Ng, K-E.; (2007) The distribution and function of the ATP-sensitive potassium channel subunit Kir6.1 in cardiac and skeletal muscle cell lines. Doctoral thesis , University of London.

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Abstract

ATP-sensitive potassium channels (KAtp) are present in the plasma membrane of a number of tissues but are also present on endomembranes such as the endoplasmic reticulum (ER) and mitochondria. They are involved in a number of physiological and pathophysiological processes and form a link between cellular metabolism and membrane excitability. Ischaemic preconditioning describes the phenomenon in which a short period of ischaemia protects against a more prolonged one. The ability of potassium channel openers such as pinacidil and nicorandil can mimic this phenomenon, with inhibitors such as glibenclamide to abolish this response, led to the suggestion that the final effector in this process was the sarcolemmal Katp channel as it was able to shorten the cardiac action potential reducing the energy requirements of the cell. However, a number of pharmacological observations were not compatible with this hypothesis as diazoxide, which does not activate the sarcolemmal channel, was able to mimic preconditioning. The focus of research then turned to the potential involvement of a KAtp channel present in the mitochondrial inner membrane called the mitoKArp channel. The molecular identification of this channel would be important and there is controversial evidence to suggest that Kiro.l may be a major component of the mitoKATP channel. I examined the hypothesis that the localisation of Kir6.1 is functionally significant in cardiac and skeletal muscle because it generates important K+ flux in intracellular membranes such as the ER and perhaps mitochondria. Co-localisation studies showed that transfected Kiro.l was located in the ER with a small but significant proportion in mitochondria. However, Kir6.1 was ER retained and not trafficked to the plasma membrane when co-expressed with its regulatory subunit, the sulphonylurea receptor SUR1. Immunofluorescent staining also detected the presence of endogenous Kir6.1 in these cell lines using antibodies specific to Kir6.1. The distribution of Kir6.1 suggests that it may play a role in reactive oxygen species (ROS) production, calcium (Ca) handling in the ER and perhaps cellular respiration in mitochondria. ROS production is often associated with KAtp channel opening and protection against cell death at reperfusion. My results showed that diazoxide induced ROS production in C2C12, HepG2 and HEK293 cell lines with glibenclamide abolishing this effect. However, in the absence of Kir6.1, the same response was still observed. This suggests that Kir6.1 is not involved in the mechanism that is responsible for ROS production. The functional role of KAtp channels were also examined in mitochondria by measuring flavoprotein and NADH autofluorescence, an index of mitochondrial redox state and mitochondrial membrane potential (Au/m) in C2C12 cells and rat ventricular myocytes. In myocytes, flavoprotein oxidation increased when cells were treated with 3-nitroproprionic acid (3-NPA) and diazoxide. Glibenclamide did not reverse this effect. However, this phenomenon was absent in C2C12 cells. Given these observations, 3-NPA and diazoxide did not affect the Avj/m in C2C12 cells whereas glibenclamide caused mitochondrial depolarisation. The Aij/m could not be measured in myocytes. A large proportion of Kir6.1 resides in the ER and I examined whether Kir6.1 would alter ATP-induced Ca2+ transients. Upon ATP stimulation, C2C12 cells released Ca2+ from internal stores via the P2Y purinergic signalling pathway. The use of dominant negatives (DN) for Kir6.1 showed that ATP-induced Ca2+ transients were affected by the absence of Kir6.1. However, on closer inspection it was revealed that the presence of eGFP to identify transfected cells seriously perturbed the Fura-2 signal. In conclusion, the KAtp sensitive channel subunit Kir6.1 is predominantly distributed in the ER with a small but significant proportion in mitochondria. I also report that pharmacological compounds such as diazoxide and glibenclamide are not always truly 'selective' for the activation and inhibition of KAtp channels. I have not identified a specific role for Kir6.1 but my data suggests that Kir6.1 is not part of the mitoKATp channel and Kir6.1 is not involved in ROS production and mitochondrial function but it may still have a role in Ca2+ handling.

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