Nitric Oxide Regulates Neuronal Activity via Calcium- Activated Potassium Channels

Nitric oxide (NO) is an unconventional membrane-permeable messenger molecule that has been shown to play various roles in the nervous system. How NO modulates ion channels to affect neuronal functions is not well understood. In gastropods, NO has been implicated in regulating the feeding motor program. The buccal motoneuron, B19, of the freshwater pond snail Helisoma trivolvis is active during the hyper-retraction phase of the feeding motor program and is located in the vicinity of NO-producing neurons in the buccal ganglion. Here, we asked whether B19 neurons might serve as direct targets of NO signaling. Previous work established NO as a key regulator of growth cone motility and neuronal excitability in another buccal neuron involved in feeding, the B5 neuron. This raised the question whether NO might modulate the electrical activity and neuronal excitability of B19 neurons as well, and if so whether NO acted on the same or a different set of ion channels in both neurons. To study specific responses of NO on B19 neurons and to eliminate indirect effects contributed by other cells, the majority of experiments were performed on single cultured B19 neurons. Addition of NO donors caused a prolonged depolarization of the membrane potential and an increase in neuronal excitability. The effects of NO could mainly be attributed to the inhibition of two types of calcium-activated potassium channels, apaminsensitive and iberiotoxin-sensitive potassium channels. NO was found to also cause a depolarization in B19 neurons in situ, but only after NO synthase activity in buccal ganglia had been blocked. The results suggest that NO acts as a critical modulator of neuronal excitability in B19 neurons, and that calcium-activated potassium channels may serve as a common target of NO in neurons

Exotic Properties of a Voltage-gated Proton Channel from the Snail Helisoma trivolvis

Voltage-gated proton channels, HV1, were first reported in Helix aspersa snail neurons. These H+ channels open very rapidly, two to three orders of magnitude faster than mammalian HV1. Here we identify an HV1 gene in the snail Helisoma trivolvis and verify protein level expression by Western blotting of H. trivolvis brain lysate. Expressed in mammalian cells, HtHV1 currents in most respects resemble those described in other snails, including rapid activation, 476 times faster than hHV1 (human) at pHo 7, between 50 and 90 mV. In contrast to most HV1, activation of HtHV1 is exponential, suggesting first-order kinetics. However, the large gating charge of ∼5.5 e0 suggests that HtHV1 functions as a dimer, evidently with highly cooperative gating. HtHV1 opening is exquisitely sensitive to pHo, whereas closing is nearly independent of pHo. Zn2+ and Cd2+ inhibit HtHV1 currents in the micromolar range, slowing activation, shifting the proton conductance–voltage (gH-V) relationship to more positive potentials, and lowering the maximum conductance. This is consistent with HtHV1 possessing three of the four amino acids that coordinate Zn2+ in mammalian HV1. All known HV1 exhibit ΔpH-dependent gating that results in a 40-mV shift of the gH-V relationship for a unit change in either pHo or pHi. This property is crucial for all the functions of HV1 in many species and numerous human cells. The HtHV1 channel exhibits normal or supernormal pHo dependence, but weak pHi dependence. Under favorable conditions, this might result in the HtHV1 channel conducting inward currents and perhaps mediating a proton action potential. The anomalous ΔpH-dependent gating of HtHV1 channels suggests a structural basis for this important property, which is further explored in this issue (Cherny et al. 2018. J. Gen. Physiol. https://doi.org/10.1085/jgp.201711968)

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Voltage-gated Ca²⁺ channels are not affected by NOC7.

<p>Ca²⁺ currents were recorded in whole-cell voltage-clamp mode. Voltage steps from a holding potential of −60 mV to +60 mV were applied in 10 mV increments. A: Representative traces of Ca²⁺ currents evoked by a voltage step from −60 mV to +10 mV before (upper), during the initial phase (middle), and during the plateau phase of treatment with NOC7 (100 µM, lower). B: Representative I–V plot of Ca²⁺ current measured at the peak amplitude and expressed as normalized Ca²⁺ current (pA/pF) before and after NOC7 application. Note that NOC7 did not have an obvious effect on Ca²⁺ currents. C: Quantification of the effect of NO on Ca²⁺ currents showing that treatment with NOC7 did not have a significant effect on normalized peak currents compared to control groups during both initial and plateau phases. Subsequent application of the Ca²⁺ channel blocker CdCl₂ (100 µM) fully eliminated Ca²⁺ currents.</p

Apamin-sensitive SK channels are responsible for the main effect of NO on membrane potential.

<p>A: Representative recording of a B19 neuron before and after treatment with apamin (5 µM). Note that apamin application led to a sustained depolarization. B: Pre-incubation with apamin (5 µM) fully blocked the plateau depolarization normally seen by treatment with NOC7 (100 µM), but a small initial depolarization was still observed. C: Quantification of the initial depolarization such as shown in A and B. Apamin caused a depolarization, but the amplitude was significantly smaller than that of NOC7 group. NOC7 after pretreatment with apamin induced a significantly smaller depolarization than NOC7 by itself. D: Quantification of the plateau depolarization showing that treatment with NOC7 or apamin resulted in a similar depolarization. Subsequent application of NOC7 in the presence of apamin did not cause any additional depolarization during the plateau phase.</p

Ca²⁺-activated K⁺ channels mediate NO-induced depolarization.

<p>A: Representative recording of a B19 neuron pretreated with a cocktail of the K⁺ channel blockers TEA (20 mM) and 4AP (5 mM), and subsequently treated with NOC7 (100 µM). Inhibition of K⁺ channels completely blocked the depolarizing effect of NOC7. B: Example of a B19 neuron pretreated with CdCl₂ (500 µM) before and after treatment with NOC7 (100 µM). CdCl₂ was used to block Ca²⁺ influx, and indirectly inhibited the activation of Ca²⁺-activated K⁺ channels. Note that NOC7 had only a small depolarizing effect on membrane potential during the initial phase, whereas any depolarization during the plateau phase was fully inhibited in the presence of CdCl₂. C: Quantification of the initial depolarization showing that pretreatment with TEA (20 mM) and 4AP (5 mM) fully blocked the depolarizing effect of NOC7, whereas CdCl₂ (500 µM) significantly inhibited the effect of NOC7 during the initial phase. D: Pretreatment with TEA and 4AP and with CdCl₂ prevented the NOC7-induced depolarization during the plateau phase.</p

IbTX-sensitive BK channels partially contribute to the initial depolarization induced by NOC7.

<p>A: Representative recording of a B19 neuron pretreated with IbTX (300 nM) and after addition of NOC7 (100 µM). Note that NOC7 after IbTX caused a sustained depolarization with similar initial and plateau amplitudes. B: Quantification of the initial depolarization showing that the amplitude of membrane depolarization was significantly reduced in the NOC7 after IbTX group compared to NOC7 by itself. C: Quantification of the plateau depolarization in response to treatment shown in A. IbTX pretreatment did not affect the depolarizing effect of NO during the plateau phase.</p

NO causes a depolarization in B19 neurons <i>in situ</i> in the presence of NOS inhibitors.

<p>A: A representative recording of a B19 neuron located within the buccal ganglion showing that treatment with NOC7 (100 µM) depolarized the membrane potential after the ganglion had been incubated in a solution containing two NOS inhibitors, L-NAME (1 mM) and 7NI (100 µM). Note that the membrane potential is enlarged at higher temporal resolution (highlighted by dashed black boxes) before and after the application of NOC7 to show the depolarization induced by NOC7. B: Quantification of maximal changes in the membrane potential. While NOC7, by itself, did not have an effect on the membrane potential of B19 neurons in intact ganglia, NOC7 was able to cause a significant depolarization, when ganglia were pretreated with L-NAME and 7NI.</p

NO induces an inward current and increases input resistance.

<p>A: Representative recording of a B19 neuron showing that treatment with NOC7 (100 µM) immediately elicited an inward current (holding potential at −50 mV). B: Quantification of the maximal NO-induced current showing that NOC7 evoked a significant inward current compared to the control group. C: Comparison of currents evoked by a voltage step of −10 mV for 0.5 s before and after treatment with NOC7 (100 µM). Note the reduction in the current after NOC7 application (indicated by dashed line). D: Quantification of normalized R_in for vehicle control and NOC7 groups. R_in was normalized to pretreatment values and is expressed in percent. NOC7 significantly increased the R_in in B19 neurons.</p