Screening Of Peoptides Activating TRPV1 Channel And The Study Of Their Structure And Functions

HN8623, HN6868 and HN3831 were new novel peptide neurotoxins isolated from the venom of the spider Haplopema hainanum. Their monoisotopic molecular mass were 8623.732Da,6869.095Da,3830.973Da respectively determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Preliminary whole-cell patch clamp recording showed that they could activate the TRPV1 channels expressed in the human embryonic kidney cells and elicited ruthenium red blockable calcium currents in a time and concentration-dependent manners. Further experiments confirmed that only 1μmol/L HN8623 could significantly activate the TRPV1 channels with the highest activity of the three molecules, but HN6868 with high concentration could persistently activate the TRPV1 channels. Partial amino acid sequence of HN8623 were determined by Edman degradation sequencing, the toxin and DKTX, a novel TRPV1 stimulant with two ICK motifs, shared 76 percent amino acid sequence homology. So HN8623 may be also a two-ICK motifs molecule with good research value. The primary structrue of HN6868 was:ACSKQIGDKCKRNCECCGKTVVCGTIYVGGKEVNQCMDKTSD NAILNGLGKGMNFIENTFSFCV, Which was also determined by Edman degradation. It was composed of 64 residues with four disulfide bonds. The most significant feature of the molecule was that it could persistently activate the TRPV1 channels and elicit large outward rectifying, ruthenium red blockable calcium currents in the human embryonic kidney cells. For this, the cultured cells were insensitive to extracellulary stimulus because of long-time excitation, so HN6868 may be a promising molecule for relieving pain drug development. The primary structrue of HN3831 was: ECRYWLGTCSKTGDCCSHLSCSPKHGWCVWDWT. It was composed of 33 residues with three disulfide bonds, so we speculated the toxin may be a typical ICK-motif molecule.Electrophysiological techniques were used to evaluate the inhibitory effect of the three molecules on all subtypes of sodium and potassium channels expressed in HEK293 cells. As a result, HN8623 and HN6867 could only activate the TRPV1 channels, but had no effect on another sodium and potassium channels. High concentration HN3831 could faintly activate the TRPV1 channels and HN3831 could also significantly inhibit Kv4.2 and Kv4.3 channels in a time and concentration-dependent manners, with IC50 values 299.6nmol/L and 114.5nmol/L respectively. Furthermore, the application of HN3831 could also change the kinetics of Kv4.2 and Kv4.3 channels through affecting the current-voltage relationship curve, the steady-state inactivation curve and recovery curve of inactivation of KV4.2 and Kv4.3 channels. Further whole-cell patch clamp experiments confirmed that HN3831 could faintly inhibit TTX-S currents with an IC50 value of 1.854μmol/L, but had no effect on TTX-R currents in adult rat dorsal root ganglion neurons.In order to further characterize the molecular mechanism of interaction between HN3831 and Kv4.3 potassium channel, alanine-scaning mutagenesis in S1-S2 and S3b-S4 regions of Kv4.3 potassium channel were performed and 19 mutants had been successfully constructed. Then patch clamp experiments were used to evaluate the inhibitory effect of HN3831 on these mutants, it was found that substitution of V282 of S3-S4 extracellular loop with Ala had led to large loss of inhibitory activity, which increased the IC50 by 5.37-fold. So it was inferred that V282 was a key amino acid residue relative to the interaction between HN3831 and Kv4.3 potassium channel.