Potassium channels comprise the most diverse group of ion channels, with more than 100 subunits being identified and classified into different channel families based on their molecular and biophysical properties (Hille, 2001; Gutman et al., 2003). It is well known that different neuron types express distinct sets of K+ channel subunits, endowing them with characteristic electrophysiological properties. Recently, it also became clear that differential distributions of distinct K+ channel subunits in various subcellular compartments add another layer of complexity to their functional impact. Hippocampal and cortical pyramidal cells (PC) express a wide variety of K+ channel subunits (http://www.brain-map.org), which might reside in distinct axo-somato-dendritic compartments. Indeed, electrophysiological experiments have identified K+ currents in PC dendrites, where they regulate the integration of synaptic inputs, control action potential (AP) back-propagation and dendritic electrogenesis (Hoffman et al., 1997; Migliore et al., 1999; Cai et al., 2004; Losonczy et al., 2008). They have also been found in axons, where they set the threshold and sculpt the shape of the APs in addition to regulating repetitive firing properties of PCs (Kole et al., 2007; Shu et al., 2007; Rasband, 2010; Bender & Trussell, 2012; Kole & Stuart, 2012). Unfortunately, small subcellular compartments such as oblique dendrites, dendritic tufts, dendritic spines, nodes of Ranvier and axon terminals are inaccessible for patch-pipette recordings, rendering their potassium currents enigmatic. Immunohistochemistry, using subunit specific antibodies, offers an alternative and powerful mean of identifying K+ channel subtypes in small subcellular compartments (Rhodes & Trimmer, 2006; Fritschy, 2008; Lorincz & Nusser, 2008b). However, ion channels at low densities could remain undetectable due to the limited sensitivity and resolution of traditional light microscopic (LM) immunolocalisation methods. Electron microscopic (EM) SDS-digested freeze-fracture replica immunolabelling technique (SDS-FRL; Fujimoto, 1995; Rash et al., 1998; Masugi-Tokita & Shigemoto, 2007) allows the detection of ion channels and receptors with a labelling efficiency close to 100% (Tanaka et al., 2005; Lorincz & Nusser, 2010). With such a sensitive and high-resolution method, we have previously determined the relative densities of Kv4.2 and Nav1.6 subunits in a large number of axo-somato-dendritic compartments of CA1 PCs (Lorincz & Nusser, 2010; Kerti et al., 2012), creating the first step towards the generation of a quantitative molecular map of the neuronal surface. Here we aim at extending our quantitative surface map of ion channels by investigating the relative densities of two delayed-rectifier (Kv1.1 and Kv2.1) and one G-protein-gated inwardly rectifying (Kir3.2) K+ channel subunits.
Previous studies, using LM immunofluorescent and peroxidase reactions, demonstrated the presence of the Kv1.1 subunit in axon initial segments (AISs), juxta-paranodal regions of myelinated axons of CA1 PCs and in the neuropil of the strata oriens (SO) and radiatum (SR) of the CA1 area (Veh et al., 1995; Rhodes et al., 1997; Monaghan et al., 2001; Lorincz & Nusser, 2008a). In contrast, the Kv2.1 subunit was found in the somata, proximal dendrites and AISs of PCs (Trimmer, 1991; Maletic-Savatic et al., 1995; Du et al., 1998; Lim et al., 2000; Misonou et al., 2004; Rhodes & Trimmer, 2006; Sarmiere et al., 2008), where it either forms clusters or has a uniform distribution depending on its phosphorylation state (Misonou et al., 2004, 2005). Potassium channels formed by the Kir3 subunits are the effectors of many G-protein-coupled receptors, and are potent regulators of neuronal excitability. Of the three main neuronal Kir3 subunits (Kir3.1-3.3; reviewed by Lujan et al., 2009; Luscher & Slesinger, 2010), the Kir3.2 subunit seems to be essential for functional channel formation (Liao et al., 1996). The Kir3-mediated K+ current is larger in the apical dendrites than in the soma of CA1 PCs (Chen & Johnston, 2005), which is consistent with the gradual increase in the strength of LM immunoreactivity for the Kir3.1, Kir3.2 and Kir3.3 subunits in distal SR and stratum lacunosum-moleculare (SLM; Ponce et al., 1996; Koyrakh et al., 2005; Kulik et al., 2006; Fernandez-Alacid et al., 2011). Although these subunits have been localised with EM immunogold methods to dendritic shafts, spines and axon terminals (Koyrakh et al., 2005; Kulik et al., 2006; Fernandez-Alacid et al., 2011), but their relative densities in different axo-somato-dendritic compartmens at various distances from the soma are still unknown.