The modulation of TRPM7 currents by nafamostat mesilate depends directly upon extracellular concentrations of divalent cations

Xuanmao Chen,Zhi-Gang Xiong,Minghua Li,John F Macdonald,Michael F Jackson,Yasuo Mori,Beverley A Orser,Tomohiro Numata

doi:10.1186/1756-6606-3-38

Abstract

Concentrations of extracellular divalent cations (Ca2+ and Mg2+) fall substantially during intensive synaptic transmission as well as during some pathophysiological conditions such as epilepsy and brain ischemia. Here we report that a synthetic serine protease inhibitor, nafamostat mesylate (NM), and several of its analogues, block recombinant TRPM7 currents expressed in HEK293T cells in inverse relationship to the concentration of extracellular divalent cations. Lowering extracellular Ca2+ and Mg2+ also evokes a divalent-sensitive non-selective cation current that is mediated by TRPM7 expression in hippocampal neurons. In cultured hippocampal neurons, NM blocked these TRPM7-mediated currents with an apparent affinity of 27 μM, as well as the paradoxical Ca2+ influx associated with lowering extracellular Ca2+. Unexpectedly, pre-exposure to NM strongly potentiated TRPM7 currents. In the presence of physiological concentrations of extracellular divalent cations, NM activates TRPM7. The stimulating effects of NM on TRPM7 currents are also inversely related to extracellular Ca2+ and Mg2+. DAPI and HSB but not netropsin, blocked and stimulated TRPM7. In contrast, mono-cationic, the metabolites of NM, p-GBA and AN, as well as protease inhibitor leupeptin and gabexate failed to substantially modulate TRPM7. NM thus provides a molecular template for the design of putative modulators of TRPM7.

Highlights

The eight members of the transient receptor potential melastatin (TRPM) group represent a subclass of nonselective cation transient receptor potential (TRP) channels [1,2]
nafamostat mesylate (NM) is a blocker of recombinant TRPM7 currents expressed in HEK293T cells Voltage ramps were employed to evoke currents from over-expressed TRPM7 channels induced in HEK293T cells
When extracellular divalents were reduced to 0.1 mM, NM inhibited outward and inward TRPM7 currents with IC50 values of 617 μM at +100 mV and 514 μM at -100 mV (n = 11-14) (Figure 1A)

Summary

Introduction

The eight members of the transient receptor potential melastatin (TRPM) group represent a subclass of nonselective cation transient receptor potential (TRP) channels [1,2]. The function of TRPM7 channels is usually attributed to one of Mg2+ homeostasis [3,4] but they are a source of entry of Ca2+ and contribute to the death of hippocampal neurons following ischemia [5,6,7]. These channels are mechano-sensitive [8], and they contribute to mechanisms controlling vesicular release of acetylcholine from sympathetic neurons [9]. We were able to use this approach to show that reducing TRPM7 currents in CA1 neurons of the rat provides substantial protection against the loss of CA1 neurons in a model of global ischemia [5,7]

Methods

Results

Conclusion