward the action potential threshold (Figure 6). This occurred because the decreased divalent concentration hyperpolarized the VGSC window current toward the membrane potential increasing sodium currents and the likelihood of action potential generation (Figure 3). Unexpectedly the deletion of CaSR modulated VGSC gating, decreasing the sensitivity of current activation to depolarization via an unidentified mechanism (Figure 3). Deletion of CaSR also indirectly affected action potential generation by modestly hyperpolarizing the membrane potential (Figure 1). While the actions of [Ca²⁺]_o on VGSCs were responsible for the vast majority of the [Ca²⁺]_o-dependent neuronal excitability, using Gd³⁺ we isolated small divalent-dependent inward currents in about half of the neurons (Figure 5). These Gd³⁺-sensitive currents presumably reflected activation of NALCN, and were unaffected by CaSR deletion, but their relatively small size compared to TTX-sensitive divalent-dependent inward currents indicate that they would be minor contributors to divalent-dependent excitability compared to VGSCs (Figures 5 and 6).

The fractions of the divalent-dependent currents and depolarizations that were sensitive to TTX were surprisingly large compared to those that were Gd³⁺-sensitive (Figures 5D and 6C) indicating the relative importance of VGSC- and NALCN-mediated contributions to divalent-dependent excitability respectively. The resistance of NALCN to TTX (Lu et al., 2007; Swayne et al., 2009) reassures that the relatively large TTX-sensitive component is due to selective block of VGSC currents. Persistent subthreshold VGSC currents have been shown to determine spiking rates in other central neurons (Taddese and Bean, 2002; Gorelova and Seamans, 2015) and so the increased VGSC currents we observed in T_0.2 are well-positioned to explain the increased action potential frequency (Figure 6). We are unable to determine from these experiments which neuronal compartment is most affected by the change in [Ca²⁺]_o (Gorelova and Seamans, 2015). However, the physiological impact of VGSC-mediated divalent-dependent excitability may be enormous overall because of the dynamic nature of [Ca²⁺]_oin vivo where it decreases from basal (1.1–1.2 mM) by 30–80% (Nicholson et al., 1978; Ohta et al., 1997; Pietrobon and Moskowitz, 2014). The overall computational effects of physiological decrements in [Ca²⁺]_o will be complex because the increased action potential generation due to changes on VGSCs (Figures 3 and 6) will be confounded by the impact of reduced Ca²⁺ entry through VACCs (Hess et al., 1986; Weber et al., 2010; Williams et al., 2012), reduced excitatory synaptic transmission (Neher and Sakaba, 2008; Vyleta and Smith, 2011), and altered CaSR-mediated signaling at the nerve terminal (Phillips et al., 2008; Chen et al., 2010; Vyleta and Smith, 2011).

It remains unclear why NALCN was the dominant effector of divalent-dependent excitability in hippocampal (Lu et al., 2010) but not neocortical neurons (Figure 6). Could our use of [Ca²⁺]_o and [Mg²⁺]_o rather than [Ca²⁺]_o alone be responsible? We changed divalents simultaneously to provide a strong stimulus to CaSR-signaling and VGSC gating, both of which are sensitive to [Ca²⁺]_o and [Mg²⁺]_o (Frankenhaeuser and Hodgkin, 1957; Brown et al., 1993). Consequently, the same pathways were expected to respond to changes in divalents or [Ca²⁺]_o alone, since the potentially confounding effects on synaptic transmission were blocked in our experiments. Another difference is that we counted spontaneous action potentials as the main measure of excitability whereas others have focused on action potentials elicited by direct injection. We used spontaneous activity to allow us to isolate the depolarization (Figure 2) that was hypothesized to arise from NALCN activation and trigger action potentials following the reduction of external divalent concentration changes (Figure 2). Spontaneous and depolarization-elicited action potentials have been recognized as forms of [Ca²⁺]_o-dependent excitability for >60 years (Frankenhaeuser and Hodgkin, 1957) and both types of activity were increased here when external divalent concentrations were decreased (Figure 1 and Figure 1—figure supplement 2). Because we observed increased excitability, despite the injection of a current to bring the steady state membrane potential back to that recorded in T_1.1, mechanisms other than a voltage-independent non-selective cation channel, like NALCN, must have been active (Figure 1 and Figure 1—figure supplement 2). Similarly, the increased spikes elicited by transient current injections in low [Ca²⁺]_o in hippocampal neurons occurred after the steady state membrane potential was set to −80 mV using a longer current injection (Lu et al., 2010). The long injection would have reversed the NALCN-mediated depolarization in low [Ca²⁺]_o and so the mechanism by which the increased excitability occurred is unclear. One possible explanation is that at low [Ca²⁺]_o NALCN could have been further activated by shorter depolarizing current injections; however, this is at odds with the lack of voltage-dependence of NALCN (Lu et al., 2010). Could NALCN be operating via a different mechanism? One possibility is that NALCN activation is enhancing excitability measured at the soma by enhancing calcium entry into nerve terminals (directly or modifying the action potential waveform and VACC activation) and strengthening excitatory synaptic transmission onto the neuron under study. This would require that the enhancement of synaptic transmission by NALCN be greater than the reduction due to reduced Ca²⁺ entry (Neher and Sakaba, 2008) but could be addressed by recording directly from terminals (Ritzau-Jost et al., 2021) or by determining if NALCN deletion has the same effect after blocking glutamatergic transmission. However, the loss of NALCN could be contributing to [Ca²⁺]_o-dependent changes in excitability independent of a depolarization based on other reports. A number of mechanisms have been postulated to explain how a persistent sodium leak into excitable cells at rest can affect excitability (Sokolov et al., 2007). Such mechanisms or other compensatory changes in neuronal function, as observed with null-mutant animals (Jun et al., 1999), could arise from the loss of NALCN and possibly contribute to the reduced sensitivity of hippocampal neurons to decreased [Ca²⁺]_o (Lu et al., 2010). Lastly, the apparent difference between the studies could reflect different properties of hippocampal and neocortical neurons. While possible it still remains unclear why the deletion of NALCN or UNC-79 completely ablated [Ca²⁺]_o-dependent excitability in hippocampal neurons (Lu et al., 2010) since these neurons contain VGSCs that retain sensitivity to changes in [Ca²⁺]_o (Isaev et al., 2012). However, if the UNC79-UNC80-NALCN pathway modulates VGSC function this could explain how loss of NALCN or UNC-79 could delete acute divalent-dependent changes in VGSC function and excitability. NALCN appears to transduce [Ca²⁺]_o- and G-protein-dependent excitability in other neurons (Philippart and Khaliq, 2018) but GPCRs other than CaSR may be involved (Kubo et al., 1998; Tabata and Kano, 2004) and under certain conditions Ca²⁺ directly blocks NALCN (Chua et al., 2020). Further characterization of the UNC79-UNC80-NALCN signaling pathway is essential given the major changes in neurological function that have been described following mutations of NALCN or upstream co-molecules such as UNC79 and UNC80 (Stray-Pedersen et al., 2016; Bourque et al., 2018; Kuptanon et al., 2019).

In a small fraction of the neocortical neurons (Figures 5 and 6) there was a modest inward current or depolarization with the lowering of extracellular divalent concentration once VGSCs had been blocked. In a few cases, they were sensitive to 10 µM Gd³⁺ consistent with a NALCN-mediated effect and those that were resistant were consistent with other divalent-dependent non-selective cation channels (Ma et al., 2012b). However, deletion of CaSR did not decrease divalent-dependent depolarizations and after membrane potential matching did not impact divalent-dependent excitability (Figures 2E and 6B). While CaSR-NALCN signaling did not contribute to divalent-dependent excitability in neocortical neurons (Figures 2 and 6) it was clear that Casr^-/- neurons were substantially less sensitive to changes [Ca²⁺]_o (Figure 1A,B). The reduced [Ca²⁺]_o sensitivity in these neurons is attributable to the combination of altered VGSC gating (Figure 3E,F) and the hyperpolarized RMP (Figure 1E). Although CaSR did not affect the amplitude of the shift in V_0.5 following the switch to T_0.2, the gating characteristics of VGSC activation was depolarized by loss of CaSR (Figure 3EF). Could CaSR stimulation activate G-proteins and regulate the V_0.5 for VGSC currents (Figure 3E,F)? In neocortical neurons, G-protein activation hyperpolarized VGSC gating and this was blocked by GDPβS (Mattheisen et al., 2018) which is inconsistent with the effect we observed here. Other possible explanations are that CaSR could regulate VGSC subunit expression or post translational modification (Cantrell et al., 1996; Zhang et al., 2019), and this may represent a compensatory mechanism similar to that observed with other mutant mouse models (Jun et al., 1999). Loss of CaSR also hyperpolarized the neocortical neurons (Figure 1I) and this may have been due to decreased function of depolarizing components or stimulation of hyperpolarizing elements. There are a number of candidate channels and pumps that have been shown to regulate the RMP in cortical neurons (Tavalin et al., 1997; Talley et al., 2001; Bean, 2007; Harnett et al., 2015; Hu and Bean, 2018). The changes in VGSC gating and RMP in Casr^-/- neurons may be attributable to homeostatic mechanisms that compensate for perturbations in network activity and have been observed in central and peripheral neurons (Turrigiano, 2008).

Overall, our studies indicate that divalent-dependent excitability in neurons is largely attributable to actions of extracellular calcium on the VGSC function. Given the dynamic nature of brain extracellular calcium, this mechanism is likely to impact neuronal signaling greatly under physiological and pathological conditions. CaSR-dependent reduction of VGSC sensitivity to membrane potential adds further complexity to extracellular calcium signaling and identifies another potential mechanism by which CaSR stimulation may influence neuronal death following stroke and traumatic brain injury (Kim et al., 2013; Hannan et al., 2018).

All data generated are in the manuscript and supporting files. Source provided for Figures 1, 2, and 6 in the manuscript.

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Author details

1. Briana J Martiszus

   1. Section of Pulmonary & Critical Care Medicine, VA Portland Health Care System, Portland, United States
   2. Department of Medicine, Division of Pulmonary & Critical Care Medicine, Oregon Health & Science University, Portland, United States

   Contribution

   Formal analysis, Investigation, Writing - original draft

   Competing interests

   No competing interests declared

2. Timur Tsintsadze

   1. Section of Pulmonary & Critical Care Medicine, VA Portland Health Care System, Portland, United States
   2. Department of Medicine, Division of Pulmonary & Critical Care Medicine, Oregon Health & Science University, Portland, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

3. Wenhan Chang

   Endocrine Research Unit, Veterans Affairs Medical Center and University of California, San Francisco, San Francisco, United States

   Contribution

   Resources, Methodology

   Competing interests

   No competing interests declared

4. Stephen M Smith

   1. Section of Pulmonary & Critical Care Medicine, VA Portland Health Care System, Portland, United States
   2. Department of Medicine, Division of Pulmonary & Critical Care Medicine, Oregon Health & Science University, Portland, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Methodology, Project administration, Writing - review and editing

   For correspondence

   smisteph@ohsu.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0331-7615

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