Aaron J. Norris

Electrical remodelling maintains firing properties in cortical pyramidal neurons lacking KCND2-encoded A-type K+ currents.

Considerable experimental evidence has accumulated demonstrating a role for voltage‐gated K+ (Kv) channel pore‐forming (α) subunits of the Kv4 subfamily in the generation of fast transient outward K+, IA, channels. Immunohistochemical data suggest that IA channels in hippocampal and cortical pyramidal neurons reflect the expression of homomeric Kv4.2 channels. The experiments here were designed to define directly the role of Kv4.2 in the generation of IA in cortical pyramidal neurons and to determine the functional consequences of the targeted deletion of Kv4.2 on the resting and active membrane properties of these cells. Whole‐cell voltage‐clamp recordings, obtained from visual cortical pyramidal neurons isolated from mice in which the KCND2 (Kv4.2) locus was disrupted (Kv4.2‐/‐ mice), revealed that IA is indeed eliminated. In addition, the densities of other Kv current components, specifically IK and Iss, are increased significantly (P < 0.001) in most (∼80%) Kv4.2‐/‐ cells. The deletion of KCND2 (Kv4.2) and the elimination of IA is also accompanied by the loss of the Kv4 channel accessory protein KChIP3, suggesting that in the absence of Kv4.2, the KChIP3 protein is targeted for degradation. The expression levels of several Kv α subunits (Kv4.3, Kv1.4, Kv2.1, Kv2.2), however, are not measurably altered in Kv4.2‐/‐ cortices. Although IA is eliminated in Kv4.2‐/‐ pyramidal neurons, the mean ±s.e.m. current threshold for action potential generation and the waveforms of action potentials are indistinguishable from those recorded from wild‐type cells. Repetitive firing is also maintained in Kv4.2‐/‐ cortical pyramidal neurons, suggesting that the increased densities of IK and Iss compensate for the in vivo loss of IA.

The sodium channel accessory subunit Navβ1 regulates neuronal excitability through modulation of repolarizing voltage-gated K+ channels

The channel pore-forming α subunit Kv4.2 is a major constituent of A-type (IA) potassium currents and a key regulator of neuronal membrane excitability. Multiple mechanisms regulate the properties, subcellular targeting, and cell-surface expression of Kv4.2-encoded channels. In the present study, shotgun proteomic analyses of immunoprecipitated mouse brain Kv4.2 channel complexes unexpectedly identified the voltage-gated Na+ channel accessory subunit Navβ1. Voltage-clamp and current-clamp recordings revealed that knockdown of Navβ1 decreases IA densities in isolated cortical neurons and that action potential waveforms are prolonged and repetitive firing is increased in Scn1b-null cortical pyramidal neurons lacking Navβ1. Biochemical and voltage-clamp experiments further demonstrated that Navβ1 interacts with and increases the stability of the heterologously expressed Kv4.2 protein, resulting in greater total and cell-surface Kv4.2 protein expression and in larger Kv4.2-encoded current densities. Together, the results presented here identify Navβ1 as a component of native neuronal Kv4.2-encoded IA channel complexes and a novel regulator of IA channel densities and neuronal excitability.

Molecular Dissection of IA in Cortical Pyramidal Neurons Reveals Three Distinct Components Encoded by Kv4.2, Kv4.3, and Kv1.4 α-Subunits

The rapidly activating and inactivating voltage-gated K+ (Kv) current, IA, is broadly expressed in neurons and is a key regulator of action potential repolarization, repetitive firing, backpropagation (into dendrites) of action potentials, and responses to synaptic inputs. Interestingly, results from previous studies on a number of neuronal cell types, including hippocampal, cortical, and spinal neurons, suggest that macroscopic IA is composed of multiple components and that each component is likely encoded by distinct Kv channel α-subunits. The goals of the experiments presented here were to test this hypothesis and to determine the molecular identities of the Kv channel α-subunits that generate IA in cortical pyramidal neurons. Combining genetic disruption of individual Kv α-subunit genes with pharmacological approaches to block Kv currents selectively, the experiments here revealed that Kv1.4, Kv4.2, and Kv4.3 α-subunits encode distinct components of IA that together underlie the macroscopic IA in mouse (male and female) cortical pyramidal neurons. Recordings from neurons lacking both Kv4.2 and Kv4.3 (Kv4.2−/−/Kv4.3−/−) revealed that, although Kv1.4 encodes a minor component of IA, the Kv1.4-encoded current was found in all the Kv4.2−/−/Kv4.3−/− cortical pyramidal neurons examined. Of the cortical pyramidal neurons lacking both Kv4.2 and Kv1.4, 90% expressed a Kv4.3-encoded IA larger in amplitude than the Kv1.4-encoded component. The experimental findings also demonstrate that the targeted deletion of the individual Kv α-subunits encoding components of IA results in electrical remodeling that is Kv α-subunit specific.

Interdependent Roles for Accessory KChIP2, KChIP3, and KChIP4 Subunits in the Generation of Kv4-Encoded IA Channels in Cortical Pyramidal Neurons

The rapidly activating and inactivating voltage-dependent outward K+ (Kv) current, IA, is widely expressed in central and peripheral neurons. IA has long been recognized to play important roles in determining neuronal firing properties and regulating neuronal excitability. Previous work demonstrated that Kv4.2 and Kv4.3 α-subunits are the primary determinants of IA in mouse cortical pyramidal neurons. Accumulating evidence indicates that native neuronal Kv4 channels function in macromolecular protein complexes that contain accessory subunits and other regulatory molecules. The K+ channel interacting proteins (KChIPs) are among the identified Kv4 channel accessory subunits and are thought to be important for the formation and functioning of neuronal Kv4 channel complexes. Molecular genetic, biochemical, and electrophysiological approaches were exploited in the experiments described here to examine directly the roles of KChIPs in the generation of functional Kv4-encoded IA channels. These combined experiments revealed that KChIP2, KChIP3, and KChIP4 are robustly expressed in adult mouse posterior (visual) cortex and that all three proteins coimmunoprecipitate with Kv4.2. In addition, in cortical pyramidal neurons from mice lacking KChIP3 (KChIP3−/−), mean IA densities were reduced modestly, whereas in mean IA densities in KChIP2−/− and WT neurons were not significantly different. Interestingly, in both KChIP3−/− and KChIP2−/− cortices, the expression levels of the other KChIPs (KChIP2 and 4 or KChIP3 and 4, respectively) were increased. In neurons expressing constructs to mediate simultaneous RNA interference-induced reductions in the expression of KChIP2, 3, and 4, IA densities were markedly reduced and Kv current remodeling was evident.

Augmentation of Kv4.2-encoded currents by accessory dipeptidyl peptidase 6 and 10 subunits reflects selective cell surface Kv4.2 protein stabilization.

Background: Somatodendritic Kv4-encoded A-Type K+ current densities are enhanced by both cytosolic K+ channel interacting proteins (KChIPs) and transmembrane dipeptidyl peptidases (DPPs). Results: DPPs selectively stabilize cell surface Kv4 protein expression, whereas KChIPs stabilize total and surface Kv4 protein expression. Conclusion: DPPs regulate Kv4-encoded current densities through mechanisms distinct from the KChIPs. Significance: Multiple mechanisms determine functional Kv4 channel densities. Rapidly activating and inactivating somatodendritic voltage-gated K+ (Kv) currents, IA, play critical roles in the regulation of neuronal excitability. Considerable evidence suggests that native neuronal IA channels function in macromolecular protein complexes comprising pore-forming (α) subunits of the Kv4 subfamily together with cytosolic, K+ channel interacting proteins (KChIPs) and transmembrane, dipeptidyl peptidase 6 and 10 (DPP6/10) accessory subunits, as well as other accessory and regulatory proteins. Several recent studies have demonstrated a critical role for the KChIP subunits in the generation of native Kv4.2-encoded channels and that Kv4.2-KChIP complex formation results in mutual (Kv4.2-KChIP) protein stabilization. The results of the experiments here, however, demonstrate that expression of DPP6 in the mouse cortex is unaffected by the targeted deletion of Kv4.2 and/or Kv4.3. Further experiments revealed that heterologously expressed DPP6 and DPP10 localize to the cell surface in the absence of Kv4.2, and that co-expression with Kv4.2 does not affect total or cell surface DPP6 or DPP10 protein levels. In the presence of DPP6 or DPP10, however, cell surface Kv4.2 protein expression is selectively increased. Further addition of KChIP3 in the presence of DPP10 markedly increases total and cell surface Kv4.2 protein levels, compared with cells expressing only Kv4.2 and DPP10. Taken together, the results presented here demonstrate that the expression and localization of the DPP accessory subunits are independent of Kv4 α subunits and further that the DPP6/10 and KChIP accessory subunits independently stabilize the surface expression of Kv4.2.

Neuronal Voltage-Gated K+ (Kv) Channels Function in Macromolecular Complexes

Considerable evidence indicates that native neuronal voltage-gated K+ (Kv) currents reflect the functioning of macromolecular Kv channel complexes, composed of pore-forming (α)-subunits, cytosolic and transmembrane accessory subunits, together with regulatory and scaffolding proteins. The individual components of these macromolecular complexes appear to influence the stability, the trafficking, the localization and/or the biophysical properties of the channels. Recent studies suggest that Kv channel accessory subunits subserve multiple roles in the generation of native neuronal Kv channels. Additional recent findings suggest that Kv channel accessory subunits can respond to changes in intracellular Ca(2+) or metabolism and thereby integrate signaling pathways to regulate Kv channel expression and properties. Although studies in heterologous cells have provided important insights into the effects of accessory subunits on Kv channel expression/properties, it has become increasingly clear that experiments in neurons are required to define the physiological roles of Kv channel accessory and associated proteins. A number of technological and experimental hurdles remain that must be overcome in the design, execution and interpretation of experiments aimed at detailing the functional roles of accessory subunits and associated proteins in the generation of native neuronal Kv channels. With the increasing association of altered Kv channel functioning with neurological disorders, the potential impact of these efforts is clear.

IA Channels Encoded by Kv1.4 and Kv4.2 Regulate Neuronal Firing in the Suprachiasmatic Nucleus and Circadian Rhythms in Locomotor Activity

Neurons in the suprachiasmatic nucleus (SCN) display coordinated circadian changes in electrical activity that are critical for daily rhythms in physiology, metabolism, and behavior. SCN neurons depolarize spontaneously and fire repetitively during the day and hyperpolarize, drastically reducing firing rates, at night. To explore the hypothesis that rapidly activating and inactivating A-type (IA) voltage-gated K+ (Kv) channels, which are also active at subthreshold membrane potentials, are critical regulators of the excitability of SCN neurons, we examined locomotor activity and SCN firing in mice lacking Kv1.4 (Kv1.4−/−), Kv4.2 (Kv4.2−/−), or Kv4.3 (Kv4.3−/−), the pore-forming (α) subunits of IA channels. Mice lacking either Kv1.4 or Kv4.2 α subunits have markedly shorter (0.5 h) periods of locomotor activity than wild-type (WT) mice. In vitro extracellular multi-electrode recordings revealed that Kv1.4−/− and Kv4.2−/− SCN neurons display circadian rhythms in repetitive firing, but with shorter periods (0.5 h) than WT cells. In contrast, the periods of wheel-running activity in Kv4.3−/− mice and firing in Kv4.3−/− SCN neurons were indistinguishable from WT animals and neurons. Quantitative real-time PCR revealed that the transcripts encoding all three Kv channel α subunits, Kv1.4, Kv4.2, and Kv4.3, are expressed constitutively throughout the day and night in the SCN. Together, these results demonstrate that Kv1.4- and Kv4.2-encoded IA channels regulate the intrinsic excitability of SCN neurons during the day and night and determine the period and amplitude of circadian rhythms in SCN neuron firing and locomotor behavior.

K+ Channel Interacting Proteins 2, 3 and 4 are Critical Components of Kv4 Channel Complexes in Cortical Pyramidal Neurons

The rapidly activating and inactivating voltage-gated K+ (Kv) current, IA, is critical for many neuronal functions, including repetitive firing and synaptic integration. Previous studies revealed that in cortical pyramidal neurons the majority of IA is encoded by Kv4.2 and Kv4.3 α-subunits. Little, however, is known about the functional roles of K+ Channel Interacting Proteins (KChIP) 1, 2, 3, and 4 in the generation of IA. Biochemical experiments revealed that KChIPs 2, 3 and 4 (2-4) co-immunoprecipitate with Kv4.2 in samples from mouse cortex suggesting roles for these three KChIPs in the generation of functional Kv4-encoded channels in cortical pyramidal neurons. Electrophysiological experiments conducted on cortical pyramidal neurons from mice (KChIP3-/-) harboring a targeted disruption of the KChIP3 locus revealed that IA densities and properties were similar to wild type neurons. Interestingly, in cortical samples from KChIP3-/- mice the protein levels of KChIP 2 and 4 were increased suggesting functional compensation for the loss of KChIP3. Similarly, in KChIP2-/- cortices KChIP3 and 4 protein levels were increased relative to wild type. Concurrently knocking down the expression of KChIPs 2-4 using RNAi constructs targeting each of the three KChIPs induced a reduction in IA density consistent with roles for KChIPs 2-4 in the generation of native Kv4-encoded IA channels. In cortical samples from Kv4.2-/- and Kv4.3-/- mice, the protein expression levels of KChIPs 2-4 were decreased. Additionally, in samples from mice lacking both Kv4.2 and Kv4.3 KChIP2-4 proteins were barely detectable. Taken together these results demonstrate that KChIPs 2-4 associate with Kv4.2 and Kv4.3 in cortical neurons, this association stabilizes KChIP proteins and, in addition, that KChIPs 2-4 are critical components of native Kv4 channels in cortical pyramidal neurons.