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Project 5: Phosphorylation-dependent trafficking of ion channels during sustained catecholamine stress

PI Göttingen: B. Schwappach; PI London: M.J. Shattock; PhD student: J. Menzel -                                finished Dr. rer. nat.

Scientific background and preliminary results

Electrical excitability of cardiomyocytes is a prerequisite for the pump function of the heart. Coordinated changes in ion permeation during the cardiac action potential (AP) require the presence of the right types and numbers of ion channels at the cell surface. Regulation of the AP, i.e. by catecholamines, enables an adaptation of cardiac function to changes in blood demand. The pertinent signal transduction cascade targets ion channels as well as many other components of cardiomyocytes. Chronic catecholamine signalling occurs during the compen­sated phase that precedes the transition to overt heart failure.

We have recently proposed a model where ion channels that follow a COPI- and 14-3-3-dependent trafficking route from the endoplasmic reticulum (ER) to the cell surface can be stalled in the Golgi apparatus and then be rapidly released upon phosphorylation of trafficking control motifs present in the channel protein (Arakel et al., J. Cell Sci. 127: 2106-2119, 2014; Kilisch et al., J Cell Sci.129:831-42, 2016). Examples for this mechanism are ATP-sensitive potassium (KATP) channels and two-pore domain (K2P) channels. KATP channels are important to render the electrical excitability of cardiomyocytes sensitive to their metabolic state and both channel types contribute to the repolarization phase of the AP. They contain trafficking control regions that can bind to a vesicular coat complex, COPI. Interaction with COPI mediates the cycling of the ion channels within the early secretory pathway (ER) and a part of the Golgi apparatus. Upon β-adrenergic stimulation PKA can phosphorylate these binding motifs and hence expedite the fully assembled, func­tional channels to the cell surface. Phosphorylation directly prevents COPI bin-ding and can, in the case of K2P channels, recruit a phosphoadaptor protein, 14-3-3, that protects the trafficking control region from dephosphorylation and thereby sustains the effect of phosphorylation.

This model is based on extensive biochemical work characterizing the relevant protein-protein interactions by several complementary methods, including the quantitative determination of the binding parameters between the K2P trafficking control regions and 14-3-3. For KATP channels, we have demonstrated the phosphorylation and changes in its cell surface expression as well as the functional consequences upon sustained β-adrenergic stimulation in isolated cardiomyocytes, Langendorff-perfused whole hearts and in mice. Overall our data demonstrate that the early secretory pathway, which is still poorly characterized in cardiomyocytes, contributes to the regulated release of ion channels to the plasma membrane. During our recent work on the project we have generated a broad array of recombinant proteins that reflect many variants of the trafficking control region of two K2P channels (TASK-1 and TASK-3) expressed in cardiomyocytes. These variants are ideal tools to identify additional binding partners of the trafficking control region since they were designed to selectively lose interactions with COPI and/or 14-3-3 proteins.

This project combines the detailed biochemical investigation of the interaction between cargo proteins (ion channels) and trafficking machinery (COPI, 14-3-3) with the analysis of endogenous ion channels under different (patho)physiological conditions in isolated cardiomyocytes and Langendorff-perfused whole hearts. Thereby, we aim to uncover regulatory mechanisms that use the cardiomyocyte secretory pathway to control cardiac excitability.

Hypotheses of the PhD project

Based on evidence obtained on KATP and K2P channels, we hypothesize that cardiomyocytes feature a general or several dedicated trafficking control complexes containing PKA and other regulatory components.

1. In the project the doctoral researcher will test the hypotheses that trafficking machinery in addition to COPI and 14-3-3 binds to the C-terminus of K2P channel subunits.

2. the PKA involved in this type of trafficking control is specifically recruited to the early secretory pathway by a dedicated AKAP (A-kinase anchoring protein).

3. chronic alterations in the cell surface expression of KATP or K2P channels occur during the compensated phase of heart failure.