Login to view PhD Thesis

Enter your username and password here in order to log in on the website:


Forgot your password?

Regulation of Kv7.1 cell surface expression

Martin Nybo Andersen

Summary

The potassium channel Kv7.1 plays with its auxiliary KCNE β-subunits an important role in various tissues in the human body. The channel is properly best known for its role in the heart where it is expressed together with KCNE1. Here the channel-complex forms the delayed rectifier potassium current IKs that contributes to the repolarization of the cardiac action potential. In addition to its role in the heart Kv7.1 is also essential for proper function of various epithelial tissues; such as the stomach where the channel is involved in gastric acid secretion and in the inner ear where the channel contributes to the release of potassium to the endolymph. The physiological relevance of the channel is emphasized by the fact that mutations in Kv7.1 can result in diseases such as Romano-Ward syndrome and Jervel and Lange-Nielsen syndrome. The outcome of both diseases is cardiac arrhythmias and the later furthermore also result in hearing loss, which emphasizes the importance of the channel in the inner ear. Mutations in Kv7.1 often result in trafficking deficiencies leading to reduced surface expression of the channel. The detailed mechanism that controls the surface expression of Kv7.1 is however still largely unknown. 

The aim of my thesis was to identify some of the mechanisms that control the surface expression of Kv7.1. To this end we used the MDCK cell system and exploited the fact that MDCK cell polarization can be controlled by a so called calcium switch assay. 

In the first part of the thesis (chapter II) we study how Kv7.1 is trafficked during the polarization of MDCK cells. We find that the channel displays a dynamic trafficking pattern. In unpolarized cell the channel localizes at the plasma membrane, but in the beginning of the polarization process channels are removed from the membrane and newly formed channels are found in the endoplasmatic reticulum (ER). Later in the polarization process the channels are released from the ER and trafficked to the basolateral membrane. This forward trafficking seems to occur as the lateral membranes are synthesizes. It thereby seems that two mechanisms are regulating the surface expression of Kv7.1 during MDCK cell polarization; an endocytotic pathway and a pathway that controls the forward trafficking of the channel. The regulator of the endocytotic pathway was in chapter IV identified as AMPK. AMPK activation was found to result in internalization of Kv7.1 and the underlying mechanism was demonstrated to be due to ubiquitinylation of the membrane expressed channels by the E3 ubiquitin ligase Nedd4-2. 

The forward trafficking was found to be influenced by PI3K and Sgk1. These two kinases were also found to influence the surface localization of Kv7.1 in polarized MDCK cells. Again the underlying viii mechanism was found to be mediated by Nedd4-2 and we found that a PI3K-Sgk1-Nedd4-2 pathway controls the localization of Kv7.1 in polarized MDCK cells (Chapter V). Also PKA activity was found to be important for the surface localization of Kv7.1 in polarized MDCK cells. We demonstrate that membrane expressed channels are endocytosed in response to PKA inhibition. This change in localization was not found to be influenced by the channels phosphorylation state (Chapter VI). Instead we find the underlying mechanism to be due to Nedd4-2 activity and we thereby demonstrate a novel function of PKA in regards to Kv7.1 activity. 

We furthermore also investigate where the assembly of the IKs complex takes place and which of the two subunits that controls the surface localization of the channel complex. We find that the channel complex is assembled post ER, most likely in the Golgi complex, and that Kv7.1 and not the β- subunit KCNE1 determines the localization of the IKs complex in MDCK cells (Chapter III). Together the five above mentioned studies present data that adds to the knowledge on mechanism controlling the surface expression of Kv7.1 as well as the assembly and trafficking of the IKs complex. Clearly, Nedd4-2 appears to be a central player connecting all three identified pathways. The upstream mediators may constitute potent mechanisms serving to adjust the Kv7.1 channel turnover and thereby potassium flux in response to different physiological stimuli such as ischemia.