A family of orthologous proteins from centipede venoms inhibit the hKir6.2 channel

Yajamana Ramu,Zhe Lu

doi:10.1038/s41598-019-50688-x

Abstract

Inhibitors targeting ion channels are useful tools for studying their functions. Given the selectivity of any inhibitor for a channel is relative, more than one inhibitor of different affinities may be used to help identify the channel in a biological preparation. Here, we describe a family of small proteins in centipede venoms that inhibit the pore (hKir6.2) of a human ATP-sensitive K+ channel (hKATP). While the traditional peptide-sequencing service gradually vanishes from academic institutions, we tried to identify the sequences of inhibitory proteins purified from venoms by searching the sequences of the corresponding transcriptomes, a search guided by the key features of a known hKir6.2 inhibitor (SpTx1). The candidate sequences were cross-checked against the masses of purified proteins, and validated by testing the activity of recombinant proteins against hKir6.2. The four identified proteins (SsdTx1-3 and SsTx) inhibit hKATP channels with a Kd of <300 nM, compared to 15 nM for SpTx1. SsTx has previously been discovered to block human voltage-gated KCNQ K+ channels with a 2.5 μM Kd. Given that SsTx inhibits hKir6.2 with >10-fold lower Kd than it inhibits hKCNQ, SsTx may not be suitable for probing KCNQ channels in a biological preparation that also contains more-SsTx-sensitive KATP channels.

Highlights

ATP-sensitive K+ (KATP) channels were discovered more than three decades ago in cardiac myocyte[1], which were subsequently found in pancreatic β cells that secret insulin[2,3,4,5]
SUR1 and Kir6.2 is normally necessary for Kir6.2 to reach the cell membrane[21]
We have shown that the venoms of multiple centipede species contain one or more proteins that target hKATP channel by blocking the hKir6.2 pore

Summary

Introduction

ATP-sensitive K+ (KATP) channels were discovered more than three decades ago in cardiac myocyte[1], which were subsequently found in pancreatic β cells that secret insulin[2,3,4,5]. We previously discovered a 54-residue venom protein, dubbed SpTx1, in the venom of Scolopendra polymorpha for its inhibitory activity against hKir6.220, a centipede species that is found to inhabit the Southwestern part of the United States. For the reason to be discussed, we tested here the approach of using the information regarding a short, functionally important region of SpTx1 to guide our identification of the sequences of additional inhibitors, which are present in the venoms of other centipede species whose transcriptome sequences are available. We verified the identified sequences by mass spectroscopy and examination of the inhibitory activities of the corresponding recombinant proteins

Methods

Results

Conclusion