Development and characterization of a monoclonal antibody blocking human TRPM4 channel

See Wee Low,Bo Chen,Bernd Nilius,Shunhui Wei,Ping Liao,Yahui Gao

doi:10.1038/s41598-021-89935-5

Abstract

TRPM4 is a calcium-activated non-selective monovalent cation channel implicated in diseases such as stroke. Lack of potent and selective inhibitors remains a major challenge for studying TRPM4. Using a polypeptide from rat TRPM4, we have generated a polyclonal antibody M4P which could alleviate reperfusion injury in a rat model of stroke. Here, we aim to develop a monoclonal antibody that could block human TRPM4 channel. Two mouse monoclonal antibodies M4M and M4M1 were developed to target an extracellular epitope of human TRPM4. Immunohistochemistry and western blot were used to characterize the binding of these antibodies to human TRPM4. Potency of inhibition was compared using electrophysiological methods. We further evaluated the therapeutic potential on a rat model of middle cerebral artery occlusion. Both M4M and M4M1 could bind to human TRPM4 channel on the surface of live cells. Prolonged incubation with TRPM4 blocking antibody internalized surface TRPM4. Comparing to M4M1, M4M is more effective in blocking human TRPM4 channel. In human brain microvascular endothelial cells, M4M successfully inhibited TRPM4 current and ameliorated hypoxia-induced cell swelling. Using wild type rats, neither antibody demonstrated therapeutic potential on stroke. Human TRPM4 channel can be blocked by a monoclonal antibody M4M targeting a key antigenic sequence. For future clinical translation, the antibody needs to be humanized and a transgenic animal carrying human TRPM4 sequence is required for in vivo characterizing its therapeutic potential.

Highlights

Transient receptor potential melastatin member 4 (TRPM4) is a calcium-activated non-selective monovalent cation channel implicated in diseases such as stroke
HEK 293 cells grown in 6-well petri dishes were transfected with 4 μg human or mouse TRPM4 plasmid using lipofectamine 2000 transfection reagent (Cat#11668019, Thermo Fisher Scientific, MA, USA). 24 h after transfection, 80 μg of total protein was resolved on 10% SDS-PAGE gels at 80 V, and electrophoretically transferred to PVDF membranes (1620177, Bio-Rad, CA, USA) at 100 V for 2 h at 4 °C
We selected an antigenic epitope which is between transmembrane segments 5 (T5) and 6 (T6) and close to the channel pore of human TRPM4 channel (Fig. 1a) for raising the blocking monoclonal antibody

Summary

Introduction

TRPM4 is a calcium-activated non-selective monovalent cation channel implicated in diseases such as stroke. We aim to develop a monoclonal antibody that could block human TRPM4 channel. We further evaluated the therapeutic potential on a rat model of middle cerebral artery occlusion Both M4M and M4M1 could bind to human TRPM4 channel on the surface of live cells. Human TRPM4 channel can be blocked by a monoclonal antibody M4M targeting a key antigenic sequence. 9-phenanthrol non-selectively inhibits transient outward, rapid delayed rectifier, and inward rectifier K + currents in h eart[18], raising concerns about its specificity to TRPM4. Another TRPM4 blocker glibenclamide is widely used to control blood glucose level in patients with diabetes mellitus type 2. More experiments are needed to evaluate its in vivo functions

Objectives

Methods

Results

Conclusion