Isolation of Human Potassium KV4.3 and functional Reconstruction of the Channel Liposomes.

Doestzada, M. (Marwah) (2015) Isolation of Human Potassium KV4.3 and functional Reconstruction of the Channel Liposomes. thesis, Medicine.

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Abstract

Human Kv4.3 channel is a voltage-gated potassium ion channel and plays a significant role in the excitation of neurons and repolarization in cardiac myocytes. Recently mutations found in Kv4.3 channels have been shown to cause the brain disease Spinocerebellar ataxia 19/22 and the heart disease Brugada syndrome. However, how mutations in this one channel can cause these two diseases is not known. To determine the exact mechanism behind these diseases it is necessary to study Kv4.3 channels at the single channel level in a controlled environment. Therefore, the aim of this thesis was to set the first step towards studies of Kv4.3 channels in a controlled environment. This was done by first expressing Kv4.3 channels in the yeast Pichia pastoris and isolating the channel using Ni-NTA chromatography. SDS-PAGE and Western blot showed expression of Kv4.3 in Pichia pastoris and purity of the isolation. Isolated channel were reconstituted (re-inserted) into preformed liposomes and channel activity was measured by patch clamping. Kv4.3 channels were also expressed in Chinese hamster ovary (CHO) cells to compare the Kv4.3 channel activity in the synthetic environment with that of the native environment of the cell. In the synthetic environment channel activity ranging between ~2.5-8.5pS were measured. Channel conductance measured from a full open level of ~3.8pS is similar to the main open level of Kv4.3 channels reported in the literature (~4pS). Conductance’s twice this level was measured in both the synthetic (~8.5pS) and the cell membrane environment (~8.8pS). Furthermore, in both the synthetic and cell membrane environment large current macroscopic currents were measured, ranging between ~16-160pS (synthetic membrane ) and ~11-250pS (cell membrane). These macroscopic currents closed in a stepwise manner and this pattern of closing was observed in both the synthetic and cell environment. Non of the currents measured in the liposomes and in the CHO cells containing Kv4.3 channels were measured in controls. In conclusion, these findings suggest that Kv4.3 channels were reconstituted into liposomes and where functional. In addition, it is suggested that the behavior of the Kv4.3 channel in the synthetic environment is similar to Kv4.3 channel activity in native cell membranes.

Item Type:	Thesis (Thesis)
Supervisor name:	Supervisor and Kocer, dr. Armagan and Faculty of Medical Science, Groningen and Department of Medical Physiology, Neuroscience
Faculty:	Medical Sciences
Date Deposited:	25 Jun 2020 10:58
Last Modified:	25 Jun 2020 10:58
URI:	https://umcg.studenttheses.ub.rug.nl/id/eprint/1834

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