Mechanosensitive Ca2 +-permeable channels in human leukemic cells: Pharmacological and molecular evidence for TRPV2

Abstract

Mechanosensitive channels are present in almost every living cell, yet the evidence for their functional presence in T lymphocytes is absent. In this study, by means of the patch-clamp technique in attached and inside-out modes, we have characterized cationic channels, rapidly activated by membrane stretch in Jurkat T lymphoblasts. The half-activation was achieved at a negative pressure of ~50mm Hg. In attached mode, single channel currents displayed an inward rectification and the unitary conductance of ~40 pS at zero command voltage. In excised inside-out patches the rectification was transformed to an outward one. Mechanosensitive channels weakly discriminated between mono- and divalent cations (PCa/PNa~1) and were equally permeable for Ca2+ and Mg2+. Pharmacological analysis showed that the mechanosensitive channels were potently blocked by amiloride (1mM) and Gd3+ (10μM) in a voltage-dependent manner. They were also almost completely blocked by ruthenium red (1μM) and SKF 96365 (250μM), inhibitors of transient receptor potential vanilloid 2 (TRPV2) channels. At the same time, the channels were insensitive to 2-aminoethoxydiphenyl borate (2-APB, 100μM) or N-(p-amylcinnamoyl)anthranilic acid (ACA, 50μM), antagonists of transient receptor potential canonical (TRPC) or transient receptor potential melastatin (TRPM) channels, respectively. Human TRPV2 siRNA virtually abolished the stretch-activated current. TRPV2 are channels with multifaceted functions and regulatory mechanisms, with potentially important roles in the lymphocyte Ca2+ signaling. Implications of their regulation by mechanical stress are discussed in the context of lymphoid cells functions.