Selezneva, Anna; (2024) The Role of Nuclear BK Channels in Macrophage Regulation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Ion channels are known to regulate cellular signalling pathways in immune cells. When studying ion channels in cells, a confounding variable is often that the ion channel can be found in numerous cellular locations. However, the majority of research in this area has focused on ion channels in the plasma membrane. Recent research has suggested that the nuclear membrane plays a significant role in immune cell activation. One possible pathway by which this may occur is via specific ion channel signalling mechanisms. While studying the function of plasma membrane located large conductance voltageand calcium-activated potassium channels (BK channels) in a model of inflammatory macrophages, the cell line RAW264.7, previous research in the laboratory also suggested the presence of a pool of intracellular BK channels in macrophages. The aim of this thesis was to investigate if functional BK channels are present in RAW264.7 macrophage nuclei and, if positive, to establish their roles in the activation of macrophages in vitro. Western blot analysis together with the pharmacology of RAW264.7 macrophage cytokine release, electrophysiological recordings and Ca2+ imaging of isolated macrophage nuclei provided evidence that a functional ion channel, with properties consistent with other BK channels, is present in the nuclear envelope. Potentiometric and Ca2+-sensitive dye imaging showed that nuclear BK channels regulate nuclear membrane potential and nuclear Ca2+ concentrations. Ryanodine receptors were found to be involved in this BK channel-regulated Ca2+ signalling from the perinuclear space into the nucleoplasm of macrophage nuclei. Downstream of nuclear Ca2+ signalling, nuclear BK channels were demonstrated to regulate CREB phosphorylation via nuclear located Ca2+ and calmodulin-dependent kinases (CaMKII and/or CaMKIV). Finally, this thesis reported that block of intracellular BK channels regulates lipopolysaccharide-induced cytokine (TNFa) release from whole cell macrophages. This thesis presents the first evidence for functional nuclear BK channels in macrophages and indicates new pathways by which gene transcription in macrophages is regulated.

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