Distinct Cell- and Layer-Specific Expression Patterns and Independent Regulation of Kv2 Channel Subtypes in Cortical Pyramidal Neurons

Abstract

The Kv2 family of voltage-gated potassium channel ␣ subunits, comprising Kv2.1 and Kv2.2, mediate the bulk of the neuronal delayed rectifier K ϩ current in many mammalian central neurons. Kv2.1 exhibits robust expression across many neuron types and is unique in its conditional role in modulating intrinsic excitability through changes in its phosphorylation state, which affect Kv2.1 expression, localization, and function. Much less is known of the highly related Kv2.2 subunit, especially in forebrain neurons. Here, through combined use of cortical layer markers and transgenic mouse lines, we show that Kv2.1 and Kv2.2 are localized to functionally distinct cortical cell types. Kv2.1 expression is consistently high throughout all cortical layers, especially in layer (L) 5b pyramidal neurons, whereas Kv2.2 expression is primarily limited to neurons in L2 and L5a. In addition, L4 of primary somatosensory cortex is strikingly devoid of Kv2.2 immunolabeling. The restricted pattern of Kv2.2 expression persists in Kv2.1-KO mice, suggesting distinct cell-and layer-specific functions for these two highly related Kv2 subunits. Analyses of endogenous Kv2.2 in cortical neurons in situ and recombinant Kv2.2 expressed in heterologous cells reveal that Kv2.2 is largely refractory to stimuli that trigger robust, phosphorylationdependent changes in Kv2.1 clustering and function. Immunocytochemistry and voltage-clamp recordings from outside-out macropatches reveal distinct cellular expression patterns for Kv2.1 and Kv2.2 in intratelencephalic and pyramidal tract neurons of L5, indicating circuit-specific requirements for these Kv2 paralogs. Together, these results support distinct roles for these two Kv2 channel family members in mammalian cortex.

Roles of specific Kv channel types in repolarization of the action potential in genetically identified subclasses of pyramidal neurons in mouse neocortex

“…2, Table 3). We previously showed that GxTx did block K v 2-mediated currents in mouse neocortical pyramidal neurons at this dose (Bishop et al 2015). These data indicate little to no role for K v 2 channels in AP repolarization in neocortical pyramidal cells (at least for a single AP in response to a brief current injection; the function of K v 2 channels becomes more important with high-frequency firing: Du et al 2000;Guan et al 2013;Johnston et al 2008;Liu and Bean 2014).…”

“…In etv1 neurons, recordings were biased toward the most superficial EGFPϩ cells, and for glt neurons recordings were biased toward deeper EGFPϩ cells. We previously showed that in somatosensory cortex, etv1 neurons have a more superficial (and narrower) expression pattern in layer 5. glt neurons are found deeper in layer 5 , but there is overlap with the etv1 distribution (Bishop et al 2015;Guan et al 2015). We switched between IR-DIC and epifluorescence to determine cell type and to obtain a gigaohm seal.…”

“…Voltage-clamp data were obtained from outside-out macropatches at 33 Ϯ 1°C (see Bishop et al 2015 for detailed methods). These patches were formed after break-in to whole cell mode by withdrawing the pipette without additional suction.…”

“…For example, consider two lines of mice that express enhanced green fluorescent protein (EGFP) in cells expressing the genes etv1 or glt (Doyle et al 2008;Gong et al 2007). In mouse somatosensory cortex, etv1 cells are a subset of IT-type neurons located primarily in superficial layer 5 and glt cells are a subset of PT-type neurons, primarily located in deep layer 5 (Bishop et al 2015;Groh et al 2010;Guan et al 2015). glt cells have narrower and more rapidly repolarizing APs than etv1 or layer 2/3 cells (Groh et al 2010;Guan et al 2015).…”

“…There are transient and persistent components to outward K ϩ currents in rodent pyramidal neurons (Bekkers 2000;Foehring and Surmeier 1993;Korngreen and Sakmann 2000;Locke and Nerbonne 1997). The persistent currents are due to strong expression of K v 1, K v 2 (the largest component), and K v 7 channels (Bekkers and Delaney 2001;Bishop et al 2015;Guan et al 2006Guan et al , 2007aGuan et al , 2007bGuan et al , 2011aGuan et al , 2011bGuan et al , 2013Guan et al , 2015Murakoshi and Trimmer 1999). In addition, Nerbonne and colleagues used genetic approaches to reveal that most of the transient, A-type current in rat neocortical cells is due to K v 4.2 and K v 4.3 channels (Carrasquillo et al 2012;Norris and Nerbonne 2010;Yuan et al 2005; see also Guan et al 2011b), with a contribution from K v 1.4 channels.…”