Kv2 Ion Channels Determine the Expression and Localization of the Associated AMIGO-1 Cell Adhesion Molecule in Adult Brain Neurons

Abstract

Voltage-gated K+ (Kv) channels play important roles in regulating neuronal excitability. Kv channels comprise four principal α subunits, and transmembrane and/or cytoplasmic auxiliary subunits that modify diverse aspects of channel function. AMIGO-1, which mediates homophilic cell adhesion underlying neurite outgrowth and fasciculation during development, has recently been shown to be an auxiliary subunit of adult brain Kv2.1-containing Kv channels. We show that AMIGO-1 is extensively colocalized with both Kv2.1 and its paralog Kv2.2 in brain neurons across diverse mammals, and that in adult brain, there is no apparent population of AMIGO-1 outside of that colocalized with these Kv2 α subunits. AMIGO-1 is coclustered with Kv2 α subunits at specific plasma membrane (PM) sites associated with hypolemmal subsurface cisternae at neuronal ER:PM junctions. This distinct PM clustering of AMIGO-1 is not observed in brain neurons of mice lacking Kv2 α subunit expression. Moreover, in heterologous cells, coexpression of either Kv2.1 or Kv2.2 is sufficient to drive clustering of the otherwise uniformly expressed AMIGO-1. Kv2 α subunit coexpression also increases biosynthetic intracellular trafficking and PM expression of AMIGO-1 in heterologous cells, and analyses of Kv2.1 and Kv2.2 knockout mice show selective loss of AMIGO-1 expression and localization in neurons lacking the respective Kv2 α subunit. Together, these data suggest that in mammalian brain neurons, AMIGO-1 is exclusively associated with Kv2 α subunits, and that Kv2 α subunits are obligatory in determining the correct pattern of AMIGO-1 expression, PM trafficking and clustering.

Epilepsy and neurobehavioral abnormalities in mice with aKCNB1pathogenic variant that alters conducting and non-conducting functions of KV2.1

“…Immunoblotting for AMIGO-1 whole brain expression revealed ≈25% lower expression in Kcnb1 R/+ and ≈50% lower expression in Kcnb1 R/R mice compared to WT littermates ( Figure 3F,G). The observed reduction of AMIGO-1 in Kcnb1 G379R mice is in contrast to our previous studies of brain sections from Kcnb1 +/and Kcnb1 -/mice (Bishop et al, 2018), in which AMIGO-1 immunolabeling was only lost in neurons that typically express high levels of KV2.1 relative to KV2.2, but was expressed normally in neurons that express KV2.2, such as CA1 pyramidal neurons ( Figure 3A).…”

“…Immunohistochemistry and immunoblotting experiments revealed that both KV2.2 and AMIGO-1 immunoreactivity was substantially lower in sections and whole brain membrane preparations from Kcnb1 R/R mice. KV2.1 and KV2.2 form heteromeric channels, with most complexes containing AMIGO-1 auxiliary subunits (Bishop et al, 2018;Kihira et al, 2010;Peltola et al, 2011a). We hypothesize that the lower KV2.2 and AMIGO-1 immunoreactivity is due to their altered subcellular localization, as both proteins were diffusely distributed throughout the ER rather than clustered at the PM (for a full discussion see (Bishop et al, 2018)).…”

“…Epifluorescence and TIRF imaging of fixed cells and image analysis was performed essentially as described (Bishop et al, 2018;Kirmiz et al, 2018b). Images were obtained with an Andor iXon EMCCD camera installed on a TIRF/widefield equipped Nikon Eclipse Ti microscope using a Nikon LUA4 laser launch with 405, 488, 561 and 647 nm lasers and a 100x PlanApo TIRF/1.49 NA objective run with NIS Elements software (Nikon).…”

“…KV2.1 is expressed at high levels in various neurons and localized to high-density clusters on the soma, proximal dendrites and axon initial segment (Bishop et al, 2015;Du et al, 1998;Sarmiere et al, 2008;Trimmer, 1991). KV2.1 clusters align with endoplasmic reticulum (ER) and plasma membrane (PM) junctions, membrane microdomains important in calcium and lipid signaling and homeostasis (Bishop et al, 2018;Bishop et al, 2015;Dickson, 2017;Du et al, 1998;Gallo et al, 2016;Henne et al, 2015;Mandikian et al, 2014;Wu et al, 2017). KV2.1 serves a structural role at EM-PM junctions independent of its potassium conducting function by binding to the ER proteins VAPA and VAPB, resulting in the recruitment of the ER to the PM (Fox et al, 2015;Johnson et al, 2018;Kirmiz et al, 2018a;Kirmiz et al, 2018b).…”

A molecular rheostat: Kv2.1 currents maintain or suppress repetitive firing in motoneurons

“…How Kv2 ion channels influence neuronal excitability is of particular interest because accumulating evidence demonstrates these exceptionally dynamic channels are targets of a remarkable range of influences that can alter their functions (Murakoshi et al 1997;Misonou et al 2004Misonou et al , 2005O'Connell et al 2006O'Connell et al , 2010Redman et al 2007;Mohapatra et al 2009;Plant et al 2011;Steinert et al 2011;Fox et al 2013). In some cultured neurons, high-density clusters of Kv2 channels are largely low or non-conducting, and it is possible that the channel proteins have additional 'non-conducting' functions (O'Connell et al 2010;Fox et al 2013;Bishop et al 2018;Kirmiz et al 2018). However, the data presented here, together with other reports (Du et al 2000;Malin & Nerbonne, 2002;Guan et al 2007Guan et al , 2013Johnston et al 2008;Mohapatra et al 2009;Liu & Bean, 2014), suggest that the electrical function of Kv2 channels in native neuronal membranes is substantial and strongly contributes to neural function.…”