Study On Regulatory Effect Of Sigma - 1 Receptor Agonist On Voltage - Gated Sodium Channel Na_v1.2

Nav1.2is a voltage-gated sodium channel that mainly expressed in central neuron, it contributes to the rising phase of the action potential in electrically excitable cells. It is also a target of local anesthetics, antiarrhythmics and anticonvulsants compounds that attenuating uncontrolled high frequency activity associated with disease states, al receptor is a two transmembrane protein located in plasma membrane and mainly expressed in central nervous and cardiovascular system. It mainly plays a modulatory role in the activity of some ion channels and in several neurotransmitter systems and its ligands were mainly used for antipsychotics, antidepressants, anticonvulsants and Parkinson’s disease or Alzheimer’s disease.(+)-SKF10047is one of the al receptor selective agonists and has been shown to modulate the Na+、K+and Ca2+channels via al receptor previously.In this study, we mainly tested the effects of (+)-SKF10047on rat brain Nav1.2by heterologous expression of the channel in HEK293T cells. Additionally, Nav1.4and Nav1.5was also tested. σ1receptor was endogenous expressed in HEK293T cells abundantly, but scarcely in COS-7cells. Western blot shows that the protein of al receptor was detected in HEK293, GC and cortical neurons, but not in COS-7. Nav1.2channel protein was also detected in GC and cortical neurons but not in HEK293T and COS-7cells.Selective al receptor agonists such as (+)-SKF10047, PRE-084, DM, and selective al receptor antagonists such as BD1047, NE-100were used to investigate the role of al receptor in Nav1.2modulation by these drugs. The channel currents in absence or presence of drugs were recorded by whole cell patch clamp recording to analysis the inhibitory effects of al receptor ligands.(+)-SKF10047in100μM reversibly inhibited Nav1.2in HEK293T cells by39.5±1.3%. In HEK293T cells,(+)-SKF10047significantly shifted the steady-state inactivation curve of INa by27.0±11.9mV towards the hyperpolarizing potential, but did not alert the steady-state activation curve.(+)-SKF10047produced a does-dependent and reversible inhibition manner in the range of0.1μM to300μM, and blocked Nav1.2channel in a use-dependent and frequency-dependent manner.More over, blocking the Gs protein by CTX (cholera toxin, Gs activator), PTX (pertussis toxin, Gi inhibitor), NF023(Gi inhibitor) and NF449(Gs inhibitor), or blocking the PKA and PKC signaling pathway by H-89, PKAi (PKA inhibitors), BIM and Go6976(PKC inhibitors), or GTPyS (G protein activator), all these treatments did not alert the effect of (+)-SKF10047on INa inhibition. Furthermore, al receptor knocked down by RNA interference or using the σ1receptor antagonists (BD1047, NE-100) or testing the INa inhibition in al receptor negative cells (COS-7) did not significantly affect the inhibitory effect of (+)-SKF10047on INa.Gene mutagenesis test shows that F1764A mutation of Nav1.2channel significantly declined the inhibitory effect of (+)-SKF10047on INa by40%approximately. The W1716C mutation did not affect the inhibitory effect of (+)-SKF10047on INa, but Y1771A mutation increased the inhibitory effect of (+)-SKF10047on INa significantly in HEK293T cells. The recovery from inactivation course of INa was delayed by presence of (+)-SKF10047, and the inhibitory effect of (+)-SKF10047on INa was shown in frequency dependent manner in the range of0.1HZ to2HZ. The5HZ trains pulse test shows that the (+)-SKF10047inhibited INa with use-dependence manner.Additionally, the INa inhibitory effect of the two other agonists of σ1receptor was also tested. The PRE-084inhibited INa by15.9±1.9%in HEK293T. The DM, a benzomorphan drug which in the same category with (+)-SKF10047, inhibited INa by65.8±2.0%in HEK293T and68.7±3.9%in COS-7. There was no significant difference between the data from two cell models. The inhibitory effects of DM on INa can not be attenuated by the al receptor antagonists (BD1047, NE-100) as well. The F1764A site mutation on Nav1.2channel significantly declined the inhibitory effect of of DM on INa from65.8±2.0%to58.6±1.9%. The activation and inactivation curve of the Nav1.2was not changed by DM. The inhibitory effect on Nav1.2of DM was consistent with the result reported by other pepole, though the INa inhibition was different which might be explained by the different experiment conditions.(+)-SKF10047was also tested on rat Nav1.4and Nav1.5channels in HEK293T and COS-7cells, and the total INa on GC and cortical neurons was also tested.INa inhibition on Nav1.4and Nav1.5channels on two cell models has no significant difference. Meanwhile, overall inward transient sodium current of rat GC and mouse cortical neuron was also inhibited by the (+)-SKF10047transiently in reversible manner.From these results, we first demonstrated that (+)-SKF10047transiently inhibits brain Nav1.2channel via σ1receptor, G-protein or PKA, PKC independent manner, but by binding to the residues of Nay1.2and blocking the channel in open or inactivated state. The DM in the same class of σ1receptor selective agonist might transiently inhibit INa with the same manner as (+)-SKF10047by binding and blocking the channel. The inhibitory effect on the current of rat Nay1.4and Nay1.5, or on the overall sodium current of neurons can be an additional evidence for that the (+)-SKF10047might transiently inhibit the VGSC generally. Other binding sites of the (+)-SKF10047on Nav1.2should be explored in further study.