Turnover of voltage-gated potassium channel Kv 1.3

Abstract

Voltage-gated K channels (Kv) is large family of channels that are expressed in both excitable and non-excitable cells. In excitable cells they contribute to the control of resting membrane potential and action potentials frequency and duration. In non-excitable tissues they are involved in many processes such as secretion to cell proliferation. Kv1.3 channel plays a key role in a wide range of physiological phenomenon. Regulation of this transmembrane protein is therefore essential for a correct function of the living cell. The balance between synthesis and degradation is highly important and must be tightly regulated. The present dissertation is focused in investigating endocytosis mechanisms of Kv1.3, as a process controlling number of the channel on the cell surface and the possible implication in cell destiny. We deciphered major endocytosis mechanisms triggered by EGF and Adenosine (ADO) in HeLa and HEK 293 heterologous cell systems as well as in native cell lines (macrophages, dendritic or neuronal precursor). These studies pointed out the impact of endocytosis in turnover and homeostasis of Kv1.3 and possible physiological relevance of these finding. Our experiments showed two different ways to control abundance of the Kv1.3 channel by EGF: via tyrosine phosphorylation and unconventional ERK1/2-dependent mechanisms. EGF triggered clathrin-dependent lysosomal degradation of Kv1.3. Moreover, this study show a high physiological relevance, pointing to EGF as a Kv1.3 inhibitor that might therefore reduce radiation-induced brain injury by targeting the key cells involved in the inflammatory process. As next, study was to investigating PMA-induced PKC-dependent endocytosis and ubiquitination. We revealed that PMA triggered PKC-dependent ubiquitin-mediated lysosomal degradation of Kv1.3. Next, we show that adenosine (ADO), which is a potent endogenous modulator, similar to PMA, stimulates PKC, thereby causing immunosuppression. PKC activation triggers down-regulation of Kv1.3 by inducing a clathrin-mediated endocytosis which targets the channel to lysosomal-degradative compartments. Therefore, the abundance of Kv1.3 at the cell surface decreases, which is clearly compatible with an effective anti-inflammatory response. This mechanism requires ubiquitination of Kv1.3, catalyzed by the E3 ubiquitin-ligase Nedd4-2. However, we discover that ADO activates both PKC and PKA signaling pathways. To further investigate the molecular mechanisms of the Kv1.3 internalization in response to ADO, we have examined the effects of PKA antagonists. Our results, for the first time, provided evidence on the effect of PKA activation on the Kv1.3 trafficking. Our findings indicated that PKA adenosine activation triggered Kv1.3 endocytosis redundantly to PKC. In addition, we put a hypothesis that PKA downregulated Kv1.3 in an ubiquitin-independent manner. In the last part of this dissertation we concentrated at molecular determinants involved in Kv1.3 ubiquitination. We found that complementary and redundant lysines participate in the ubiquitin-dependent PKC and PKA regulation of Kv1.3.