Channel Surface Expression Research Articles

EP News: Basic and Translational

Electrocardiographic changes and arrhythmias in Brugada syndrome (BrS) remain controversial. Mutations in the Na channel gene SCN5A and its regulatory proteins have been linked to BrS. Aiba et al (Circ Cardiovasc Genet 2014;7:249, PMID 24795344) studied the properties of a BrS SCN5A mutation of a protein kinase A (PKA) consensus phosphorylation site, R526H. In vitro PKA phosphorylation was detected in the I-II linker of wild-type (WT) Na channels, but not in R526H or S528A mutants. Surface expression and peak Na current (INa) densities of R526H and S528A channels were reduced as compared with WT channels. Infection of rat cultures with WT and mutant channels increased conduction velocity compared with controls, but PKA stimulation significantly increased peak INa and conduction velocity only in WT cultures. With the intracellular application of NADH, both WT and mutant INa decrease, but this effect is reversed by PKA stimulation in WT channels but not in mutant channels. The authors conclude that the BrS mutation R526H is associated with a reduction in basal INa and a failure of PKA stimulation to augment the current, thus predisposing to arrhythmias in patients with BrS.

Polarized axonal surface expression of neuronal KCNQ potassium channels is regulated by calmodulin interaction with KCNQ2 subunit.

KCNQ potassium channels composed of KCNQ2 and KCNQ3 subunits give rise to the M-current, a slow-activating and non-inactivating voltage-dependent potassium current that limits repetitive firing of action potentials. KCNQ channels are enriched at the surface of axons and axonal initial segments, the sites for action potential generation and modulation. Their enrichment at the axonal surface is impaired by mutations in KCNQ2 carboxy-terminal tail that cause benign familial neonatal convulsion and myokymia, suggesting that their correct surface distribution and density at the axon is crucial for control of neuronal excitability. However, the molecular mechanisms responsible for regulating enrichment of KCNQ channels at the neuronal axon remain elusive. Here, we show that enrichment of KCNQ channels at the axonal surface of dissociated rat hippocampal cultured neurons is regulated by ubiquitous calcium sensor calmodulin. Using immunocytochemistry and the cluster of differentiation 4 (CD4) membrane protein as a trafficking reporter, we demonstrate that fusion of KCNQ2 carboxy-terminal tail is sufficient to target CD4 protein to the axonal surface whereas inhibition of calmodulin binding to KCNQ2 abolishes axonal surface expression of CD4 fusion proteins by retaining them in the endoplasmic reticulum. Disruption of calmodulin binding to KCNQ2 also impairs enrichment of heteromeric KCNQ2/KCNQ3 channels at the axonal surface by blocking their trafficking from the endoplasmic reticulum to the axon. Consistently, hippocampal neuronal excitability is dampened by transient expression of wild-type KCNQ2 but not mutant KCNQ2 deficient in calmodulin binding. Furthermore, coexpression of mutant calmodulin, which can interact with KCNQ2/KCNQ3 channels but not calcium, reduces but does not abolish their enrichment at the axonal surface, suggesting that apo calmodulin but not calcium-bound calmodulin is necessary for their preferential targeting to the axonal surface. These findings collectively reveal calmodulin as a critical player that modulates trafficking and enrichment of KCNQ channels at the neuronal axon.

Depletion of 14-3-3γ reduces the surface expression of Transient Receptor Potential Melastatin 4b (TRPM4b) channels and attenuates TRPM4b-mediated glutamate-induced neuronal cell death.

BackgroundTRPM4 channels are Ca2+-activated nonselective cation channels which are deeply involved in physiological and pathological conditions. However, their trafficking mechanism and binding partners are still elusive.ResultsWe have found the 14-3-3γ as a binding partner for TRPM4b using its N-terminal fragment from the yeast-two hybrid screening. Ser88 at the N-terminus of TRPM4b is critical for 14-3-3γ binding by showing GST pull-down and co-immunoprecipitation. Heterologous overexpression of 14-3-3γ in HEK293T cells increased TRPM4b expression on the plasma membrane which was measured by whole-cell recordings and cell surface biotinylation experiment. Surface expression of TRPM4b was greatly reduced by short hairpin RNA (shRNA) against 14-3-3γ. Next, endogenous TRPM4b-mediated currents were electrophysiologically characterized by application of glutamate and 9-phenanthrol, a TRPM4b specific antagonist in HT-22 cells which originated from mouse hippocampal neurons. Glutamate-induced TRPM4b currents were significantly attenuated by shRNAs against 14-3-3γ or TRPM4b in these cells. Finally, glutamate-induced cell death was greatly prevented by treatment of 9-phenanthrol or 14-3-3γ shRNA.ConclusionThese results showed that the cell surface expression of TRPM4 channels is mediated by 14-3-3γ binding, and the specific inhibition of this trafficking process can be a potential therapeutic target for glutamate-induced neuronal cell death.

Chapter 3: Molecular basis for the therapeutic effectiveness of botulinum neurotoxin type A.

The utility of botulinum neurotoxin type A (BoNT/A) for treating overactive muscles and endocrine glands is attributable to a unique conflation of properties honed to exploit and inactivate synaptic transmission. Specific, high-affinity coincident binding to gangliosides plus an intraluminal loop of synaptic vesicle protein 2 (SV2) by the heavy chain (HC) of BoNT/A confers selectivity for presynaptic nerve terminals and subsequent uptake by endocytosis. Upon vesicle acidification, the HC forms a channel for transmembrane transfer of the light chain to the cytosol, as observed by single channel recordings. The light chain is a Zn(2+) -dependent endoprotease that cleaves and inactivates SNAP-25, thereby blocking exocytotic release of transmitters, a discovery that revealed the pivotal role of the latter in synaptic vesicle fusion. A di-leucine motif in BoNT/A light chain stabilizes this protease, contributing to its longevity inside nerves. The ubiquity of SV2 and SNAP-25 has prompted re-evaluation of the nerve types susceptible to BoNT/A. In urology, there is emerging evidence that BoNT/A blocks neuropeptide release from afferent nerves, exocytosis of acetylcholine and purines from efferent nerves, and possibly ATP release from the urothelium. Suppression by BoNT/A of the surface expression of nociceptor channels on bladder afferents might also contribute to its improvement of urological sensory symptoms.

Brain-derived Neurotrophic Factor (BDNF) Induces Sustained Intracellular Ca2+ Elevation through the Up-regulation of Surface Transient Receptor Potential 3 (TRPC3) Channels in Rodent Microglia

Microglia are immune cells that release factors, including proinflammatory cytokines, nitric oxide (NO), and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) is important for microglial functions such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we sought to examine the underlying mechanism of BDNF-induced sustained increase in [Ca(2+)]i in rodent microglial cells. We observed that canonical transient receptor potential 3 (TRPC3) channels contribute to the maintenance of BDNF-induced sustained intracellular Ca(2+) elevation. Immunocytochemical technique and flow cytometry also revealed that BDNF rapidly up-regulated the surface expression of TRPC3 channels in rodent microglial cells. In addition, pretreatment with BDNF suppressed the production of NO induced by tumor necrosis factor α (TNFα), which was prevented by co-adiministration of a selective TRPC3 inhibitor. These suggest that BDNF induces sustained intracellular Ca(2+) elevation through the up-regulation of surface TRPC3 channels and TRPC3 channels could be important for the BDNF-induced suppression of the NO production in activated microglia. We show that TRPC3 channels could also play important roles in microglial functions, which might be important for the regulation of inflammatory responses and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders.

The Cav1.2 N terminus contains a CaM kinase site that modulates channel trafficking and function.

The L-type voltage-gated calcium channel Cav1.2 and the calcium-activated CaM kinase cascade both regulate excitation transcription coupling in the brain. CaM kinase is known to associate with the C terminus of Cav1.2 in a region called the PreIQ-IQ domain, which also binds multiple calmodulin molecules. Here we identify and characterize a second CaMKII binding site in the N terminus of Cav1.2 that is formed by a stretch of four amino residues (cysteine-isoleucine-serine-isoleucine) and which regulates channel expression and function. By using live cell imaging of tsA-201 cells we show that GFP fusion constructs of the CaMKII binding region, termed N2B-II co-localize with mCherry-CaMKII. Mutating CISI to AAAA ablates binding to and colocalization with CaMKII. Cav1.2-AAAA channels show reduced cell surface expression in tsA-201 cells, but interestingly, display an increase in channel function that offsets the trafficking deficit. Altogether our data reveal that the proximal N terminus of Cav1.2 contains a CaMKII binding region which contributes to channel surface expression and function.

Genetically induced dysfunctions of Kir2.1 channels: implications for short QT3 syndrome and autism-epilepsy phenotype.

Short QT3 syndrome (SQT3S) is a cardiac disorder characterized by a high risk of mortality and associated with mutations in Kir2.1 (KCNJ2) channels. The molecular mechanisms leading to channel dysfunction, cardiac rhythm disturbances and neurodevelopmental disorders, potentially associated with SQT3S, remain incompletely understood. Here, we report on monozygotic twins displaying a short QT interval on electrocardiogram recordings and autism–epilepsy phenotype. Genetic screening identified a novel KCNJ2 variant in Kir2.1 that (i) enhanced the channel's surface expression and stability at the plasma membrane, (ii) reduced protein ubiquitylation and degradation, (iii) altered protein compartmentalization in lipid rafts by targeting more channels to cholesterol-poor domains and (iv) reduced interactions with caveolin 2. Importantly, our study reveals novel physiological mechanisms concerning wild-type Kir2.1 channel processing by the cell, such as binding to both caveolin 1 and 2, protein degradation through the ubiquitin–proteasome pathway; in addition, it uncovers a potential multifunctional site that controls Kir2.1 surface expression, protein half-life and partitioning to lipid rafts. The reported mechanisms emerge as crucial also for proper astrocyte function, suggesting the need for a neuropsychiatric evaluation in patients with SQT3S and offering new opportunities for disease management.

A mutation causing Brugada syndrome identifies a mechanism for altered autonomic and oxidant regulation of cardiac sodium currents.

The mechanisms of the electrocardiographic changes and arrhythmias in Brugada syndrome (BrS) remain controversial. Mutations in the sodium channel gene, SCN5A, and regulatory proteins that reduce or eliminate sodium current (INa) have been linked to BrS. We studied the properties of a BrS-associated SCN5A mutation in a protein kinase A (PKA) consensus phosphorylation site, R526H. In vitro PKA phosphorylation was detected in the I-II linker peptide of wild-type (WT) channels but not R526H or S528A (phosphorylation site) mutants. Cell surface expression of R526H and S528A channels was reduced compared with WT. Whole-cell INa through all channel variants revealed no significant differences in the steady-state activation, inactivation, and recovery from inactivation. Peak current densities of the mutants were significantly reduced compared with WT. Infection of 2D cultures of neonatal rat ventricular myocytes with WT and mutant channels increased conduction velocity compared with noninfected cells. PKA stimulation significantly increased peak INa and conduction velocity of WT but not mutant channels. Oxidant stress inhibits cardiac INa; WT and mutant INa decreases with the intracellular application of reduced nicotinamide adenine dinucleotide (NADH), an effect that is reversed by PKA stimulation in WT but not in R526H or S528A channels. We identified a family with BrS and an SCN5A mutation in a PKA consensus phosphorylation site. The BrS mutation R526H is associated with a reduction in the basal level of INa and a failure of PKA stimulation to augment the current that may contribute to the predisposition to arrhythmias in patients with BrS, independent of the precipitants.

Sorting nexin 27 regulation of G protein-gated inwardly rectifying K⁺ channels attenuates in vivo cocaine response.

The subcellular pathways that regulate G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels are important for controlling the excitability of neurons. Sorting nexin 27 (SNX27) is a PDZ-containing protein known to bind GIRK2c/GIRK3 channels, but its function in vivo is poorly understood. Here, we investigated the role of SNX27 in regulating GIRK currents in dopamine (DA) neurons of the ventral tegmental area (VTA). Mice lacking SNX27 in DA neurons exhibited reduced GABABR-activated GIRK currents but had normal Ih currents and DA D2R-activated GIRK currents. Expression of GIRK2a, an SNX27-insensitive splice variant, restored GABABR-activated GIRK currents in SNX27-deficient DA neurons. Remarkably, mice with significantly reduced GABABR-activated GIRK currents in only DA neurons were hypersensitive to cocaine and could be restored to a normal locomotor response with GIRK2a expression. These results identify a pathway for regulating excitability of VTA DA neurons, highlighting SNX27 as a promising target for treating addiction.

A590T mutation in KCNQ1 C-terminal helix D decreases IKs channel trafficking and function but not Yotiao interaction

KCNQ1 encodes the α subunit of the voltage-gated channel that mediates the cardiac slow delayed rectifier K+ current (IKs). Here, we report a KCNQ1 allele encoding an A590T mutation [KCNQ1(A590T)] found in a 39-year-old female with a mild QT prolongation. A590 is located in the C-terminal α helical region of KCNQ1 that mediates subunit tetramerization, membrane trafficking, and interaction with Yotiao. This interaction is known to be required for the proper modulation of IKs by cAMP. Since previous studies reported that mutations in the vicinity of A590 impair IKs channel surface expression and function, we examined whether and how the A590T mutation affects the IKs channel. Electrophysiological measurements in HEK-293T cells showed that the A590T mutation caused a reduction in IKs density and a right-shift of the current–voltage relation of channel activation. Immunocytochemical and immunoblot analyses showed the reduced cell surface expression of KCNQ1(A590T) subunit and its rescue by coexpression of the wild-type KCNQ1 [KCNQ1(WT)] subunit. Moreover, KCNQ1(A590T) subunit interacted with Yotiao and had a cAMP-responsiveness comparable to that of KCNQ1(WT) subunit. These findings indicate that the A590 of KCNQ1 subunit plays important roles in the maintenance of channel surface expression and function via a novel mechanism independent of interaction with Yotiao.

Selective inhibition of meningeal nociceptors by botulinum neurotoxin type A: Therapeutic implications for migraine and other pains

BackgroundMeningeal and other trigeminal nociceptors are thought to play important roles in the initiation of migraine headache. Currently, the only approved peripherally administered chronic migraine prophylactic drug is onabotulinumtoxinA. The purpose of this study was to determine how botulinum neurotoxin type A (BoNT-A) affects naïve and sensitized meningeal nociceptors.Material and methodsUsing electrophysiological techniques, we identified 43 C- and 36 Aδ-meningeal nociceptors, and measured their spontaneous and evoked firing before and after BoNT-A administration to intracranial dura and extracranial suture-receptive fields.ResultsAs a rule, BoNT-A inhibited C- but not Aδ-meningeal nociceptors. When applied to nonsensitized C-units, BoNT-A inhibited responses to mechanical stimulation of the dura with suprathreshold forces. When applied to sensitized units, BoNT-A reversed mechanical hypersensitivity. When applied before sensitization, BoNT-A prevented development of mechanical hypersensitivity. When applied extracranially to suture branches of intracranial meningeal nociceptors, BoNT-A inhibited the mechanical responsiveness of the suture branch but not dural axon. In contrast, BoNT-A did not inhibit C-unit responses to mechanical stimulation of the dura with threshold forces, or their spontaneous activity.DiscussionThe study provides evidence for the ability of BoNT-A to inhibit mechanical nociception in peripheral trigeminovascular neurons. These findings suggest that BoNT-A interferes with neuronal surface expression of high-threshold mechanosensitive ion channels linked preferentially to mechanical pain by preventing their fusion into the nerve terminal membrane.

Intravascular pressure stimulates functional KV1.5 surface expression in mesenteric artery smooth muscle cells (1077.4)

Intravascular pressure stimulates arterial depolarization, leading to myocyte voltage‐dependent K+ (KV) channel activation that opposes vasoconstriction. We tested the hypothesis that membrane potential regulates smooth muscle cell KV channel surface expression to modulate Kv channel functionality in mesenteric arteries. Q‐PCR indicated predominant expression of KV1.5 and 2.1 in mesenteric arteries. Contrary to expectations, 70% of total and surface KV1.5 protein was lost within 3 hrs in non‐pressurized arteries. In contrast, KV1.5 or 2.1 mRNA and total and surface KV2.1 protein did not change over the same time course. Bafilomycin, a lysosome inhibitor, and MG‐132, a proteasome inhibitor, both prevented KV1.5 loss. Depolarization (30 mM K+) also inhibited KV1.5 loss and this was blocked by nimodipine, a voltage‐dependent Ca2+ channel blocker. Patch‐clamp studies indicated that KV currents sensitive to DPO‐1, a Kv1.5 inhibitor, were 60% smaller in myocytes isolated from arteries kept at zero pressure than controls. Kv current loss was also blocked by bafilomycin. We also show that physiological intravascular pressure maintains functional Kv1 channel activity in arteries. In summary, data indicate that intravascular pressure inhibits KV1.5 channel degradation to maintain functional surface expression of these proteins in arterial myocytes.Grant Funding Source: NHLBI/NIH

Brugada Syndrome Disease Phenotype Explained in Apparently Benign Sodium Channel Mutations

Brugada syndrome (BrS) is an arrhythmogenic disorder that has been linked to mutations in SCN5A, the gene encoding for the pore-forming α-subunit of the cardiac sodium channel. Typically, BrS mutations in SCN5A result in a reduction of sodium current with some mutations even exhibiting a dominant-negative effect on wild-type (WT) channels, thus leading to an even more prominent decrease in current amplitudes. However, there is also a category of apparently benign (atypical) BrS SCN5A mutations that in vitro demonstrates only minor biophysical defects. It is therefore not clear how these mutations produce a BrS phenotype. We hypothesized that similar to dominant-negative mutations, atypical mutations could lead to a reduction in sodium currents when coexpressed with WT to mimic the heterozygous patient genotype. WT and atypical BrS mutations were coexpressed in Human Embryonic Kidney-293 cells, showing a reduction in sodium current densities similar to typical BrS mutations. Importantly, this reduction in sodium current was also seen when the atypical mutations were expressed in rat or human cardiomyocytes. This decrease in current density was the result of reduced surface expression of both mutant and WT channels. Taken together, we have shown how apparently benign SCN5A BrS mutations can lead to the ECG abnormalities seen in patients with BrS through an induced defect that is only present when the mutations are coexpressed with WT channels. Our work has implications for risk management and stratification for some SCN5A-implicated BrS patients.

Nedd4‐2 regulates surface expression and may affect N ‐glycosylation of hyperpolarization‐activated cyclic nucleotide‐gated (HCN)‐1 channels

HCN channels are important regulators of neuronal excitability. The proper function of these channels is governed by various mechanisms, including post-translational modifications of channel subunits. Here, we provide evidence that ubiquitination via a ubiquitin ligase, neuronal precursor cell expressed developmentally downregulated (Nedd)-4-2, is involved in the regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. We identified a PY motif (L/PPxY), the characteristic binding motif for Nedd4-2 in the C terminus of the HCN1 subunit, and showed that HCN1 and Nedd4-2 interacted both in vivo (rat hippocampus, neocortex, and cerebellum) and in vitro [human embryonic kidney 293 (HEK293) cells], resulting in increased HCN1 ubiquitination. Elimination of the PY motif reduced, but did not abolish, Nedd4-2 binding, which further involved a stretch of ∼100 aa downstream in the HCN1 C terminus. Coexpression of Nedd4-2 and HCN1 drastically reduced the HCN1-mediated h-current amplitude (85-92%) in Xenopus laevis oocytes and reduced surface expression (34%) of HCN1 channels in HEK293 cells, thereby opposing effects of tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b)-(1a-4), an auxiliary subunit that promotes HCN1 surface expression. Regulation may further include N-glycosylation of HCN1 channels, which is significantly enhanced by TRIP8b(1a-4), but may be reduced by Nedd4-2. Taken together, our data indicate that Nedd4-2 plays an important role in the regulation of HCN1 trafficking and may compete with TRIP8b(1a-4) in this process.

Deletion of α-subunit exon 11 of the epithelial Na+ channel reveals a regulatory module

Epithelial Na(+) channel (ENaC) subunits (α, β, and γ) found in functional complexes are translated from mature mRNAs that are similarly processed by the inclusion of 13 canonical exons. We examined whether individual exons 3-12, encoding the large extracellular domain, are required for functional channel expression. Human ENaCs with an in-frame deletion of a single α-subunit exon were expressed in Xenopus oocytes, and their functional properties were examined by two-electrode voltage clamp. With the exception of exon 11, deletion of an individual exon eliminated channel activity. Channels lacking α-subunit exon 11 were hyperactive. Oocytes expressing this mutant exhibited fourfold greater amiloride-sensitive whole cell currents than cells expressing wild-type channels. A parallel fivefold increase in channel open probability was observed with channels lacking α-subunit exon 11. These mutant channels also exhibited a lost of Na(+) self-inhibition, whereas we found similar levels of surface expression of mutant and wild-type channels. In contrast, in-frame deletions of exon 11 from either the β- or γ-subunit led to a significant loss of channel activity, in association with a marked decrease in surface expression. Our results suggest that exon 11 within the three human ENaC genes encodes structurally homologous yet functionally diverse domains and that exon 11 in the α-subunit encodes a module that regulates channel gating.

Is There a Contribution of Both Cav1.4 and Cav1.3 L-Type Calcium Channels to Retinal Synaptic Transmission?

Cav1.3 and Cav1.4 L-type calcium channels were previously both shown to be expressed in the retina. Whereas Cav1.4 channels are predominantly expressed in the outer plexiform layer (OPL) at photoreceptor ribbon synapses, reports on the distribution pattern of Cav1.3 channels in the retina are controversial. One study reported the uniform expression across all retinal cell layers, and others showed accumulation in photoreceptor inner segments or the OPL or the inner nuclear layer or the ganglion cell (GC) layer of the retina. Mutations in the pore-forming α1-subunit, found in patients diagnosed with Congenital Stationary Night Blindness type 2 (CSNB2), result in impaired signaling between photoreceptor cells and second-order neurons. Exemplary, we report the functional consequences of the novel CaV1.4 mutation GV found in an Austrian family. Biophysical analysis of GV channels in whole-cell patch-clamp experiments revealed a reduced current density ([pA/pF]: wt: 12.8±1.4, n=18; GV: 3.7±1.0, n=7; p<0.001, 15 mM Ca2+ used as charge carrier) due to decreased surface expression of functional channels, expected to lead to impaired retinal signaling. In contrast, the contribution of Cav1.3 channels to synaptic transmission is rather ambiguous. We therefore performed multi electrode (MEA) analyses of light-dark evoked GC activity in Cav1.3-/- mice retinas. Retinas were excised from adult wildtype (wt) and Cav1.3-/- mice (red-light, carbogen-equilibrated Ames) and mounted ganglion-cell-side-down (via nitrocellulose-menbrane) on a MEA array. Preliminary data showed a prolongation of GC response latencies in Cav1.3-/- compared to wt supporting the idea that Cav1.3 channels also contribute to synaptic transmission. However qRT-PCR analysis of Cav1.4-/- retinas showed a significant upregulation of Cav1.3-mRNA, thus an alternative role for signal transduction might also be suggested. Support: Austrian Science Funds (FWF), P22526:AK; SFB F44 (F44020): JS, AK, (F44060): GJO, Medical University Vienna.

Cholesterol Regulation of Atrial GIRk Channels

In recent years, cholesterol emerged as a major regulator of ion channel function. The most common effect of cholesterol on ion channels is a decrease in channel activity. Here we focus on G-protein gated inwardly rectifying potassium (GIRK or Kir3) channels that play an important role in regulating membrane excitability in cardiac, neuronal and endocrine cells. We have recently shown that unexpectedly cholesterol enrichment up-regulates GIRK activity in atrial myocytes. In accordance, we also observed elevated GIRK currents in cholesterol-enriched Xenopus oocytes expressing the GIRK1/GIRK4 heteromers, the two pore-forming subunits expressed in the heart. In this study, we addressed two questions: (1) is there a correlation between cholesterol and atrial GIRK currents in diet-induced hypercholesterolemia in-vivo and (2) what is the biophysical basis of cholesterol-induced increase in GIRK activity. Our results show that feeding rats high-cholesterol diet for 21-22 weeks resulted in ∼2.5-fold increase in serum LDL levels without any change in HDL and ∼1.8-fold increase in cholesterol level in the atrial tissue. Furthermore, this increase results in up to 3-fold increase in atrial GIRK currents. We also demonstrate here that cholesterol enrichment in-vitro has no effect on the surface expression of the GFP-tagged GIRK channels expressed in Xenopus oocytes, as measured by fluorescent microscopy. This observation was confirmed in HEK293 cells using TIRF microscopy. Most importantly, using planar lipid bilayers we show that cholesterol significantly increases the open probability of the GIRK channels. No change was observed in the unitary conductance. Thus, taken together, our data indicate that up-regulation of GIRK channels by cholesterol is not a result of an increase in their surface expression but is due to the increase in their open probability.

Shaker Kv Channel's Sugar Remotion in Real-Time

Voltage-gated K+ channels (Kv) are tetramers, each subunit containing six transmembrane segments (S1-S6). These segments make two functionally and structurally independent domains: an ion conduction pore (S5,S6), and voltage-sensor domains (S1-S4). Like many transmembrane proteins, Kv channels undergo extensive posttranslational modifications, with a significant percentage of the mature protein mass being composed of glycan moieties. Shaker Kv channels are glycosylated at two asparagine residues (N259 and N263) located in the first extracellular loop. Mutating asparagine residues to glutamine abolishes glycosylation but does not prevent cell surface expression of functional channels. Using the same population of Shaker Kv channels, we studied the function and toxin binding before and after deglycosylation by PNGase F. Binding properties of Hanatoxin, GxTx and Agitoxin remained unaltered by removing glycosylation moieties, suggesting that sugars attached to the channels are not intimately interacting with regions of the voltage sensor domains and the pore where these toxins bind. We also studied ionic and gating currents, before and after deglycosylation with PNGase F. Opening kinetics were substantially slowed down by deglycosylation, as reported previously in many members of the Kv family. Total charge distribution mediated by the voltage sensors of the non-conductive W434F mutant Shaker channels shifted only ∼3-4 mV towards positive potentials after deglycosylation. Overall our data provides direct proof to understand how glycosylation contributes to Kv channel function.

Dipeptidyl peptidase 10 (DPP10(789)): a voltage gated potassium channel associated protein is abnormally expressed in Alzheimer's and other neurodegenerative diseases.

The neuropathological features associated with Alzheimer's disease (AD) include the presence of extracellular amyloid-β peptide-containing plaques and intracellular tau positive neurofibrillary tangles and the loss of synapses and neurons in defined regions of the brain. Dipeptidyl peptidase 10 (DPP10) is a protein that facilitates Kv4 channel surface expression and neuronal excitability. This study aims to explore DPP10789 protein distribution in human brains and its contribution to the neurofibrillary pathology of AD and other tauopathies. Immunohistochemical analysis revealed predominant neuronal staining of DPP10789 in control brains, and the CA1 region of the hippocampus contained strong reactivity in the distal dendrites of the pyramidal cells. In AD brains, robust DPP10789 reactivity was detected in neurofibrillary tangles and plaque-associated dystrophic neurites, most of which colocalized with the doubly phosphorylated Ser-202/Thr-205 tau epitope. DPP10789 positive neurofibrillary tangles and plaque-associated dystrophic neurites also appeared in other neurodegenerative diseases such as frontotemporal lobar degeneration, diffuse Lewy body disease, and progressive supranuclear palsy. Occasional DPP10789 positive neurofibrillary tangles and neurites were seen in some aged control brains. Western blot analysis showed both full length and truncated DPP10789 fragments with the later increasing significantly in AD brains compared to control brains. Our results suggest that DPP10789 is involved in the pathology of AD and other neurodegenerative diseases.

Interactions of KChIP4a and its mutants with Ca2+ or Kv4.3 N-terminus by affinity capillary electrophoresis

The specific binding of auxiliary Kv channel-interacting proteins (KChIPs) to the N terminus of Kv4 pore-forming α-subunits results in modulation of gating properties, surface expression, and subunit assembly of Kv4 channels. However, the interactions between KChIPs and Kv4 remain elusive. Thus, affinity capillary electrophoresis (ACE) was employed to quantitatively evaluate the interactions between KChIPs and Kv4.3 N terminus (KvN) and between KChIP4a/related mutants and Ca2+ for the first time. The mobility ratio, derivatives calculated from the mobility shift method, was used to deduce the binding constants (Kb). As a result, the binding constants for KChIP4a/KvN and KChIP1/KvN complexes were (8.32±1.66)×106Lmol–1 and (5.26±0.71)×106Lmol–1, respectively. In addition, in the presence of calcium (10μmolL–1), the binding constant of KChIP4a/KvN increased to (6.72±1.66)×107Lmol–1. In addition, the binding constant of KChIP4a with Ca2+ was (7.1±1.5)×107Lmol–1. Besides, studies on the effect of truncated mutants revealed that the third EF hand of KChIP4a was related to high-affinity binding with Ca2+, and the integrity of the molecular structure of KChIP4a was important for Ca2+ binding. This method profits from small samples, rapid analysis, and simple operation without being time-consuming.