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Cell-Based Reporter Release Assay to Determine the Activity of Calcium-Dependent Neurotoxins and Neuroactive Pharmaceuticals

Cell-Based Reporter Release Assay to Determine the Activity of Calcium-Dependent Neurotoxins and Neuroactive Pharmaceuticals

Article Cell-Based Reporter Release Assay to Determine the Activity of Calcium-Dependent and Neuroactive Pharmaceuticals

Andrea Pathe-Neuschäfer-Rube, Frank Neuschäfer-Rube * and Gerhard P. Püschel

Department of Nutritional Biochemistry, Institute of Nutritional Science, University of Potsdam, 14469 Potsdam, Germany; [email protected] (A.P.-N.-R.); [email protected] (G.P.P.) * Correspondence: [email protected]; Tel.: +49-33200-88-569

Abstract: The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent release. In this functional assay, a reporter is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a -mediated activation of the Gq-coupled muscarinic- receptor and by the Ca2+-channel forming α-. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine and α-latrotoxin-mediated release by the Ca2+-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and level.

 In accordance with the expression profile a depolarization-stimulated luciferase release by a high  K+-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser + Citation: Pathe-Neuschäfer-Rube, A.; extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K -stimulated Neuschäfer-Rube, F.; Püschel, G.P. luciferase release. In summary, the newly established cell-based assay may represent a versatile tool Cell-Based Reporter Release Assay to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which to Determine the Activity of mediate their activity by the modulation of calcium-dependent neurotransmitter release. Calcium-Dependent Neurotoxins and Neuroactive Pharmaceuticals. Toxins Keywords: cell-based assay; neurotoxins; muscarinic acetylcholine receptor; voltage-dependent 2021, 13, 247. https://doi.org/ calcium channels; VGCC 10.3390/toxins13040247 Key Contribution: The current study provides evidence that a new assay is suitable to measure the Received: 17 February 2021 activity of muscarinic acetylcholine and antagonists, α-latrotoxin and inhibitors of voltage- Accepted: 29 March 2021 dependent calcium-channels. The assay tests the liberation of a reporter enzyme from neurosecretory Published: 30 March 2021 vesicles. The reporter enzyme acts as a surrogate for neurotransmitter release.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- 1. Introduction iations. Disorders in neurotransmitter release are key features of severe neuronal diseases like Parkinson, chronic pain, and depression [1–3]. Neurotoxins and neuroactive pharma- ceutical substances which affect neurotransmitter release are highly interesting tools in the treatment of neuronal diseases. Bacterial neurotoxins such as botulinum toxins and Copyright: © 2021 by the authors. tetanus toxins block neurotransmitter release by the highly specific proteolytic inactivation Licensee MDPI, Basel, Switzerland. This article is an open access article of target snare SNAP-25, syntaxin and synaptobrevin (VAMP), which are essential distributed under the terms and for the fusion of neurosecretory vesicles with the plasma membrane [4]. On the other conditions of the Creative Commons hand, neurotransmitter release can also be modulated by neurotoxins and neuroactive Attribution (CC BY) license (https:// pharmaceuticals without affecting the activity of these target proteins. 2+ creativecommons.org/licenses/by/ The key step in neurotransmitter release is the surge in the intracellular Ca - 4.0/). concentration that triggers the fusion of neurosecretory vesicles with the plasma membrane

Toxins 2021, 13, 247. https://doi.org/10.3390/toxins13040247 https://www.mdpi.com/journal/toxins Toxins 2021, 13, 247 2 of 13

of the presynaptic cell [5–7]. The increase of intracellular calcium concentration can be provoked either by an influx of extracellular calcium along the concentration gradient by opening Ca2+-channels or by the release from intracellular stores like the sarcoplasmatic reticulum. The entry of extracellular calcium can be stimulated by a depolarization- mediated opening of voltage gated calcium channels (VGCC) or by a mediated de novo formation of Ca2+-channels. Activation of Gq-coupled GPCR on the surface of the presynaptic cell will lead to activation of phospholipase C, the formation of second messenger IP3 and therefore release of calcium from intracellular stores. In addition to proteolytic bacterial neurotoxins, other neurotoxins and neuroactive pharmaceuticals which stimulate or inhibit neurotransmitter release by modulation of intra- cellular Ca2+-concentration are of particular interest in the treatment of neuronal diseases. For this reason, derived neurotoxins and synthetic compounds which inhibit the activity of VGCCs or modulate the activation or inhibition of the Gq-coupled muscarinic acetylcholine receptor system were screened for their potential to modulate neurotrans- mitter release [3,8,9]. The biological activity of these neurotoxins is measured in vivo by lethality assays or in vitro cell culture assays by measuring the modulation of intracellular calcium concentration using fluorogenic calcium dyes. A third approach is the direct quantification of in cell culture supernatants. Whereas the mouse assay is ethically problematic, determination of the intracellular calcium concentration in cells might not directly reflect the neurotoxic potential of a compound. On the other hand, measurement of neurotransmitters in cell culture supernatants is time consuming and not suitable for high throughput screening. In this study, a completely different approach to measure the biologic activity of a variety of agonists, antagonists and toxins was used: The neuronal cell line SIMA was stably transfected with a plasmid coding for Gaussia princeps luciferase (GLuc), which was N-terminally extended with the leader sequence of human proopiomelanocortin (hPOMC1- 26) that redirects the GLuc into neuro-secretory vesicles. From these vesicles, GLuc was released upon depolarization of the cells into the cell culture supernatant together with neurotransmitters. In proof of principle, it was recently shown that the depolarization- dependent release was efficiently inhibited by BoNT/A and to a lesser extent by BoNT/B and tetanus toxin [10,11]. The goal of this study was to analyze whether this assay is also suitable to detect the activity of other neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. To this end, the modulation of GLuc release by the muscarinic acetylcholine receptor carbachol and its antagonist atropine, as well as the Ca2+-channel forming neurotoxin α-latrotoxin and inhibitors of VGCCs was analyzed in SIMA-hPOMC1-26-GLuc cells.

2. Results 2.1. Suitability for Testing Compounds Leading to Neurotransmitter Release by an Increase of Intracellular Ca2+-Concentration from Intracellular Stores To assess the suitability of the luciferase release assay for testing compounds which pro- voke neurotransmitter release because of an increase of intracellular calcium-concentration from intracellular pools, cells were stimulated with the M3-muscarinic acetylcholine recep- tor agonist carbachol. Activation of the M3 receptor leads to an activation of Gq protein and therefore an IP3-mediated release of Ca2+ from the endoplasmic reticulum. When cells were incubated with a non-depolarizing Na+-containing buffer, a certain amount of luciferase activity was released into the cell culture supernatant. (Figure1A). This unspecific release was significantly increased by stimulation with 100 µM and 1000 µM carbachol. At a concentration of 1000 µM carbachol luciferase release into the medium was nearly identical to the release stimulated by a K+-containing depolarizing buffer, which was 3–4-fold higher than the unspecific release. Carbachol did not influence luciferase release induced by K+-depolarization buffer. To test if the stimulation of luciferase release was specific for the activation of the muscarinic acetylcholine receptor, cells were treated with the muscarinic acetylcholine receptor antagonist atropine before and during the carbachol Toxins 2021, 13, 247 3 of 14

which was 3–4-fold higher than the unspecific release. Carbachol did not influence lucif- Toxins 2021, 13, 247 erase release induced by K+-depolarization buffer. To test if the stimulation of luciferase3 of 13 release was specific for the activation of the muscarinic acetylcholine receptor, cells were treated with the muscarinic acetylcholine receptor antagonist atropine before and during stimulation.the carbachol While stimulation. 500 µM atropineWhile 500 did µM not a influencetropine did the not luciferase influence release the luciferase by K+-mediated release + depolarization,by K -mediated the depolarization, carbachol-stimulated the carbachol release-stimulated was completely release abolished was completely by atropine abol- (Figureished by1B). atropine (Figure 1B).

FigureFigure 1. 1.Carbachol-dependent Carbachol-dependent stimulationstimulation ofof luciferaseluciferase release.release. SIMASIMA cellscells stablystably expressingexpressinghPOMC1-26 hPOMC1-26 GLucGLuc werewere culturedcultured andand differentiateddifferentiated as described described in in the the methods methods section. section. After After removing removing the the medium, medium, cells cells were were washed washed with withfresh freshmedium medium and incubated and incubated in differentiation in differentiation medium medium in the in absence the absence or presence or presence of 500 of µM 500 atropineµM atropine for 10 for min. 10 min.Cells Cells were + + werethen thenincubated incubated for three for three minutes minutes with with non- non-depolarizingdepolarizing (Na (Na, control)+, control) or depolarizing or depolarizing (K , stimulated) (K+, stimulated) balanced balanced salt solu- salt tion in the presence of different carbachol concentrations (A) or 1 mM carbachol −/+ 500 µM atropine (B). Cell culture solution in the presence of different carbachol concentrations (A) or 1 mM carbachol −/+ 500 µM atropine (B). Cell culture supernatants were centrifuged and luciferase activity was determined in the cell culture supernatants. Values are means supernatants were centrifuged and luciferase activity was determined in the cell culture supernatants. Values are means ± ± SEM of at least three independent experiments. Statistics: Student’s t-test for unpaired samples, a: > control buffer with- SEMout carbachol: of at least threeb: > control independent buffer experiments.with the respective Statistics: carbachol Student’s concentration; t-test for unpaired p < 0.05. samples, a: > control buffer without carbachol: b: > control buffer with the respective carbachol concentration; p < 0.05. Thus, the cell-based assay was suitable to determine the neurotransmitter release Thus, the cell-based assay was suitable to determine the neurotransmitter release stimulated by a liberation of calcium from intracellular stores. stimulated by a liberation of calcium from intracellular stores.

2.2.2.2. SuitabilitySuitability forfor TestingTesting Compounds Compounds Leading Leading to to Neurotransmitter Neurotransmitter Release Release by by an an Increase Increase of of 2+ 2+ IntracellularIntracellular CaCa2+-Concentration-Concentration by Ca2+--Channel-FormingChannel-Forming N Neurotoxinseurotoxins InIn additionaddition toto depolarization depolarization oror Gq-coupled Gq-coupled receptor receptor stimulation, stimulation, neurotransmitter neurotransmitter 2+ releaserelease can can also also be be activated activated by by the the action action of of Ca Ca2+ pore forming LTX-neurotoxinsLTX-neurotoxins producedproduced byby black-widowblack-widow spiders from from the the latrodectus family family [12 [].12 The]. Themammalian mammalian-specific-specific α-latro-α- latrotoxintoxin (α-LTX) (α-LTX) is a is relatively a relatively big big protein protein (1381 (1381 AA) AA) which which can can bind bind the presynapticpresynaptic cellcell 2+ adhesionadhesion protein protein neurexin, neurexin, leading leading to theto the formation formation of a of new a new Ca2+ -channelCa -channel in the in membrane the mem- ofbrane the presynapticof the presynaptic cells. Since cells. this Since Ca this2+-channel Ca2+-channel is permanently is permanently open, open, extracellular extracellular Ca2+ canCa2+ enter can enter the cell the along cell along the concentration the concentration gradient, gradient, leading leading to neurotransmitter to neurotransmitter release re- andlease therefore and therefore the permanent the permanent depolarization depolarization of postsynaptic of postsynaptic cells. cells. For thisFor reason,this reason, the potentialthe potential of α of-LTX α-LTX to stimulateto stimulate luciferase luciferase release release from from SIMA-hPOMC1-26-GLuc SIMA-hPOMC1-26-GLuc cells cells waswas tested.tested.α α-LTX-LTX significantlysignificantly and and dose-dependently dose-dependently increasedincreased luciferase luciferase releaserelease under under controlcontrol conditions conditions up up from from 0.1 0.1 nM nMα α-LTX-LTX (Figure (Figure2 A).2A). At At a a concentration concentration of of 10 10 nM, nM,α α-LTX-LTX stimulatedstimulated luciferaseluciferase releaserelease intointo thethe mediummedium waswas asas highhigh asasthe the release release stimulated stimulatedby by a a KK++-containing-containing depolarizationdepolarization bufferbuffer (3-fold).(3-fold). SimilarSimilar toto carbachol,carbachol,α α-LTX-LTX did did not not influence influence luciferaseluciferase release release induced induced by by K K++-depolarization.-depolarization. ToTo testtest ifif thethe αα-LTX-mediated-LTX-mediated stimulation stimulation ofof luciferaseluciferase release release was was Ca Ca2+2+-dependent,-dependent, cellscells werewere treatedtreated with with the the Ca Ca2+2+-chelator-chelator EGTAEGTA duringduring thethe stimulationstimulation byby KK++-depolarization-depolarization oror byby αα-LTX.-LTX. WhereasWhereas EGTAEGTA diddid notnot reduce reduce luciferase release under control conditions, both the K+-dependent depolarization and α-LTX-mediated release was completely blocked by EGTA (Figure2B).

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Toxins 2021, 13, 247 4 of 13 luciferase release under control conditions, both the K+-dependent depolarization and α- LTX-mediated release was completely blocked by EGTA (Figure 2B).

FigureFigure 2.2.α α-Latrotoxin-dependent-Latrotoxin-dependent stimulationstimulation ofof luciferaseluciferase release.release. SIMASIMA cellscells stablystably expressingexpressing hPOMC1-26hPOMC1-26 GLucGLuc werewere culturedcultured andand differentiated as as described described in in the the methods methods section. section. After After removing removing the themedium, medium, cells cells were were washed washed with withfresh freshmedium medium and incubat and incubateded in differentiation in differentiation medium medium for 10 for min. 10 Cells min. Cellswere werethen incubated then incubated for five for minutes five minutes with non with-depo- non- +, + depolarizinglarizing (Na (Na control)+, control) or depolarizing or depolarizing (K , stimulated) (K+, stimulated) balanced balanced salt solution salt solution in the in presence the presence of different of different α-latrotoxinα-latrotoxin con- centrations (A) or 5 nM α-latrotoxin −/+ 10 mM EGTA (B). Cell culture supernatants were centrifuged, and luciferase concentrations (A) or 5 nM α-latrotoxin −/+ 10 mM EGTA (B). Cell culture supernatants were centrifuged, and luciferase activity was determined in the cell culture supernatants. Values are means ± SEM of at least three independent experi- activity was determined in the cell culture supernatants. Values are means ± SEM of at least three independent experiments. ments. Statistics: Student’s t-test for unpaired samples, a: > control buffer without α-latrotoxin: b: > control buffer with the α Statistics:respectiveStudent’s α-latrotoxin t-test concentration; for unpaired p < samples, 0.05. a: > control buffer without -latrotoxin: b: > control buffer with the respective α-latrotoxin concentration; p < 0.05. Thus, the cell-based assay was also suitable to determine the neurotransmitter release Thus, the cell-based assay was also suitable to determine the neurotransmitter release stimulated by the entry of extracellular Ca2+ via Ca2+-channel forming α-LTX. stimulated by the entry of extracellular Ca2+ via Ca2+-channel forming α-LTX.

2.3.2.3. SuitabilitySuitability forfor TestingTesting CompoundsCompounds LeadingLeading toto anan InhibitionInhibition ofof NeurotransmitterNeurotransmitter Release Release by by 2+ BlockingBlocking Voltage-GatedVoltage-Gated CaCa2+--ChannelsChannels (VGCC) ManyMany neurotoxins and and neuroactive neuroactive compounds compounds act act as asinhibit inhibitorsors of voltage of voltage-gated--gated-cal- calciumcium channels channels (VGCC). (VGCC). Voltage Voltage-gated-gated calcium calcium channels channels are are activated activated by by potential-- mediatedmediated depolarization. depolarization. Therefore, calcium influx influx triggers triggers synaptic vesicle exocytosis exocytosis leadingleading toto releaserelease ofof excitatoryexcitatory neurotransmittersneurotransmitters [[1313].]. VGCCsVGCCs cancan bebe classifiedclassified basedbased onon theirtheir voltagevoltage activationactivation characteristicscharacteristics asas highhigh oror low-voltagelow-voltage activatedactivated channelschannels [[1414].]. TheThe VGCCsVGCCs cancan bebe furtherfurther subdividedsubdivided basedbased onon theirtheir structuralstructural similaritiessimilarities ofof thethe channel-channel- formingforming αα11-subunit-subunit (Cav1,(Cav1, Cav2 and Cav3)Cav3) oror theirtheir sensitivitysensitivity to bebe blockedblocked byby pharma-pharma- ceuticalceutical agents (L, N, P/Q, R R and and T T-type).-type). Collectively, Collectively, the the high high-voltage-voltage VGCCs VGCCs include include L- L-(Cav1.1,(Cav1.1, Cav1.2, Cav1.2, Cav1.3, Cav1.3, CaV1.4), CaV1.4), P/Q P/Q-(Cav2.1),-(Cav2.1), N-(Cav2.2) N-(Cav2.2) and and R-(Cav2.3) R-(Cav2.3) type type channels, chan- nels,while while the low the-voltage low-voltage VGCCs VGCCs include include T-type T-type (Cav3.1, (Cav3.1, Cav3.2, Cav3.2, Cav3.3) Cav3.3) channels. channels. The high The- high-voltagevoltage VGCCs VGCCs typically typically form form hetero hetero multimers multimers that thatconsist consist of the of thechannel channel-forming-forming α1- αsubunit1-subunit along along with with auxiliary auxiliary β, α2δ,β, α 2andδ, and γ-subunits.γ-subunits. BeforeBefore the potential of of VGCC VGCC inhibitors inhibitors to to block block luciferase luciferase release release was was assessed assessed in inSIMA SIMA-hPOMC1-26-GLuc-hPOMC1-26-GLuc cells, cells, the theexpression expression profile profile of VGCC of VGCC channel channel-forming-forming α1-sub-α1- subunitsunits was wasanalyzed analyzed in the in reporter the reporter cell line cell both line at boththe mRNA at the and mRNA protein and level protein (Figure level 3). (FigureqPCR analysis3) . qPCR revealed analysis that revealed SIMA-hPOMC1 that SIMA-hPOMC1-26-Gluc-26-Gluc cells express cells VGCC express in the VGCC descend- in theing descending order: CaV1.3 order: (L-type) CaV1.3 = CaV2.2 (L-type) (N =- CaV2.2type) >> (N-type) CaV1.1 >> (L- CaV1.1type) > (L-type) CaV1.4 (L >- CaV1.4type) = (L-type)CaV3.1 (T = CaV3.1-type) > (T-type) CaV2.3 > (R CaV2.3-type) (R-type)> CaV3.3 > CaV3.3(T-type) (T-type) = CaV1.2 = CaV1.2 (L-type) (L-type) > CaV2.1 > CaV2.1 (P/Q- (P/Q-type)type) (Figure (Figure 3A). On3A). the On protein the protein level, level,only antibodies only antibodies against against the α1 the-subunitsα1-subunits CaV1.3 CaV1.3(L-type) (L-type) und CaV2.2 und CaV2.2(N-type) (N-type) detected detected proteins proteins of the estimated of the estimated molecular molecular weight weightaround around170–180 170–180 kDa by kDaWestern by Western blot analysis blot analysis in lysates in lysatesof SIMA of-hPOMC1 SIMA-hPOMC1-26-GLuc-26-GLuc cells (Figure cells (Figure3B). Therefore, it seems that SIMA-hPOMC1-26-GLuc cells mainly express VGCC from the L-type and N-type.

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Toxins 2021, 13, 247 5 of 13 3B). Therefore, it seems that SIMA-hPOMC1-26-GLuc cells mainly express VGCC from the L-type and N-type.

Figure 3. Expression of CaV in the reporter cell line. The expression of the CaVs was determined in differentiated SIMA cells Figure 3. Expression of CaV in the reporter cell line. The expression of the CaVs was determined in differentiated SIMA stably expressing hPOMC1-26 GLuc. (A) Total RNA was isolated and relative mRNA expression of CaV was determined cells stably expressing hPOMC1-26 GLuc. (A) Total RNA was isolated and relative mRNA expression of CaV was deter- by RT-qPCR as described in the methods section. Values are means ± SEM of three independent mRNA preparations. mined by RT-qPCR as described in the methods section. Values are means ± SEM of three independent mRNA prepara- (Btions.) CaVprotein (B) CaVprotein expression expression was determined was determined in three in three independently independently produced produ cellced lysatescell lysates (L1, (L1 L2, andL2 and L3) L3 with) with specific specific antibodiesantibodies by by Western Western blot. blot.

In line with the VGCC expression profile K -depolarization-stimulated luciferase In line with the VGCC expression profile K++-depolarization-stimulated luciferase re- releaselease was was significantly significantly inhibited byby thethe L-typeL-typeVGCC VGCC inhibitors inhibitors nifedipine nifedipine and and verapamil verapamil whichwhich were were used used as as antihypertensive antihypertensive agents agents [15 [15].]. Nifedipine Nifedipine inhibited inhibited luciferase luciferase release release with an EC50 of 33 nM (Figure4A). The inhibition was significant from 10 nM and higher, with an EC50 of 33 nM (Figure 4A). The inhibition was significant from 10 nM and higher, and maximal at a concentration of 10 µM (Figure4A). Similar to nifedipine, verapamil and maximal at a concentration of 10 µM (Figure 4A). Similar to nifedipine, verapamil inhibited luciferase release with an EC50 of 79 nM which was significant from 100 nM and inhibited luciferase release with an EC50 of 79 nM which was significant from 100 nM and higher, and maximal at a concentration of 10 µM (Figure4B). higher, and maximal at a concentration of 10 µM (Figure 4B). At a concentration of 10 µM both nifedipine and verapamil decreased K+-stimulated luciferase release to the level of the unspecific release. Therefore, it appears that a major part of K+-depolarization stimulated GLuc release was L-type VGCC dependent and SIMA- hPOMC1-26-GLuc is a useful tool for the screening of neuronal L-type VGCC modulators. The VGCC expression profile of SIMA-hPOMC1-26-GLuc also showed high expression of N-type VGCC, CaV2.2. In line with this, N-type VGCC inhibitors ω- GVIA and MVIIA from marine cone snails decreased K+-depolarization stimulated luciferase release significantly and maximally at a concentration of 1 nM (Figure5).

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Figure 4. Inhibition of luciferase release by L-Type VGCC inhibitors. SIMA cells stably expressing hPOMC1-26 GLuc were cultured and differentiated as described in the methods section. After removing the medium, cells were washed with fresh medium and incubated in differentiation medium with different concentrations of L-type VGCC inhibitors nifedipine (A) or verapamil (B) for 10 min. Cells were then incubated for three minutes with non-depolarizing (Na+, control) or depolar- izing (K+, stimulated) balanced salt solution in the presence of different nifedipine (A) or verapamil (B) concentrations. Cell culture supernatants were centrifuged, and luciferase activity was determined in the cell culture supernatants. Values are means ± SEM of at least three independent experiments. Statistics: Student’s t-test for unpaired samples, a: > control buffer without nifedipine or verapamil: c: < control or stimulation buffer without nifedipine or verapamil; p < 0.05.

At a concentration of 10 µM both nifedipine and verapamil decreased K+- stimulated Figure 4. InhibitionInhibition of of luciferase luciferaseluciferase release release release by by L L-Type-Type to VGCC the level inhibitors. of the unspecificSIMA cells stablyrelease. expressing Therefore, hPOMC1 hPOMC1-26 it appears-26 GLuc that were a major cultured and differentiated asaspart described of K+- indepolarization the methods section. stimulated After removing GLuc release the medium, was Lcells-type were VGCC washed dependent with fresh and medium and incubatedincubated inin differentiationdiSIMAfferentiation-hPOMC1 medium medium-26- withGLuc with different different is a useful concentrations concentrations tool for the of of screening L-type L-type VGCC VGCC of neuronal inhibitors inhibitors nifedipineL -nifedipinetype VGCC (A )(A or )mod- or verapamil (B) for 10 min. Cells were then incubated for three minutes with non-depolarizing (Na+, control) or depolar- verapamil (B) for 10 min. Cellsulators. were then incubated for three minutes with non-depolarizing (Na+, control) or depolarizing izing (K+, stimulated) balanced salt solution in the presence of different nifedipine (A) or verapamil (B) concentrations. (K , stimulated) balanced salt solutionThe VGCC in the expression presence of differentprofile of nifedipine SIMA-hPOMC1 (A) or verapamil-26-GLuc (B also) concentrations. showed high Cell expres- Cell+ culture supernatants were centrifuged, and luciferase activity was determined in the cell culture supernatants. Values cultureare means supernatants ± SEM of at were least centrifuged,sion three of independent N- andtype luciferase VGCC, experim activityCaV2.2.ents. Statistics: was In determinedline Studentwith this, in’s thet- testN cell- typefor culture unpaired VGCC supernatants. inhibitorssamples, a: Values > -controlconotoxins are meansbuffer without± SEM ofnifedipine at least three orGVIA verapamil: independent and MVIIAc: < experiments. control from or stimulationmarine Statistics: cone Student’sbuffer snails without t-testdecreased fornifedipine unpaired K+-depolarization or samples,verapamil; a: >p control

Figure 5. Inhibition of luciferase release by N N-Type-Type VGCC inhibitors. SIMA cells stably expressing expressing hPOMC1-26hPOMC1-26 GLucGLuc werewere cultured cultured and and differentiated differentiated as as described described in in the the methods methods section. section. After After remov- removing the medium, cells were washed with fresh medium and incubated in differentiation ing the medium, cells were washed with fresh medium and incubated in differentiation medium with medium with different concentrations of N-type VGCC inhibitors - GVIA or -cono- different concentrations of N-type VGCC inhibitors ω-conotoxin GVIA or ω-conotoxin MVIIA for toxin MVIIA for 10 min. Cells were then incubated for three minutes with non-depolarizing (Na+, + 10control min., Cellsnot shown were then) or depolarizing incubated for (K three+, stimulated) minutes with balanced non-depolarizing salt solution (Na in the, control, presence not of shown) dif- or depolarizing (K+, stimulated) balanced salt solution in the presence of different ω-conotoxin concentrations. Cell culture supernatants were centrifuged, and luciferase activity was determined in the cell culture supernatants. Values are means ± SEM of at least three independent experi- ments. Statistics: Student’s t-test for unpaired samples, a: > control buffer without ω-conotoxins: Figurec: < stimulation 5. Inhibition buffer of luciferase without ω release-conotoxins; by N-Typep < 0.05. VGCC inhibitors. SIMA cells stably expressing hPOMC1-26 GLuc were cultured and differentiated as described in the methods section. After removingBy contrast, the medium, at 1 cells nM were neither washedω-conotoxin with fresh affectedmedium unspecificand incubated luciferase in differentiation release (not mediumshown). with In different contrast concentrations to the L-type of VGCCN-type inhibitorsVGCC inhibitors nifedipine -conotoxin and verapamil,GVIA or -cono- which toxinshowed MVIIA a 55% for 10 reduction min. Cells of were luciferase then incubated release for at three a concentration minutes with ofnon 10-depolarizingµM, maximal (Na+ω, - controlconotoxin-induced, not shown) or inhibition depolarizing was (K only+, stimulated) 30% of Kbalanced+-stimulated salt solution luciferase in the release presence(Figure of dif-5) .

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ferent -conotoxin concentrations. Cell culture supernatants were centrifuged, and luciferase ac- tivity was determined in the cell culture supernatants. Values are means ± SEM of at least three independent experiments. Statistics: Student’s t-test for unpaired samples, a: > control buffer with- out -conotoxins: c: < stimulation buffer without -conotoxins; p < 0.05.

By contrast, at 1 nM neither -conotoxin affected unspecific luciferase release (not Toxins 2021, 13, 247 7 of 13 shown). In contrast to the L-type VGCC inhibitors nifedipine and verapamil, which showed a 55% reduction of luciferase release at a concentration of 10 µM, maximal - conotoxin-induced inhibition was only 30% of K+-stimulated luciferase release (Figure 5). Therefore, it it appears appears that that a apart part of of K K+-depolarization+-depolarization stimulated stimulated luciferase luciferase release release was was N- typeN-type VGCC VGCC dependent dependent and and SIMA SIMA-hPOMC1-26-GLuc-hPOMC1-26-GLuc cells cells are are a a suitable suitable tool tool for for the screeningscreening of neuronal N N-type-type VGCC modulators. InIn contrastcontrast to to L-type L-type and and N-type N-type VGCC VGCC the the expression expression level level of T-type of T- VGCCstype VGCCs in SIMA- in SIMAhPOMC1-26-GLuc-hPOMC1-26-GLuc cells was cells low was (Figure low (Figure3). Surprisingly, 3). Surprisingly, K +-depolarization K+-depolarization stimulated stimu- latedluciferase luciferase release release was also was significantly also significantly inhibited inhibited by the by T-type the T VGCC-type VGCC inhibitors inhibitors trimetha- tri- methadionedione and zonisamide, and zonisamide, which arewhich used are in theused therapy in the oftherapy neuronal of neuronal diseases such diseases as Parkinson such as + Parkinsonand epilepsy and [16 epilepsy]. Both trimethadione[16]. Both trimethadione and zonisamide and zonisamide inhibited K inhibited-depolarization K+-depolari- medi- zationated luciferase mediated release luciferase significantly release significantly from 50 nM from and higher,50 nM and maximal higher, at am concentrationaximal at a con- of centration50 µM and of left 50 theµM unspecific and left the release unspecific unaffected release (Figure unaffected6A,B). (Figure 6A,B).

Figure 6. InhibitionInhibition of of luciferase luciferase release release by by T-Type T-Type VGCC VGCC inhibitors. inhibitors. SIMA SIMA cells cells stably stably expressing expressing hPOMC1 hPOMC1-26-26 GLuc GLucwere werecultured cultured and differentiated and differentiated as descri asbed described in the methods in the methods section. section.After removing After removing the medium, the cells medium, were cellswashed were with washed fresh medium and incubated in differentiation medium with different concentrations of T-type VGCC inhibitors trimethadione with fresh medium and incubated in differentiation medium with different concentrations of T-type VGCC inhibitors (A) or zonisamide (B) for 10 min. Cells were then incubated for three minutes with non-depolarizing (Na+, control) or trimethadione (A) or zonisamide (B) for 10 min. Cells were then incubated for three minutes with non-depolarizing (Na+, depolarizing (K+, stimulated) balanced salt solution in the presence of different trimethadione (A) or zonisamide (B) con- centrations.control) or depolarizing Cell culture (K+,supernatants stimulated) were balanced centrifuged salt solution, and luciferase in the presence activity of was different determined trimethadione in the cell (A) culture or zonisamide super- (natants.B) concentrations. Values are Cellmeans culture ± SEM supernatants of at least three were independent centrifuged, experiments. and luciferase Statistics: activity wasStudent determined’s t-test for in theunpaired cell culture sam- ples,supernatants. a: > control Values buffer are without means trimethadione± SEM of at least or zonisamide: three independent c: < control experiments. or stimulat Statistics:ion buffer Student’s without t-test trimethadione for unpaired or zonisamide;samples, a: > p control < 0.05. buffer without trimethadione or zonisamide: c: < control or stimulation buffer without trimethadione or zonisamide; p < 0.05. Similar to the -conotoxins GVIA and MVIIA, trimethadione inhibited only 30% of the stimulatedSimilar to theluciferaseω-conotoxins release GVIA (Figure and 6A MVIIA,,B). In trimethadionecontrast to trimethadione, inhibited only zonisamide 30% of the wasstimulated toxic for luciferase SIMA-hPOMC1 release (Figure-26-GLuc6A,B). cells In at contrasta concentration to trimethadione, of 5 mM and zonisamide strongly wassup- pressedtoxic for unspecific SIMA-hPOMC1-26-GLuc and stimulated cells luciferase at a concentration release (Figure of 5 6B). mM Therefore, and strongly while suppressed expres- sionunspecific level of and T-type stimulated VGCC luciferasewas low at release the mRNA (Figure and6B). protein Therefore, level, while it seemed expression that a s levelmall partof T-type of K+- VGCCdepolarization was low stimulated at the mRNA luciferase and protein release level, was itT- seemedtype VGCC that dependent a small part and of + SIMAK -depolarization-hPOMC1-26- stimulatedGLuc cells luciferasecan also be release used for was the T-type analysis VGCC of T dependent-type mediators. and SIMA- hPOMC1-26-GLucAccording to their cells canvery also low be expression used for the level, analysis both ofthe T-type P/Q-type mediators. inhibitor agatoxin from Accordingthe spider Agelonopsis to their very aperta low expression (Figure 7A) level, and both the R the-type P/Q-type VGCC inhibitor agatoxinSNX-482 fromfrom the the spider spider Agelonopsis aperta gigas ( (FigureFigure 7B)7A) did and not the inhibit R-type the VGCC luciferase inhibitor release SNX-482 under controlfrom the and spider stimulated Hysterocrates conditions. gigas (Figure7B) did not inhibit the luciferase release under control and stimulated conditions. To exclude cytotoxicity, the impact of all VGCC inhibitors at the highest concentration employed in the release assays was tested in the AlamarBlue assay, based on the conversion of resazurin to the fluorogenic resorufin by viable cells. None of the toxins reduces cell viability (Supplementary Figure S1). Toxins 2021, 13, 247 8 of 13 Toxins 2021, 13, 247 8 of 14

FigureFigure 7. 7.No No inhibition inhibition of of luciferase luciferase release release by by P/Q P/Q and and R-Type R-Type VGCC VGCC inhibitors. inhibitors. SIMA SIMA cells cells stably stably expressing expressing hPOMC1-26 hPOMC1- GLuc26 GLuc were were cultured cultured and and differentiated differentiated as describedas described in thein the methods methods section. section. After After removing removing the the medium, medium, cells cells werewere washedwashed with with fresh fresh medium medium and and incubated incubated in in differentiation differentiation medium medium with with different different concentrations concentrations of of P/Q-type P/Q-typeVGCC VGCC inhibitorinhibitor agatoxin agatoxin ( A(A)) or or R-type R-type VGCC VGCC inhibitor inhibitor SNX-482 SNX-482 ( B(B)) for for 10 10 min. min. Cells Cells were were then then incubated incubated for for three three minutes minutes with with non-depolarizing (Na+, control) or depolarizing (K+, stimulated) balanced salt solution in the presence of different agatoxin non-depolarizing (Na+, control) or depolarizing (K+, stimulated) balanced salt solution in the presence of different agatoxin (A) or SNX-482 (B) concentrations. Cell culture supernatants were centrifuged, and luciferase activity was determined in (A) or SNX-482 (B) concentrations. Cell culture supernatants were centrifuged, and luciferase activity was determined in the cell culture supernatants. Values are means ± SEM of at least three independent experiments. Statistics: Student’s t-test ± thefor cellunpaired culture samples, supernatants. a: > control Values buffer are means withoutSEM agatoxin of at leastor SNX482; three independent p < 0.05. experiments. Statistics: Student’s t-test for unpaired samples, a: > control buffer without agatoxin or SNX482; p < 0.05. To exclude cytotoxicity, the impact of all VGCC inhibitors at the highest concentra- 3. Discussion tion employed in the release assays was tested in the AlamarBlue assay, based on the con- 3.1.version Suitability of resazurin of the Assay to the for fluorogenic Compounds resorufin Stimulating by viable Neurotransmitter cells. None Releaseof the toxins reduces cell viabilityMuscarinic (Suppl acetylcholineementary Fig receptor:ure S1). In two former projects, release of the reporter enzyme GLuc was stimulated by a high K+-depolarization buffer and entry of extracellular Ca3. 2+Discussioninto the reporter cell line [10,11]. One aim of this study was to analyze if GLuc can also 2+ be3.1. released Suitability by aof Ca the A-increasessay for C fromompounds intracellular Stimulating pools. Neurotransmitter Muscarinic acetylcholine Release receptors, a member of class I, seven transmembrane G-protein-coupled receptors (GPCRs), comprise five distinctMuscarinic subtypes, acetylcholine denoted receptor: as muscarinic In two M1, former M2, M3,projects, M4, andrelease M5 of receptors the reporter [17–20 en-]. + Whereaszyme GLuc M2 was and stimulated M4 receptors by area high Gi-coupled K -depolarization and inhibit buffer cAMP-formation, and entry of M1,extracellular M3 and 2+ M5Ca receptors into the arereporter coupled cell to line Gq [ and10,11 increased]. One aim intracellular of this study calcium was concentrationto analyze if GLuc from thecan 2+ sarcoplasmaticalso be released reticulum by a Ca via-increase phospholipase from intracellular C activation pools. and Muscarinic IP3-formation. acetylcholine Carbachol, re- aceptors, non-selective a member muscarinic of class I, acetylcholine seven transmembrane receptor agonist, G-protein increases-coupled intracellular receptors (GPCRs), calcium concentrationcomprise five distinct and neurotransmitter subtypes, denoted release as muscarinic [21]. In line M1, with M2, this,M3, M4, carbachol and M5 provoked receptors the[17– release20]. Whereas of the reporterM2 and M4 enzyme receptors GLuc are from Gi-coupled SIMA-hPOMC1-26-GLuc and inhibit cAMP- cellsformation, in a dose- M1, dependentM3 and M5 manner receptors and are the coupled release wasto Gq completely and increas blockeded intracellular by the muscarinic calcium concentration acetylcholine receptorfrom the antagonistsarcoplasmatic atropine. reticulum At high via phospholipase concentrations, C carbachol-mediated activation and IP3-formation. GLuc release Car- reachedbachol, thea non same-selective level as muscarinic GLuc release acetylcholine stimulated receptor by high Kagonist,+-depolarization. increases intracellular calciumAs dysfunctionconcentration in and the neurotransmitter system release has been[21]. identifiedIn line with in this, various carbachol neuronal pro- diseases,voked the such release as Parkinson of the reporter and epilepsyenzyme [GLuc22,23 ],from antagonists SIMA-hPOMC1 of the muscarinic-26-GLuc cells system in a remaindose-dependent of great interest manner as and potential the release lead was CNS completely drug substances. blocked Theby the tropane muscarinic alkaloids ace- scopolaminetylcholine receptor and antagonist atropine. are widely At used high as concentrations, carbachol drugs [24-].mediated GLuc hasrelease also reached been used the in same the treatment level as GLuc of motion release sickness stimulated for a by long high time K+ [-25depolarization.]. The drawbacks of scopolamineAs dysfunction are the in the manifold cholinergic central system and peripheralhas been identified nervous in system various side neuronal effects, dis- as scopolamineeases, such as is Parkinson not receptor and subtypeepilepsy specific. [22,23], antagonists Development of the of newmuscarinic selective system and potentremain muscarinicof great int acetylcholineerest as potential receptor lead antagonistsCNS drug substances. either by de The novo tropane synthesis alkaloids or screening scopola- ofmine animal and venomshyoscyamine is of noteare widely in the used treatment as anticholinergic of neuronal diseasesdrugs [24 [26]. ].Scopolamine Screening forhas anti-cholinergicalso been used in drugs the treatment includes the of motion structure sickness guided for development a long time of[25 M3]. The receptor drawbacks specific of antagonistsscopolamine [27 are] and the themanifold identification central and of M1 peripheral . M3 receptornervous selectivesystem side drugs effects, [28]. as New sco- antagonistspolamine is werenot receptor analyzed subtype either specific. by radioligand-binding Development of new studies selective with and the potent membrane mus- ofcarinic M1-5 acetylcholine receptor transfected receptor CHOantagonists cells oreither by functionalby de novo studies synthesis (IP-formation or screening and of ani- ß- arrestinmal recruitment) is of note with in receptor-overexpressingthe treatment of neuronal CHO diseases and HEK293 [26]. Screening cells. However, for anti these-cho- models might not directly mirror the real functional read-out of anticholinergic drugs, the

Toxins 2021, 13, 247 9 of 13

modulation of neurotransmitter release. To overcome this problem, our cell-based SIMA- hPOMC1-26-GLuc cell model may be a useful tool to verify basic screening of muscarinic acetylcholine receptor antagonist because a.) receptor expression level and expression profile is more similar to neuronal cells than in CHO or HEK293 cells and b.) the last functional step of , the neurotransmitter release, is determined in the SIMA hPOMC1-26-GLuc cell model. α-latrotoxin: The second aim of the study was to analyze whether calcium-channel forming toxins can stimulate GLuc release from SIMA-hPOMC1-26-GLuc cells instead of high-K+- depolarization buffer. α-Latrotoxin (α-LTX), a neurotoxin from black widow spider triggers neurotransmitter release by synaptic vesicle exocytosis from presy- naptic nerve terminals. It is the main toxic component in the venom of black widow spiders, whose bite leads to , a syndrome consisting of muscle pain, abdominal cramps and raised blood pressure [29]. α-LTX has been an extremely useful tool in the analysis of synaptic signal transmission as α-LTX acts very selectively on presynaptic nerve terminals [12]. The action of α-LTX is mediated by two distinct mechanisms: First, α-LTX can bind to the receptor molecule , leading to insertion of α-LTX in the plasma membrane. This stimulates exocytosis of classical neurotransmitters such as glutamate and acetylcholine in a calcium-independent manner 12]. Second, α−LTX interacts with the protein neurexin to form a permanently open Ca2+-channel, which leads to the release of catecholamines in a calcium-dependent manner [12]. In the present study, in low nM concentrations α-LTX stimulated release of the reporter GLuc under control conditions, but did not affect GLuc release by high K+-depolarization (Figure2). At the highest concentration used (5 nM), α-LTX mediated GLuc release did not differ from high K+-depolarization induced release. As the α-LTX stimulated GLuc release was completely blocked by EGTA, the mechanism of α-LTX dependent GLuc release might reflect neurexin and calcium-dependent release of catecholamines rather than latrophilin and Ca2+-independent release of classical neurotransmitters. Interestingly, α-LTX not only stimulates a massive exocytosis of neurotransmitters but also causes an acute and complete degeneration of motor axon terminals, followed by a rapid recovery [30]. By contrast, botulinum toxins induce a long-lasting paralysis without nerve-terminal degeneration. In a former study, it was shown that injection of α-LTX in mouse muscles which were paralyzed with BoNT/A accelerates the recovery of neurotransmission from several months to a few days [31]. This interplay of both toxins can bring more insights into the mechanisms of peripheral human pathologies due to degeneration of motor axon terminals. As the SIMA-hPOMC1-26-Gluc cell line can easily measure the action of BoNT and α-LTX, it might be a useful tool to analyze the interaction of both toxins in their regulation of neurotransmitter release.

3.2. Suitability of the Assay for Compounds Inhibiting Neurotransmitter Release The last aim of the study was to analyze if the GLuc release from SIMA-hPOMC1-26- GLuc cells by high K+-depolarization can be inhibited by VGCC inhibitors rather than by BoNTs, which has previously been demonstrated [11]. Voltage dependent Ca2+-channels are a group of voltage-gated ion channels with a permeability for Ca2+-ions. They are formed as a complex of different subunits: α1, α2δ, β1-4, and γ, where the α1subunit forms the ion conducting pore. According to their calcium pore forming α1-subunit (CaV) VGCCs can be classified in several types, the L-type (CaV1.1–CaV1.4), P/Q-type (CaV2.1), N-type (CaV2.2), R-type (CaV2.3) and T-type (CaV3.1–CaV3.3). They can be discriminated by their inhibition by different neurotoxins and neuro-pharmaceutical inhibitors. N-type VGCC are interesting therapeutic targets for the treatment of nociceptive pain whereas T-type VGCCs channel blockers are used as antiepileptic drugs [16,32]. L-Type VGCC inhibitors such as dihydropyridines have been used as antihypertensive agents for a long time, but block both CaV1.2 and CaV1.3 VGCCs [33]. The newer Cav1.3 VGCCs specific dihydropyridine derivate isradipine was shown to be neuroprotective in a mouse model Toxins 2021, 13, 247 10 of 13

of Parkinson disease [34] and was discussed as a potential strategy for the treatment of Alzheimer disease [35]. As VGCCs are interesting targets for the treatment of neuronal diseases, SIMA- hPOMC1-26-Gluc cells were first analyzed for VGCC expression. The highest expression on the mRNA and protein level was measured for L-type CaV1.3 and N-type CaV2.2, whereas T-type CaV3.1 and L-type CaV1.1 were detected only on the mRNA level. The CaV expression profile was nearly identical to the profile in the neuroblastoma cell line SH- SY5Y [36]. In line with the expression profile of L-Type inhibitors verapamil and nifedipine, N-type inhibitors ω-conotoxins GVIA and MVIIA, and T-type inhibitors zonisamide and trimethadione inhibited GLuc release induced by high-K+-depolarization. In contrast to the high expression of N-type CaV2.2, inhibitors of L-type CaV showed the strongest inhibition of GLuc release, whereas ω-conotoxins, as well as zonisamide and trimethadione, were less active. Since SIMA-hPOMC1-26-GLuc cells do not express L-type CaV1.1, CaV1.2 and CaV1.4, the cell-based assay may be suitable for functional screening for CaV1.3 inhibitors.

3.3. Cytotoxicity of Compounds Inhibiting Neurotransmitter Release Cytotoxicity could affect the assay in two ways: Cell lysis could increase the non- specific release. This would yield false positive results in assays, which test, for example, calcium channel activators. On the other hand, cytotoxicity could non-specifically interfere with the fusion of neuro-secretory vesicles. This would yield false positive results, for example, for calcium channel blockers. However, at the maximal concentration used in the release assays, none of the VGCC inhibitors tested showed cytotoxic side effects.

4. Conclusions In conclusion, the newly established cell-based assay may represent a versatile tool for the analysis of neurotoxins and neuroactive pharmaceuticals which act by the modulation of intracellular calcium-concentration. The applications range from compounds which stimulate neurotransmitter release to inhibiting compounds.

5. Materials and Methods 5.1. Materials All chemicals were purchased from commercial sources indicated throughout the text. Oligonucleotides were custom-synthesized by Eurofins Operon (Ebersberg, Germany) or Biolegio (Nijmegen, The Netherlands). Neurotoxins and neuroactive pharmaceuticals used: carbachol, atropine, verapamil, nifedipine, thrimethadione and zonisamide were from Sigma-Aldrich (Taufkirchen, Germany); α-Latrotoxin (ALX-630-027) was from Enzo Life Science (Lörrach, Germany); ω-conotoxins GVIA and MVIIA were from Alamone labs (Jerusalem, Israel); agatoxin and SNX-482 were from tebu-bio (Offenbach, Germany). Antibodies used were: CaV1.1 (sc-514685), CaV1.2 (sc-398433), CaV1.3 (sc-515679), CaV1.4 (sc-517005) and CaV2.2 (sc-271010) were from SantaCruz Biotechnology (Heidelberg, Ger- many) and CaV3.1 (Acc-021) was from Alamone Labs.

5.2. Cell Culture Generation of the stably transfected neuroblastoma cell line SIMA hPOMC1-26 GLuc has been described previously [10]. Non-transfected SIMA cells were originally from DSMZ, (Braunschweig, Germany). Cells were cultured in RPMI 16040 medium supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), 2 mM stable L-alanyl-L-glutamine and penicillin (100 U/mL)/streptomycine (100 µg/mL) as antibiotics.

5.3. Luciferase Release from Cells Treated with Neurotoxins or NeuroActive Pharmaceuticals For release experiments SIMA-hPOMC1-26-GLuc cells were differentiated in poly l-lysine coated 96-well plates (5 × 103–5 × 104 cells/well) with differentiation medium (RPMI 1640 supplemented with 1 x B27 supplement, 1 x N2 supplement, 2 mM L-alanyl-L-glutamine, 1 mM non-essential amino-acids, 10 mM 4-(2-hydroxyethyl)-1- Toxins 2021, 13, 247 11 of 13

piperazineethanesulfonic acid (HEPES) and penicillin (100 U/mL)/streptomycin (100 µg/mL)) for 96 h with a medium change after 48 h. Subsequently, cells were preincu- bated with 100 µL fresh medium in the absence or presence of VGCC inhibitors for 10 min at 37 ◦C. The medium was aspirated and GLuc release was stimulated with 100 µL/well con- trol (20 mM Hepes pH 7.4, 136 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl2 and 1.25 mM MgSO4) or depolarization buffer (20 mM Hepes pH 7.4, 40.7 mM NaCl, 100 mM KCl, 1.25 mM CaCl2 and 1.25 mM MgSO4) in the absence or presence of carbachol or α-latrotoxin for 3 min or 5 min (α-latrotoxin) at 37 ◦C. The supernatant was transferred into reaction vials and centrifuged at 100× g for 3 min to remove detached cells. To determine GLuc activity 20 µL of the supernatant was mixed with 100 µL luciferase substrate solution and the luminescence was measured using Fluostar Optima. GLuc release was normalized to GLuc activity in remaining lysed cells and the mean of GLuc activity in untreated control and stimulated cells was set to 100% (AU).

5.4. Real-Time RT-PCR Total RNA from differentiated SIMA hPOMC1-26-GLuc cells was isolated using peqGold Total RNA Kit (Peqlab, Germany). 1–2 µg total RNA was reverse transcribed into cDNA using an oligo dT as a primer and an M-MuLV Reverse Transcriptase (Thermo Scientific, Darmstadt, Germany). Hot start real-time PCR for the quantification of each transcript was carried using 2 x Maxima SybrGreen qPCR mix (Thermo Scientific), 0.25 µM of each primer and 2.5–5 µL of cDNA, which was diluted 1:10. PCR was performed with an initial enzyme activation step at 95 ◦C for 10 min, followed by 42 cycles of denaturation at 95 ◦C for 30 sec, annealing at 57 ◦C for 30 sec and extension at 72 ◦C for 1 min in a real-time DNA thermal cycler (CFX96™, 10 µL reaction volume, BIO-RAD; Munich, Germany). The oligonucleotides used are listed in Table1. The expression levels of VGCC were calculated relative to GAPDH as a reference gene.

Table 1. Oligonucleotide primers used for realtime qPCR.

Gene Forward Reverse GAPDH 50-TGATGACATCAAGAAGGTGG 50-TTACTCCTTGGAGGCCATGT CaV 1.1 50-ACCATTGAGGAAGAGGCAGC 50-CATAGGCGACATTGGCGTTG CaV 1.2 50-TGCCCTTGCATCTGGTTCAT 50-ATCAAGACCGCTTCCACCAG CaV 1.3 50-CCCAGGCAGAAACATCGACT 50-CTGCCATGATCTGTTGCTGC CaV 1.4 50-CTTGGTGGAGGCTGTGCTTA 50-TATTGAGCAGTTGGGGAGGG CaV 2.1 50-CCTGAGCATGACCACCCAAT 50-CATGTGCTCTCGGCCCTC CaV 2.2 50-TACAAGACGGCCAACTCCTC 50-TCAGGGAGGACACGTAGGAA CaV 2.3 50-AGACGCTCACTTTCGAAGCA 50-TTGTTGACAGCCCCACACAT CaV 3.1 50-GCTGGATGAGCAGAGGAGAC 50-ATCTTTCTTTGGGGAGGGCG CaV 3.2 50-CTCAGGGCTTCCTGGACAAG 50-CCGTCCAAGAAAGGGTCTCC CaV 3.3 50-GAAGAGATGAGGGTCGCAGG 50-GCCAGAATCCCAGAGCATCA Accession numbers for the genes were: GAPDH (AB062273), CaV 1.1 (NM_000069.2), CaV 1.2 (NM_199460.3), CaV 1.3 (NM_000720.3), CaV 1.4 (NM_005183.3), CaV 2.1 (NM_000068.3), CaV 2.2 (NM_000718.3), CaV 2.3 (NM_001205293.1), CaV 3.1 (BC110995.1), CaV 3.2 (NM_021098.2), CaV 3.3 (NM_021096.3).

5.5. Western Blot Analysis SIMA hPOMC1-26-GLuc cells were lysed in Lämmli sample buffer (80 mM Tris/HCl pH 6.8, 2% (w/v) SDS, 5% (w/v) glycerol, 0.025% w/v bromophenol blue and 5% (v/v 2-mercatoethanol) homogenized by sonication. Insoluble material was removed by cen- trifugation (10,000× g, 15 min, 4 ◦C). Proteins were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked in 5% non-fat dry milk in 20 mM Tris, 136 mM NaCl and 0.1% (v/v) TWEEN 20 (Polyoxyethylenesorbi- tan monolaurate, TBS/Tween) for 1 h at room temperature and incubated with anti CaV antibodies in TBS/Tween containing 5% bovine serum albumin overnight at 4 ◦C and a horseradish-peroxidase-conjugated anti-rabbit or anti-mouse IgG for 2 h at room tem- Toxins 2021, 13, 247 12 of 13

perature. Visualization of immune complexes was performed using chemoluminescence reagent Clarity Western ECL (BIO-RAD, Feldkirchen, Germany).

5.6. Cytotoxicity Assay For the determination of overall cytotoxicity differentiated SIMA-hPOMC1-26-GLuc cells were preincubated with 100 µL fresh medium in the absence or presence of VGCC inhibitors for 10 min at 37 ◦C. The medium was aspirated and 100 µL/well resazurin medium (90% (v/v) differentiation medium + 10% (v/v) 1 mg/mL resazurin in PBS) was added. Fluorescence of resorufin, liberated from the chromogen by vital cells only was determined in the Fluostar Optima microreader (BMG-Labtech, Ortenberg, Germany) with 530 nm excitation and 590 nm emission wavelength filters. The increase of fluorescence was monitored every 30 min for 2 h, the slope of the fluorescence increase was determined in the linear part.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/toxins13040247/s1, Figure S1 Cytotoxicity assay with SIMA-hPOMC1-26-GLuc cells and VGCC inhibitors. Differentiated SIMA-hPOMC1-26-GLuc cells were preincubated with 100 µL fresh medium in the absence or presence of VGCC inhibitors or 0.1% (v/v) Triton X-100 for 10 min at 37 ◦C. The medium was aspirated and 100 µL/well resazurin containing medium was added. Fluorescence of resorufin, generated by resazurin reduction by vital cells only was determined in the Fluostar Optima microreader with 530 nm excitation and 590 nm emission wavelength filters. The increase of fluorescence was monitored every 30 min for 2 h, the slope of the fluorescence increase was determined in the linear part. Data are means ± SEM of 3-8 independent determinations performed in triplicate. Statistics: Student’s t-test for unpaired samples. a: < naive, p < 0.05. Author Contributions: A.P.-N.-R. and F.N.-R. performed the experiments, analyzed data and con- tributed to writing the manuscript. G.P.P. planned the study and wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This project was partially funded by EFRE-Staf Grant 85000915 and the BMBF grant 013L0132A. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Original data and Excel files are available on request. Acknowledgments: The technical assistance of Ines Kahnt is gratefully acknowledged. Conflicts of Interest: The authors declare no conflict of interest.

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