K_V4 Channels Link Cognitive Decline and Cardiac Dysfunction During Aging

Voltage-dependent potassium channels (K_V4) are expressed in the brain and the heart where they regulate cognitive function and cardiac output. Aging is associated with increased tendency for cardiovascular disease and cognitive decline but the link between those debilitating diseases is missing. Here we show that K_V4 channels could possibly tie cardiovascular diseases and neuronal dysfunction and demonstrate a collection of functional tools from Alomone Labs to investigate this possibility.

Introduction

Structure

Potassium channels are a large and diverse protein superfamily that are typically grouped based on their structure and mode of activation²³. The voltage-gated potassium (K_V) channels encompass a large portion of this protein superfamily and are further divided into 12 subfamilies based on sequence homology to their Drosophila orthologs (Shaker, Shab, Shal and Shaw)^18,23. The Shal-type potassium channels also known as K_V4.1, K_V4.2 and K_V4.3 (in mammals) are highly expressed in the brain and the heart where they regulate numerous physiological functions including neuronal excitability and cardiac pacemaking respectively². In the context of aging and its related diseases this article will mainly focus on these types of potassium channels.

The Shal-type potassium channels, share a prototypical structure with other potassium channels such as the cytoplasmic carboxy terminal, T1 assembly domain, a six α-helical transmembrane domain (S1-S6) and the pore loop (P-Loop), which is selective for potassium ions². The helical S4 segment is unique among other S subunits as it is considered to be the voltage sensor due to dense clusters of positively charged arginine and lysine residues². Upon membrane depolarization, the S4 domain mediates protein conformational change and opens the channel for potassium ion flux¹⁸.

The regulation over K_V channel activity is a complex and multi-step process that depends on many factors including protein-protein interactions, cell signaling events, transcriptional and post translational activities as will be demonstrated in detail below.

Transcriptional regulation

K_V4 expression is influenced by numerous factors including changes in transcriptional activity. Argenziano et al. found that testosterone controls K_V4.3 expression in the heart¹. Using Anti-K_V4.3 Antibody (#APC-017), the authors showed that Finasteride and Flutamide (drugs preventing androgen signaling through different mechanisms) decrease K_V4.3 expression in the right ventricle of rat heart, suggesting a role for testosterone and androgen signaling in potassium channel expression¹.

Micro RNA (miRNA) miR-223-3p is highly expressed/upregulated in a rat model of acute myocardial ischemia (AMI)¹². Its expression was shown to be inversely proportional to that of K_V4.2 as determined by western blot analysis using Anti-K_V4.2 Antibody (#APC-023). The possibility that miR-223-3p negatively regulates K_V4.2 expression was tested and validated by expressing the miRNA in primary neonatal rat ventricular myocytes. Indeed, K_V4.2 suppression by miR-223-3p was abolished by the administration of Antagomir to silence miR-223-3p12. The effect of miR-223-3p seemed to be specific to K_V4.2 as protein levels of other K_V channels, namely K_V4.3, were not altered by the miRNA¹².

In the nervous system cleaved GLP-1 peptide stimulates long term potentiation (LTP) signal in hippocampal nerves⁵. Western blot analysis of hippocampal lysates using Anti-K_V4.2 Antibody showed decreased expression of the protein following chronic administration of GLP-1 (9-36) in mice which was associated with increased LTP signals⁵.

Subcellular Trafficking and Localization

The spatial distribution of potassium channels is important for proper cellular functions¹⁰. This is especially true for cells with complex structures such as neurons¹⁰. The possible role of TRPC6 channel in regulating K_V4 channel distribution was recently addressed. TRPC6 knockdown resulted in a decrease of cell membrane associated K_V4.3 and an increase in cytosolic abundance of the protein9 as evident by western blot of cytosolic/membrane fractions of hippocampal tissue homogenates using Anti-K_V4.3 Antibody⁹. In addition, immunohistochemical staining of rat brain sections using Anti-K_V4.3 Antibody showed that rats administered with TRPC6 siRNA displayed reduced K_V4.3 clusters in dentate gyrus cells and parvalbumin (PV) positive GABAergic interneurons (Figure 1)⁹, supporting the possible role of TRPC6 in regulating K_V4.3 subcellular distribution.

A novel interaction between the glycoprotein Nectin-2α and K_V4.2 was shown to tune K_V4.2 localization to specialized plasma membrane regions of adjacent somata of cholinergic neurons. This interaction was demonstrated by high resolution electron microscopy using Anti-K_V4.2 Antibody¹⁶. Furthermore, genetic ablation of Nectin-2α in mice reduced K_V4.2 fluorescent signal at the apical membrane of cholinergic neurons, confirming that Nectin-2α-K_V4.2 interaction is important for K_V4.2 localization.

K_V4 Regulation by Post-Translational Modifications

Glycosylation is a common post translational modification important for numerous biological processes including protein folding in the endoplasmic reticulum (ER) and protein trafficking to the plasma membrane². Recently, Endie et al., investigated the effects of syalic acid (a negatively charged sugar) modifications on K_V channels in mouse ventricular myocytes⁶. Using transgenic mice lacking the sialyltransferase ST3Gal4, the authors demonstrated a delayed potassium outward (I_to) current that consequently delayed ventricular repolarization period as evident by prolonged QT intervals⁶. To investigate potential mechanisms, the authors used Anti-K_V4.2 Antibody and Anti-K_V1.5 (KCNA5) Antibody (#APC-004) to compare protein expression by western blot. Whereas, no change in protein expression was evident, the authors suggest that sialic acid modification of K_V channels is a key step in finetuning K_V channels activity⁶.

K_V4 Interacting Proteins

The diversity in K_V channel activity can be influenced by protein-protein interactions². For example, Turnow et al., demonstrate that K_V4.3- DPP10a interaction regulates transient potassium currents (I_to)²¹. Using immunofluorescence, they provide evidence for K_V4.3-DPP10a co-localization using Anti-K_V4.3 Antibody in human atrial myocytes and Chinese Hamster Ovaries (CHO)²¹. Furthermore, using functional assays they demonstrate this interaction is physiologically relevant since potassium currents were not visualized in the absence of DPP10a in CHO²¹.

Similarly, Wang et al., demonstrated the interaction between K_V4.2-KChIP3- DPP10a in rat neocortical brain sections and olfactory bulbs using Anti- K_V4.2 Antibody and suggest this protein complex mediates sub-threshold potassium currents in vivo²².

K_V4 Channels in Cardiovascular Diseases

Potassium channels regulate the electrical driving force for normal cardiac function⁷. In particular, they act to restore membrane polarization and counterbalance depolarizing ions such as sodium and calcium⁷.

Impaired cardiac electrical activity is frequently observed following myocardial hypertrophy, which is an adaptive response to cardiac dysfunction. M3 muscarinic receptors are members of cholinergic receptors that innervate cardiac cells and control their electrical function. Chen et al., examined whether M3 over-expression could alleviate the adverse electrical signal after cardiac hypertrophy³. To test this hypothesis the authors generated transgenic mice, over-expressing M3 muscarinic receptors. Using Anti-M3 Muscarinic Receptor Antibody (#AMR-006), the authors confirmed the over-expression of M3 receptors by western blot and observed that that cardiac electrical activity was comparable to sham controls following transverse aortic constriction model³. To identify a potential mechanism by which M3 over-expression restores cardiac function, the authors examined the expression of various channels, which control potassium outflow and hence could potentially re-establish normal cardiac pace³. They found that M3 muscarinic receptors elevated K_ir2.1 expression but no change was observed in K_V4.3, as determined using Anti-K_V4.3 Antibody³.

Recently, a genetic survey in a small cohort of Chinese population identified a missense mutation within K_V4.3 gene (KCND3) associated with atrial fibrillation (AF)⁸. This mutation leads to Thr 361 to Ser (Thr→Ser) replacement in K_V4.3 protein. To delve deeper into the mechanisms leading to atrial fibrillation, Huang et al., expressed wild type K_V4.3 or its mutated counterpart, together with KChIp2 in HEK293T cells. Using this system, they discovered that Thr→Ser mutation increases total K_V4.3 expression and increases its membrane localization, as determined by western blot with Anti-K_V4.3 Antibody (Figure 2). In addition, the authors measured potassium currents and discovered that Thr→Ser is associated with K_V4.3 gain of function⁸.

In contrast, Cheng et al., demonstrated that K_V4.3 over-expression in cardiac myocytes protected mice from heart failure⁴. Specifically, using Anti-K_V4.3 Antibody, the authors showed the association between elevated K_V4.3 expression and decreased phosphorylation of calmodulin-dependent protein kinase (CaMKII) and suggested this protective effect to be due to calcium homeostasis⁴.

Recent studies have uncovered novel mutations within K_V interacting proteins that can have debilitating effects on cardiac function such as the Brugada syndrome, which causes irregular heart beats⁷. Accordingly, Portero et al., identified a mutation in K_Vβ2 associated with the syndrome¹⁴. Using an in vitro expression system, the authors were able to confirm that Arg to Gln replacement does not affect K_V4.3 expression as seen by western blot using Alomone Labs’ respective antibody, but rather affects cardiac electrophysiology in a manner that has yet to be uncovered.

Similarly, Tsai et al., identified KChIP1 copy number variants to be a strong genetic predictor for AF in Taiwanese population²⁰. To investigate how KChIP1 is involved in AF, the authors undertook protein-protein interaction studies in adult rat hearts using Anti-KChIP1 (KCNIP1) Antibody (#APC-141) in pull-down assays and re-probing with antibodies against the potassium channels K_V4.2 and K_V4.3 or the calcium channel with Anti-Ca_V1.2 (CACNA1C) Antibody (#ACC-003)²⁰. Unexpectedly, none of these proteins were detected, indicating a different mechanism by which KChIP1 regulates cardiac performance. The authors tried a genetic approach in which they silenced KChIP1 in atrial cell line, HL-1 and found a significant change in potassium currents and membrane depolarization, suggesting that KChIP1 itself regulates potassium outward currents²⁰.

K_V4 Channels in Neuronal Dysfunction

Cognitive decline due to aging is associated with CA1 and CA3 pyramidal neuron malfunction in the hippocampus¹⁷. Simkin et al., speculated that the increased firing in CA3 pyramidal neurons contributing to cognitive decline in aged brains may be caused by K_V channels¹⁷. Administration of Phrixotoxin-1 (#STP-700), a selective and potent blocker of K_V4.2 and K_V4.3 channels significantly improved the electrophysiological activity of CA3 neurons in old rats (Figure 3). Additional results indicated that K_V4.2 inhibition could possibly slow down neurodegenerative processes during aging¹⁷.

Parkinson’s Disease is another example of progressive neurodegenerative process where α-synuclein accumulation can harm vulnerable neurons such as the substantia nigra (SN) dopaminergic neurons¹⁹. In genetically engineered mice, bearing A53T mutation in α-synuclein protein, selective high firing rates in SN dopaminergic neurons (DA) were observed¹⁹. Comparable with the in vivo model, isolated neurons from mutated α-synuclein DA neurons displayed increased firing rates in a pattern that suggested a shift in pacemaker currents. Voltage-gated K_V4 channels were previously shown to control dopaminergic neuron pacemaking. Application of Phrixotoxin-2 (#STP-710), a specific K_V4 channel blocker completely hindered the difference in electrophysiological recordings between the control group and α-synuclein mutant neurons (Figure 4A)¹⁹. In addition, immunohistochemical staining of mouse brain sections using Anti-K_V4.3 Antibody showed that K_V4.3 expression is higher in substantia nigra of mice bearing the mutation in α-synuclein (Figure 4B). This suggests that altered K_V4 activity or expression underlies this phenotypic difference¹⁹.

Different cell types respond differently to elevated α-synuclein levels¹¹. For example, the dorsal motor nucleus of the vagus (DMV) nerve can tolerate high levels of α-synuclein levels and display milder apoptosis compared to the SN dopaminergic neurons¹¹. Furthermore, α-synuclein accumulation alters electrical activity in SN dopaminergic neurons, but has no evident effect on DMV neurons¹¹. In accordance, immunohistochemical staining of mouse brain sections bearing the A53T mutation in α-synuclein protein shows similar K_V4.3 levels and expression pattern when compared to wildtype¹¹.

Pharmacological Approaches to Studying K_V4 Channels

Over the past decade, the list of small molecule/peptide inhibitors that target K_V4 channels has grown substantially²³. These tools, can now be exploited to discover novel functions and biological processes in which K_V4 channels are involved. For example, Phrixotoxin-1 and AmmTx3 Toxin (#STA- 305) peptide toxin blockers for K_V4.3 and K_V4.2 were used to study how toxin transient potassium currents affect preBotzeiger type-1 neurons rhythmicity and their impact on breathing¹³.

In a similar way Heteropodatoxin-2 (#STH-340), a specific K_V4.2 blocker was used to uncover a novel mechanism of synaptic plasticity in tuft dendrites of layer 5 pyramidal neurons¹⁵ where low frequency electrical stimulation in tuft dendrites induced long term potentiation activity.

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