MAPK-dependent actin cytoskeletal reorganization underlies BK channel activation by insulin Dervla O’Malley and Jenni Harvey Neurosciences Institute, Division of Pathology & Neuroscience, Ninewells Hospital & Medical School, University of Dundee, Dundee DD1 9SY, UK Keywords: hippocampus, hyperexcitability, insulin signalling, rat Abstract Numerous brain regions are enriched with insulin and insulin receptors, and several lines of evidence indicate that insulin is an important modulator of neuronal function. Indeed, recent studies have demonstrated that insulin inhibits hippocampal epileptiform-like activity, in part by activating large-conductance Ca 2+ -activated potassium (BK) channels. Moreover, the mitogen-activated protein kinase (MAPK) signalling cascade has been found to couple insulin to BK channel activation. However, the cellular events downstream of MAPK that underlie this action of insulin are unknown. Here we demonstrate that in hippocampal neurons, BK channel activation by insulin is blocked by actin filament stabilization, suggesting that this process is dependent on the actin cytoskeleton. Stabilizing actin filaments also markedly attenuated the ability of insulin to inhibit the aberrant hippocampal synaptic activity evoked following Mg 2+ removal. Insulin also promoted rapid reorganization of fluorescently labelled polymerized actin filaments; an action that was prevented by inhibitors of MAPK activation. Moreover, in parallel studies, insulin increased the level of phospho-MAPK immunostaining in hippocampal neurons. These data are consistent with BK channel activation by insulin involving MAPK-dependent alterations in actin dynamics. This process may have important implications for the role of insulin in regulating hippocampal excitability. Introduction It is well established that insulin regulates energy metabolism via its actions on peripheral glucose homeostasis. Insulin also enters the CNS, where it acts on hypothalamic neurons to regulate energy balance (Schwartz et al., 2000). However, insulin and insulin receptors are expressed in many brain regions, including the hippocampus, cerebellum, cortex and olfactory bulb (Folli et al., 1994). During development and in adult tissue, insulin receptors have a distinct and highly regionalized expression pattern (Heidenreich et al., 1988). However, there is still a lack of knowledge about the precise role of insulin in neuronal function. Several lines of evidence implicate insulin in hippocampal associative learning and memory processes (Wickelgren, 1998). Indeed, patients with diabetes with insulin deficiency or resistance often display cognitive impairments (Gispen & Biessels, 2000). In rodent models of diabetes, deficits have been observed in spatial memory tasks (Gispen & Biessels, 2000). Furthermore, alterations in hippocampal insulin receptor levels have been found following spatial memory tasks (Zhao & Alkon, 2001). Insulin also modulates the surface expression of N-methyl-d-aspartate (NMDA), AMPA and c-aminobutyric acid (GABA) A receptors (Wan et al., 1997; Skeber- dis et al., 2001; Zhou et al., 2001). Another cellular target for insulin are large-conductance Ca 2+ -activated K + (BK) channels, as recom- binant and native BK channel activity is enhanced by insulin (O’Malley & Harvey, 2004). One functional consequence of this is attenuation of hippocampal hyperexcitability, as insulin inhibits epileptiform-like activity in a hippocampal culture model (O’Malley et al., 2003). The insulin receptor is a receptor tyrosine kinase, which once stimulated promotes phosphorylation of insulin receptor substrate (IRS) proteins that recruit SH2-domain kinases such as phosphoinositide 3-kinase (PI 3-kinase; Cantrell, 2001). PI 3-kinase promotes phos- phorylation of phosphoinositides on the 3-position resulting predom- inately in PtdIns(3,4,5)P3, which is utilized by various second messengers including protein kinase B, p38 mitogen-activated protein kinase and stress-activated protein kinase 2 (Cantrell, 2001). Insulin can also stimulate the Ras-Raf-mitogen-activated protein kinase (MAPK) cascade. In hippocampal neurons, the MAPK pathway plays a key role in insulin modulation of learning and memory processes (Zhao et al., 1999). BK channel activation and inhibition of hippocampal epilepti- form-like activity by insulin also involves a MAPK-dependent process (O’Malley et al., 2003; O’Malley & Harvey, 2004). The activity of numerous ion channels, including BK (Huang et al., 2002; Piao et al., 2003; O’Malley et al., 2005), ATP-sensitive K + (K ATP ; Terzic & Kurachi, 1996; Harvey et al., 2000) and NMDA channels (Rosenmund & Westbrook, 1993), are modulated by alterations in actin dynamics (Janmey, 1998). Moreover, activation of K ATP channels by insulin and leptin is dependent on actin filament dynamics (Harvey et al., 2000; Mirshamsi et al., 2004). Reorganization of actin filaments is also pivotal for BK channel activation by leptin (O’Malley et al., 2005). However, it is not clear whether insulin activation of BK channels and inhibition of hippocampal epileptiform-like activity is dependent on the actin cytoskeleton. Here we show that MAPK-dependent reorganiza- tion of actin filaments underlies BK channel activation by insulin. Moreover, the ability of insulin to inhibit hippocampal epileptiform-like activity is also driven by alterations in actin dynamics. Correspondence: Dr J. Harvey, as above. E-mail: j.z.harvey@dundee.ac.uk Received 9 August 2006, accepted 7 December 2006 European Journal of Neuroscience, Vol. 25, pp. 673–682, 2007 doi:10.1111/j.1460-9568.2007.05347.x ª The Authors (2007). 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