Klonierung und funktionelle Charakterisierung des 'long transient receptor channel 2', LTRPC2, einem Mitglied der Familie kalziumpermeabler TRP-Kationenkanäle

Abstract

It has been proposed that transient receptor potential channels (TRP-channels) participate in ligand gated Ca^(2+) entry in non-excitable cells. To analyze the physiological relevance of TRP-channels human neutrophile granulocyte were chosen as a model for non-excitable cells. The activation of neutrophiles is accompanied by membrane depolarisation and by a rise of the [Ca^(2+)]i. Until now the identity of the proteins which initiate the activation of neutrophile granulocytes is not known. Among the TRP-channels, the LTRPC2 is predominantly expressed by neutrophile granulocytes. To investigate the relevance of LTRPC2 for the function of neutrophile granulocytes, LTRPC2 was cloned by the RT-PCR technique and was then heterologously transfected in CHO- and in HEK 293 cells for functional characterisation. Two splice variants of the LTRPC2 were identified in human neutrophile granulocytes. These splice forms were used to construct four alternative variants with deletions at the N-terminal and/or C-terminal domain (LTRPC2; LTRPC2-deltaC; LTRPC2-deltaN; LTRPC2-deltaN-deltaC) using an appropriate cloning strategy. Fluorometric measurments of the [Ca^(2+)]i and measurment of ionic currents of heterologously transfected cells with patch clamp revealed no activation of the LTRPC2 variants in dependence of an intracellular Ca^(2+) store depletion. This stands in contrast to the classical TRP-proteins. Since NAD^(+) and its metabolite ADP-ribose have recently been reported to activate LTRPC2, I analysed the response of LTRPC2 variants to NAD^(+) and ADP-ribose. Whereas no activation by NAD^(+) was observed in any LTRPC2 variant, ADP-ribose exerted a stimulating effect exclusively on the full-lenght LTRPC2. Since ADP-ribose can be formed from NAD^(+), and NAD^(+) is elevated during oxidative stress, it was tested wether the various LTRPC2 channel variants mediate cation influx in response to an oxidant. Therefore ion currents and the [Ca^(2+)]i were measured after stimulating transfected cells with H2O2 as a model of oxidative stress. A stimulation with H2O2 could be observed in cells transfected with full-length LTRPC2 or transfected with the variant LTRPC2-deltaC. Thus the activation of LTRPC2 by H2O2 is independent of ADP-ribose because the splice variant LTRPC2-deltaC was exclusively activated by H2O2, while full-length LTRPC2 could be activated by ADP-ribose as well. Since intracellular application of the antioxidant mannitol prevented a stimulation of LTRPC2 and LTRPC2-deltaC by H2O2, it can be concluded that H2O2 does not act directly on the channel but by intracellular oxidation reactions. It is possible that the activation of LTRPC2 plays a role for the stimulation of neutrophile granulocytes during inflammation, when neutrophiles become activated by H2O2