| Neuronal nicotinic acetylcholine receptors (NNRs) form a heterogeneous family of ion channels that are differently expressed in many regions of the central nervous system (CNS) and peripheral nervous system. These different receptor subtypes, which have characteristic electrophysiological, pharmacological and biophysical properties, have a pentameric structure consisting of the homomeric or heteromeric combination of 12 different subunits (α2-α10,β2-β4). Over recent years, these molecules have attracted the attention of academic and industrial scientists because of their relevance to understanding brain functions and their possible involvement in various pathologies. They also have been regarded as important therapeutic targets for many cholinergic disorders. Thus, further research into the electrophysiological properties of different subtypes of NNRs would benefit not only the understanding of the changes of cholinergic signals in different physiological or pathological conditions, but also the discovery of drugs that targeting various cholinergic disorders.Benthiactzine (benzilate thio S-2-diethylamino ethyl ester hydrochloride), which has both antimuscarinic and antinicotinic effects, was our newly developed organophosphate (OP) poisoning antidote. Previously, we have shown that: (1) under conditions of OP poisoning, desensitized NNRs were not in a nonfunctional state as traditionally thought, but could still mediate OP toxicity by facilitating mAChRs functions, which suggests the significance of benthiactzine's antinicotinic effects in theory; (2) benthiactzine was much weaker than atropine in binding to mAChRs expressed in 14 different tissues or to five different mAChR subtypes in a specific [3H]quinuclidinyl benzilate-binding assay, while afforded better protection than atropine against the most lethal ChEI, VX or sarin, in a mouse model , suggesting the significance of benthiactzine's antinicotinic effects in practice. Thus, the characterization of pharmacological properties of benthiactzine's antagonism against NNRs would be of great significance not only for revealing the molecular mechanisms of its antidotal effect, but also for guiding future antidote discovery. This study may also provide new intervention strategy for other cholinergic disorders. For above purposes, in the present study, patch-clamp whole-cell recording technique was used to identify and characterize benthiactzine's antinicotinic effect on three typical NNRs subtypes:α4β2,α4β4, andα7, which are expressed heterogenously in SH-EP1 cells. Theα4β2 andα7 are the predominant subtypes in the CNS, and theα4β4 is also distributed in many important parts of the brain. In this study, the electrophysiological properties of different functional states of three different NNRs subtypes was characterized, based on which the pharmacological properties of benthiactzine's antagonism against three different NNRs subtypes was evaluated and through which site benthiactzine interact with the three NNRs subtypes was concluded. At last, we tried to explore a new way to evaluate the effects of cholinergic agents by investigate their effects on the different functional states of NNRs.Main findings from this study are as follows:Part I Electrophysiological Study of Different Functional States ofα4β2,α4β4, andα7 NNRs which Are Expressed Heterogenously in SH-EP1 CellsBy investigating the electrophysiological properties of different functional states ofα4β2,α4β4, andα7-NNRs, this part of study established the electrophysiological technique station for following drug evaluation.1. Identification of the SH-EP1 Cells Expressingα4β2,α4β4, andα7-NNRsThe SH-EP1 cells cultured in this study were morphologically similar as previously reported by others and expressed expected NNRs subunits.α4β2,α4β4 andα7-NNRs inward currents could be induced by rapid application of agonists and showed their unique electrophysiological properties, suggesting the expression of functional NNRs by SH-EP1 cells.2. Electrophysiological properties of the activation ofα4β2,α4β4, andα7- NNRsThe open states of neuronalα4β2,α4β4, andα7-NNRs and their transitions have distinct characteristics.α7-NNRs was activated the fastest and also decayed the fastest;α4β4-NNRs was activated the slowest and almost didn't decay;α4β2-NNRs was activated faster thanα4β4-NNRs and slower thanα7-NNRs, and decayed in two phases.The peak of the currents mediated by this three types of NNRs are all dose-dependent. EC50 for them are:α4β2: nicotine 3.1±0.7μmol/L;α4β4: nicotine 1.1±0.6μmol/L;α7: choline 814. 6±209.0μmol/L.The inward currents of all this three types of receptors are voltage-dependent and have inward rectification property. Their inward rectification order:α7 >α4β2 >α4β4. 3. Electrophysiological properties of the desensitization ofα4β2,α4β4, andα7- NNRsInward currents were elicited by rapid application of agonist in all the three types of SH-EP1 cells.α7-NNRs desensitized the fastest and deepest in the three whileα4β4-NNRs the slowest and lightest during prolonged stimulation.α4β2-NNRs desensitized the fastest in the three during repetitive stimulation whileα4β4 andα7-NNRs didn't have obvious progressing desensitization.4. Electrophysiological properties of the reactivation ofα4β2-NNRsWe discovered thatα4β2-NNRs can be reactivated after the removal of high concentration of agonist. When the membrane potential was clamped at–60 mV, an inward current was elicited by rapid application of 1 mmol/L nicotine in SH-EP1 cells. This current reached peak rapidly and then desensitized fast when the application of agonist was prolonged. However, after the removal of nicotine, an additional inward current reoccurred. This phenomenon didn't occur when the concentration of nicotine was lower than 100μmol/L.Part II The Pharmacological Properties of Benthiactzine's Antagonism againstα4β2,α4β4, andα7 Subtype of NNRs which Are Expressed Heterogenously in SH-EP1 CellsBased on the electrophysiological technique established in the first part of study, the feature of benthiactzine's effect on different NNRs subtypes and the molecular mechanisms underlying benthiactzine's effect were investigated in the part of study. To identify benthiactzine's action site on different types of NNRs, the investigation of its effect on desensitization, voltage and use-dependent were included in this part of study.1. Benthiactzine Blocks NNRs-Mediated Whole-Cell CurrentsNNRs-mediated whole-cell current responses were elicited by the EC50 doses of nicotine (3μmol/L forα4β2 and 1μmol/L forα4β4) or choline (10 mmol/L forα7, choline concentration used here is 10 times higher than EC50 to elicit large enough currents to facilitate analysis and reduce error), and currents were analyzed to fit for decay time constant (tau;τ), peak current (Ip), and steady-state current (Is).Relative to the control whole-cell current responses elicited during the challenge exposure to nicotine alone, nicotine-induced inward currents mediated viaα4β2 orα4β4-NNRs in the same SH-EP1 cell were reduced if assessed during coapplication with benthiactzine. The effects of benthiactzine applied at different concentrations during 4-s nicotine exposure showed concentration dependence of the functional block. The concentration–response profiles for inhibition by benthiactzine of peak whole-cell responses onα4β2 andα4β4-NNRs gave IC50 values of 4.9 and 7.5μmol/L, respectively. In contrast, the choline-induced inward currents mediated viaα7 nAChRs in the same SH-EP1 cell were reduced much less if assessed during coapplication with benthiactzine; the IC50 value was 92.2μmol/L. These data indicate that benthiactzine is a more potent antagonist toα4β2 andα4β4 NNRs than toα7 NNRs. We also found that benthiactzine deepened the degree of decay (Is/Ip; the smaller the value, the deeper the decay degree) ofα4β2,α4β4, orα7-NNRs currents; the half-maximal effective concentration (EC50) values were 0.8, 0.5, and 18.7μmol/L, respectively. Benthiactzine also accelerated the decay course (tau; the bigger the value, the longer the course) of these three NNRs; the EC50 values were 1.1, 0.4, and 1009.7μmol/L, respectively.2.Benthiactzine Blocks NNRs-Mediated Currents in a Time- and Concentration- Dependent MannerThe blockade of benthiactzine on NNRs was reversible. Three minutes after washout of 100μmol/L of benthiactzine, the peak currents ofα4β2,α4β4, andα7 NNRs recovered to 89.6%±6.2%, 80.4%±8.3%, and 86.3%±12.0%, respectively. Ten minutes after washout of 100μmol/L of benthiactzine, the peak currents ofα4β4 andα7 NNRs had recovered almost completely, whereas theα4β2 NNRs-mediated current recovered to only 89.2%±3.5%.3. Voltage Dependence of Benthiactzine Block of NNRs FunctionThe voltage dependence of the blocking effect of benthiactzine onα4β2,α4β4, andα7 NNRs was explored at different holding potentials (from–120 to 0 mV) in 30-mV steps. Exposure to benthiactzine (100μmol/L) reduced the peak amplitude of currents mediated viaα4β2 orα4β4 NNRs, and the magnitude of this reduction was more pronounced at hyperpolarized potentials. Benthiactzine blocked theα4β2 andα4β4 currents by 94%±2% and 93%±2% at–120 mV, and by 78%±4% and 61%±5% at 0 mV, respectively. In contrast to the voltage dependence of inhibition observed forα4β2 andα4β4 NNRs, inhibition ofα7 NNRs by benthiactzine exhibited much weaker voltage dependence across the range of potentials tested (30%±5% at–120 mV and 33%±4% at 0 mV).4. Use Dependence of Benthiactzine Block of NNRs FunctionWe also investigated whether benthiactzine produces a use-dependent blockade of these three nAChR subtypes. Under conditions where the whole-cell current responses of SH-EP1 NNRs cells to repetitive applications of nicotine for 4 s at 3-min intervals showed no significant response rundown (data not shown), four repetitive applications of nicotine (3μmol/L forα4β2 and 1μmol/L forα4β4) in the continuous presence of 100μmol/L benthiactzine caused a gradual reduction in the nicotinic responses. However, 9 min of benthiactzine pretreatment without repeated application of nicotine, caused less inhibition of the nicotine-induced response. That is, under conditions where the initial peak current response to nicotine in the presence of coapplied benthiactzine was reduced relative to the response to nicotine alone by the same amount (9.3%±2.4 % or 10.7%±1.9 % forα4β2; 15.0%±3.0 % or 14.0%±2.0 % forα4β4; P > 0.05; n≥4), the response to nicotine challenge after 9 min of 100μmol/L benthiactzine exposure during repeated nicotine challenges was only 2.1%±0.3 % (α4β2) or 2.5%±0.3 % (α4β4) of the control value. This compares with 3.8 %±0.4 % (α4β2) or 6.0%±0.5 % (α4β4) of the control value for the response to nicotine challenge after 9 min of benthiactzine exposure in the absence of intervening nicotine challenges (P < 0.05 forα4β2 and P < 0.01 forα4β4; n≥4). These results indicate that benthiactzine-mediated block ofα4β2 andα4β4 NNRs function is use dependent, while benthiactzine-mediated blockade ofα7 NNRs function is not use dependent.PartⅢThe Pharmacological Properties of Benthiactzine's Antagonism against Different Functional States ofα4β2-NNRsIn the second part of study, by following the widely accepted experimental procedure, we investigated benthiactzine's antagonism properties on three typical NNRs subtypes. In this part of study, we tried to explore a new way to evaluate the effects of cholinergic agents by investigate their effects on the different functional states of NNRs.1. Benthiactzine Blocks the Transformation ofα4β2-NNRs from Closed State to Activated StateThe whole cell current traces for responses to 3μmol/L nicotine alone reached a peak rapidly, indicating a fast kinetics of transformation from closed state to activated state, whereas in the presence of 100μmol/L benthiactzine the peak was significantly cut down, indicating that much fewer receptors opened. The number of activated receptors (represented by the peak values of elicited currents) in the presence of 100μmol/L benthiactzine was only 8.2±3.4 % (n = 5) of the activatable receptors. The activatable receptors was blocked by benthiactzine with an IC50 value of 4.9μmol/L.2.Benthiactzine Blocks the Transformation ofα4β2-NNRs from Activated State to Desensitized StateThe application of nicotine elicited an inward current viaα4β2-NNRs, this current reached a peak rapidly. After that, the current decayed because the prolonged nicotine binding induced these receptors to transform from activated state to desensitized refractory state. But when 0.1μmol/L benthiactzine was coapplied, the extent of decay was profounder. Our previous study showed that benthiactzine is an open channel blocker ofα4β2-NNRs (unpublished data). After the receptor opens, benthiactzine may act by a steric mechanism which means that the benthiactzine molecules physically block the ion channels ofα4β2-NNRs, and prevents receptors transforming into desensitized state. Thus the additional decayed currents reflect the undesensitized activated receptors. This part of receptors was calculated to be 9.7±4.1 % (n = 5) of the receptor number in the absence of benthiactzine. The kinetics of benthiactzine's block on desensitizedα4β2-NNRs can be described by the following function: Nopen-blocked / Nactivated = [(A1e-t/τ1 + A2e-t/τ2 + C1)-(A3e-t/τ3 + A4e-t/τ4 + C2)] / IP.3.Benthiactzine Blocks the Reactivatedα4β2-NNRsOur studies examined whole-cell current responses ofα4β2-NNRs to nicotine applied as 4-s pulses at 3-min intervals at concentrations between 100 nmol/L and 1 mmol/L. Inward hump currents evident during drug washout were observed for whole cell currents induced by 1 mmol/L nicotine. Coapplication of 100μmol/L benthiactzine with 1 mmol/L nicotine not only reduced the peak value of activated current but also diminished the reactivated hump current. In the presence of 100μmol/L benthiactzine, the nicotine elicited peak currents were reduced to 56±8 % (P < 0.05, n = 4) of that in the absence of benthiactzine.From above observations, it could be concluded:1. The open states ofα4β2,α4β4, andα7-NNRs had distinct characteristics and the inward currents of all these three types of receptors were dose and voltage-dependent and had inward rectification property. Their properties of desensitization during sustained or repeated stimulation of agonist were also different from each other.2. Benthiactzine inhibitedα4β2,α4β4, andα7-NNRs reversibly. The inhibition effect was more potent onα4β2 andα4β4 subtype than onα7 subtype. The inhibition effect onα4β2 andα4β4 subtype accelerated the desensitization progress of receptors and had voltage-dependent and use-dependent, suggesting benthiactzine interacting with the receptors in the central pore as an open-channel blocker. The inhibition effect onα7 subtype accelerated the desensitization progress of receptors, but had no voltage- dependent and use-dependent, suggesting benthiactzine interacting with the receptors in the allosteric site that outside and away from the cell membrane.3. Benthiactzine inhibited the activated, desensitized, and reactivated functional states ofα4β2-NNRs, the predominant subtype of NNRs in the brain.
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