| Abstract
| - Capsaicin receptor channels (TRPV1) are nonselective cation channels that integrate multiplenoxious stimuli in sensory neurons. In an effort to identify new inhibitors of these channels we screeneda venom library for activity against TRPV1 channels and found robust inhibitory activity in venom fromAgelenopsis aperta, a north American funnel web spider. Fractionation of the venom using reversed-phase HPLC resulted in the purification of two acylpolyamine toxins, AG489 and AG505, which inhibitTRPV1 channels from the extracellular side of the membrane. The activity of AG489 was characterizedfurther, and the toxin was found to inhibit TRPV1 channels with a Ki of 0.3 μM at −40 mV. Inhibitionof TRPV1 channels by AG489 is strongly voltage-dependent, with relief of inhibition at positive voltages,consistent with the toxin inhibiting the channel through a pore-blocking mechanism. We used scanningmutagenesis throughout the TM5−TM6 linker, a region thought to form the outer pore of TRPV1 channels,to identify pore mutations that alter toxin affinity. Four mutants dramatically decrease toxin affinity andseveral mutants increase toxin affinity, consistent with the notion that the TM5−TM6 linker forms theouter vestibule of TRPV1 channels and that AG489 is a pore blocker.
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