The Kv2.1 voltage-gated K+ channel is widely expressed throughout mammalian brain, where it contributes to dynamic activity-dependent regulation of intrinsic neuronal excitability. Here we show that somatic plasma membrane Kv2.1 clusters are juxtaposed to clusters of intracellular ryanodine receptor (RyR) Ca2+ -release channels in mouse brain neurons, most prominently in medium spiny neurons (MSNs) of the striatum. Electron microscopy-immunogold labeling shows that in MSNs, plasma membrane Kv2.1 clusters are adjacent to subsurface cisternae, placing Kv2.1 in close proximity to sites of RyR-mediated Ca2+ release. Immunofluorescence labeling in transgenic mice expressing green fluorescent protein in specific MSN populations reveals the most prominent juxtaposed Kv2.1:RyR clusters in indirect pathway MSNs. Kv2.1 in both direct and indirect pathway MSNs exhibits markedly lower levels of labeling with phosphospecific antibodies directed against the S453, S563, and S603 phosphorylation site compared with levels observed in neocortical neurons, although labeling for Kv2.1 phosphorylation at S563 was significantly lower in indirect pathway MSNs compared with those in the direct pathway. Finally, acute stimulation of RyRs in heterologous cells causes a rapid hyperpolarizing shift in the voltage dependence of activation of Kv2.1, typical of Ca2+ /calcineurin-dependent Kv2.1 dephosphorylation. Together, these studies reveal that striatal MSNs are distinct in their expression of clustered Kv2.1 at plasma membrane sites juxtaposed to intracellular RyRs, as well as in Kv2.1 phosphorylation state. Differences in Kv2.1 expression and phosphorylation between MSNs in direct and indirect pathways provide a cell- and circuit-specific mechanism for coupling intracellular Ca2+ release to phosphorylation-dependent regulation of Kv2.1 to dynamically impact intrinsic excitability.

KCNE5 is an X-linked gene encoding KCNE5, an ancillary subunit to voltage-gated potassium (K_V) channels. Human KCNE5 mutations are associated with atrial fibrillation (AF)- and Brugada syndrome (BrS)-induced cardiac arrhythmias that can arise from increased potassium current in cardiomyocytes. Seeking to establish underlying molecular mechanisms, we created and studied Kcne5 knockout ( Kcne5^-/0) mice. Intracardiac ECG revealed that Kcne5 deletion caused ventricular premature beats, increased susceptibility to induction of polymorphic ventricular tachycardia (60 vs. 24% in Kcne5^+/0 mice), and 10% shorter ventricular refractory period. Kcne5 deletion increased mean ventricular myocyte K_V current density in the apex and also in the subpopulation of septal myocytes that lack fast transient outward current ( I_to,f). The current increases arose from an apex-specific increase in slow transient outward current-1 ( I_Kslow,1) (conducted by K_V1.5) and I_to,f (conducted by K_V4) and an increase in I_Kslow,2 (conducted by K_V2.1) in both apex and septum. Kcne5 protein localized to the intercalated discs in ventricular myocytes, where K_V2.1 was also detected in both Kcne5^-/0 and Kcne5^+/0 mice. In HL-1 cardiac cells and human embryonic kidney cells, KCNE5 and K_V2.1 colocalized at the cell surface, but predominantly in intracellular vesicles, suggesting that Kcne5 deletion increases I_K,slow2 by reducing K_V2.1 intracellular sequestration. The human AF-associated mutation KCNE5-L65F negative shifted the voltage dependence of K_V2.1-KCNE5 channels, increasing their maximum current density >2-fold, whereas BrS-associated KCNE5 mutations produced more subtle negative shifts in K_V2.1 voltage dependence. The findings represent the first reported native role for Kcne5 and the first demonstrated Kcne regulation of K_V2.1 in mouse heart. Increased K_V current is a manifestation of KCNE5 disruption that is most likely common to both mouse and human hearts, providing a plausible mechanistic basis for human KCNE5-linked AF and BrS.-David, J.-P., Lisewski, U., Crump, S. M., Jepps, T. A., Bocksteins, E., Wilck, N., Lossie, J., Roepke, T. K., Schmitt, N., Abbott, G. W. Deletion in mice of X-linked, Brugada syndrome- and atrial fibrillation-associated Kcne5 augments ventricular K_V currents and predisposes to ventricular arrhythmia.

The Kv2 family of voltage-gated potassium channel α subunits, comprising Kv2.1 and Kv2.2, mediate the bulk of the neuronal delayed rectifier K(+) 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, phosphorylation-dependent 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.

Significance statement

Neurons within the neocortex are arranged in a laminar architecture and contribute to the input, processing, and/or output of sensory and motor signals in a cell- and layer-specific manner. Neurons of different cortical layers express diverse populations of ion channels and possess distinct intrinsic membrane properties. Here, we show that the Kv2 family members Kv2.1 and Kv2.2 are expressed in distinct cortical layers and pyramidal cell types associated with specific corticostriatal pathways. We find that Kv2.1 and Kv2.2 exhibit distinct responses to acute phosphorylation-dependent regulation in brain neurons in situ and in heterologous cells in vitro. These results identify a molecular mechanism that contributes to heterogeneity in cortical neuron ion channel function and regulation.