TRPM2 Oxidation Activates Two Distinct Potassium Channels in Melanoma Cells through Intracellular Calcium Increase.

Loretta Ferrera,Sara Bertelli,Caterina A M La Porta,Maria Rita Fumagalli,Giovanni Zifarelli,Alessia Remigante,Cristiana Picco,Michael Pusch,Raffaella Barbieri,Paola Gavazzo,Paolo Zuccolini

doi:10.3390/ijms22168359

Abstract

Tumor microenvironments are often characterized by an increase in oxidative stress levels. We studied the response to oxidative stimulation in human primary (IGR39) or metastatic (IGR37) cell lines obtained from the same patient, performing patch-clamp recordings, intracellular calcium ([Ca2+]i) imaging, and RT-qPCR gene expression analysis. In IGR39 cells, chloramine-T (Chl-T) activated large K+ currents (KROS) that were partially sensitive to tetraethylammonium (TEA). A large fraction of KROS was inhibited by paxilline—a specific inhibitor of large-conductance Ca2+-activated BK channels. The TEA-insensitive component was inhibited by senicapoc—a specific inhibitor of the Ca2+-activated KCa3.1 channel. Both BK and KCa3.1 activation were mediated by an increase in [Ca2+]i induced by Chl-T. Both KROS and [Ca2+]i increase were inhibited by ACA and clotrimazole—two different inhibitors of the calcium-permeable TRPM2 channel. Surprisingly, IGR37 cells did not exhibit current increase upon the application of Chl-T. Expression analysis confirmed that the genes encoding BK, KCa3.1, and TRPM2 are much more expressed in IGR39 than in IGR37. The potassium currents and [Ca2+]i increase observed in response to the oxidizing agent strongly suggest that these three molecular entities play a major role in the progression of melanoma. Pharmacological targeting of either of these ion channels could be a new strategy to reduce the metastatic potential of melanoma cells, and could complement classical radio- or chemotherapeutic treatments.

Highlights

Ion channels are specialized membrane proteins involved in several physiological functions, such as electrical signaling, signal transduction, and transport of ions small molecules across biological membranes in response to specific stimuli [1,2]
In order to investigate the electrophysiological responses of primary melanoma IGR39 cells to oxidative reagents in the whole-cell patch-clamp configuration, we applied the mild oxidant Chl-T
We focused on TRPM2 because it is known to be activated by oxidation [65]

Summary

Introduction

Ion channels are specialized membrane proteins involved in several physiological functions, such as electrical signaling, signal transduction, and transport of ions small molecules across biological membranes in response to specific stimuli [1,2]. The chemical and physical changes caused by methionine oxidation drastically alter the functions of ion channels, and the effect of oxidative stress on ion channels could become an essential part of a potential pathogenic mechanism in cancer cells [16,18] Both the abnormal production of ROS and alterations in ion channel activity can promote many processes causing development and progression in a tumor cell, such as cellular proliferation, evasion of apoptosis or anoikis, metastasis, and angiogenesis [19,20]

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Results

Conclusion