Rectifier Channels Research Articles

The intrinsic and synaptic responsiveness of a new realistic Purkinje cell model

The latest discoveries on Purkinje cell (PC) physiology suggest that the mechanisms of PCs intrinsic excitability have to be revisited. Starting from available models [1], we have constructed a new PC model in Python-NEURON, which explicitly accounts for the Axon Initial Segment (AIS) [2-4] and a part of the axon including the first node of Ranvier (RVN). The fast Na+ channels are located in AIS, soma with initial dendrite and RVN [4]. The K+ delayed rectifier channels are located only in the soma. The Ca2+ and Ca2+-dependent K+ channels, including SK2, as well as intracellular Ca2+ dynamics have been updated [5]. The new model configuration now generates simple spike (SS) firing reproducing the experimental input-output curve[6]. SSs initiate in AIS and then back-propagate into the soma decaying sharply inside the dendritic tree. Activation of parallel fiber (pf) generates a short burst followed by a pause caused by Stellate cells. Following a complex spike (CS), SS activity is interrupted independently of the inhibitory synaptic input. Interestingly, the model can shift its state from silent to autorhythmic (configuring a bistable behavior) upon transient current injection or activation of CFs. The pf and granule cell ascending axon (aa) synapses have been modeled using a stochastic release mechanism activating AMPA synaptic receptors. The facilitation and depression profiles of pf and aa synapses faithfully reproduce the experimental data. This model provides a valuable tool to further investigate the Purkinje cell function in cerebellar network models.

Principles of Cardiac Electric Propagation and Their Implications for Re-entrant Arrhythmias

The study of clinical electrophysiology essentially comprises examining how electric excitation develops and spreads through the millions of cells that constitute the heart. Given the enormous number of cells in a human heart, there is an extremely large number of possible ways that the heart can behave. We encounter rhythms across the spectrum from the organized and orderly behavior of sinus rhythm through repetitive continuous excitation (via reentry) in structurally defined circuits like atrial flutter and, finally, the complex, dynamic, and disorganized behavior of fibrillation. Despite these myriad possibilities, one can apply a basic understanding of the principles of propagation to predict how cardiac tissue will behave under varied circumstances and in response to various manipulations. In this article, we review the principles of propagation and how these can be used to understand reentry of all degrees of complexity. We use these principles to explain the mechanisms by which antiarrhythmic medications and ablation can terminate and prevent reentry. This article is not intended to be an exhaustive description of the physiology of cardiac propagation, rather, it is meant to capture the essence of propagation with sufficient detail to provide an intuitive feel for the interplay of the physiological features relevant to propagation. The figures and videos used in this article were created using a computational model of cardiac propagation (VisibleEP LLC, Colchester, VT). It is a hybrid between a physics-based and cellular automaton model. The model incorporates the fundamental features of propagation without modeling individual ion channels.1 The model manifests several relevant emergent properties, for example, electrotonic interactions, restitution of action potential duration, and conduction velocity as well as source–sink balance–dependent propagation. ### Cell Excitation A cell becomes excited when the balance of inward and outward currents passes a critical point after which inward currents exceed outward and an action potential ensues. …

IDENTIFICATION OF THE ATP SENSITIVE POTASSIUM CHANNEL ISOLATED FROM MAMMAL AND AVIAN SKELETAL MUSCLE

The ATP sensitive potassium channels are heteromultimers (octameric complexes) of two types of subunits, the inward rectifier potassium channel (Kir6.x) and the sulfonylureas receptor (SUR). It has been shown that mitochondrial ATP sensitive potassium channel (mitoKATP) plays an important role in protecting muscle during fatigue. The molecular composition of the mitoKATP channel has been the focus of several studies and the results have been controversial, mainly in the type of the rectifier channel, so the goal this study was to identify by Western blot the structural composition of mitoKATP channels from both mammalian and avian skeletal muscle. A previous result shows the presence of the subunit Kir 6.1 and Kir 6.2 in both types of tissues. However more experiments are needed to determine what subunit is more abundant.

Incretin-stimulated interaction between β-cell Kv1.5 and Kvβ2 channel proteins involves acetylation/deacetylation by CBP/SirT1

The incretins, GIP (glucose-dependent insulinotropic polypeptide) and GLP-1 (glucagon-like peptide-1) are gastrointestinal hormones conferring a number of beneficial effects on β-cell secretion, survival and proliferation. In a previous study, it was demonstrated that delayed rectifier channel protein Kv2.1 contributes to β-cell apoptosis and that the prosurvival effects of incretins involve Kv2.1 PTMs (post-translational modifications), including phosphorylation and acetylation. Since Kv1.5 overexpression was also shown to stimulate β-cell death, the present study was initiated in order to determine whether incretins modulate Kv1.5α-Kvβ2 interaction via PTM and the mechanisms involved. GIP and GLP-1 reduced apoptosis in INS-1 β-cells (clone 832/13) overexpressing Kv1.5, and RNAi (RNA interference)-mediated knockdown of endogenous Kv1.5 attenuated apoptotic β-cell death. Both GIP and GLP-1 increased phosphorylation and acetylation of Kv1.5 and its Kvβ2 protein subunit, leading to their enhanced interaction. Further studies demonstrated that CBP [CREB (cAMP-response-element-binding protein)-binding protein]/SirT1 mediated acetylation/deacetylation and interaction between Kvβ2 and Kv1.5 in response to GIP or GLP-1. Incretin regulation of β-cell function therefore involves the acetylation of multiple Kvα and Kvβ subunits.

Kir2.4 Surface Expression and Basal Current Are Affected by Heterotrimeric G-Proteins

Kir2.4, a strongly rectifying potassium channel that is localized to neurons and is especially abundant in retina, was fished with yeast two-hybrid screen using a constitutively active Gαo1. Here, we wished to determine whether and how Gαo affects this channel. Using transfected HEK 293 cells and retinal tissue, we showed that Kir2.4 interacts with Gαo, and this interaction is stronger with the GDP-bound form of Gαo. Using two-electrode voltage clamp, we recorded from oocytes that were injected with Kir2.4 mRNA and a combination of G-protein subunit mRNAs. We found that the wild type and the inactive mutant of Gαo reduce the Kir2.4 basal current, whereas the active mutant has little effect. Other pertussis-sensitive Gα subunits also reduce this current, whereas Gαs increases it. Gβγ increases the current, whereas m-phosducin, which binds Gβγ without affecting the state of Gα, reduces it. We then tested the effect of G-protein subunits on the surface expression of the channel fused to cerulean by imaging the plasma membranes of the oocytes. We found that the surface expression is affected, with effects paralleling those seen with the basal current. This suggests that the observed effects on the current are mainly indirect and are due to surface expression. Similar results were obtained in transfected HEK cells. Moreover, we show that in retinal ON bipolar cells lacking Gβ3, localization of Kir2.4 in the dendritic tips is reduced. We conclude that Gβγ targets Kir2.4 to the plasma membrane, and Gαo slows this down by binding Gβγ.

Molecular Mechanisms Underlying the Apoptotic Effect of KCNB1 K+ Channel Oxidation

Potassium (K(+)) channels are targets of reactive oxygen species in the aging nervous system. KCNB1 (formerly Kv2.1), a voltage-gated K(+) channel abundantly expressed in the cortex and hippocampus, is oxidized in the brains of aging mice and of the triple transgenic 3xTg-AD mouse model of Alzheimer's disease. KCNB1 oxidation acts to enhance apoptosis in mammalian cell lines, whereas a KCNB1 variant resistant to oxidative modification, C73A-KCNB1, is cytoprotective. Here we investigated the molecular mechanisms through which oxidized KCNB1 channels promote apoptosis. Biochemical evidence showed that oxidized KCNB1 channels, which form oligomers held together by disulfide bridges involving Cys-73, accumulated in the plasma membrane as a result of defective endocytosis. In contrast, C73A-mutant channels, which do not oligomerize, were normally internalized. KCNB1 channels localize in lipid rafts, and their internalization was dynamin 2-dependent. Accordingly, cholesterol supplementation reduced apoptosis promoted by oxidation of KCNB1. In contrast, cholesterol depletion exacerbated apoptotic death in a KCNB1-independent fashion. Inhibition of raft-associating c-Src tyrosine kinase and downstream JNK kinase by pharmacological and molecular means suppressed the pro-apoptotic effect of KCNB1 oxidation. Together, these data suggest that the accumulation of KCNB1 oligomers in the membrane disrupts planar lipid raft integrity and causes apoptosis via activating the c-Src/JNK signaling pathway.

Functional Assessment of Crystallization-Optimized G Protein-Coupled Receptors using Ion Channel-Coupled Receptors

Ion Channel-Coupled Receptors (ICCRs) are artificial ligand-gated ion channels created by genetic fusion of G protein-coupled receptors (GPCRs) to a K+ inward rectifier channel (Kir6.2) such that the channel is a direct reporter of the receptor conformational changes. This concept has been validated with 4 prototypical GPCRs: the M2 muscarinic, the D2L dopaminergic, the β2 adrenergic and the opsin receptors (Moreau et al., Nature Nanotech 3:620 2008, Caro et al. PLoS ONE 6:e18226 2011, Caro et al. PLoS ONE 7:e43766 2012). Voltage-clamp recordings showed that ICCRs detect the agonist- and antagonist-bound states of the receptor via direct physical coupling. This GPCR-channel communication proceeds without any involvement of G proteins and the electrical signal amplitude is correlated with the ligand concentration.The intrinsic instability of the GPCRs has proved a challenge to crystallographic studies. A successful approach, introduced in 2007 by Cherezov et al (Science. 318:1258) and subsequently applied to obtain 12 GPCR structures, consists in the insertion of the T4 phage lysozyme domain in the 3rd intracellular loop of the receptors. However, this modification abolishes G protein binding and prohibits related functional assays. Current characterization of crystallization-optimized GPCR(T4L) is performed by radiolabeled ligand assays or localized FRET techniques. Requiring no biochemical steps, ICCRs are an alternative tool to functionally characterize modified GPCRs that are unable to bind or activate G proteins and not amenable to most GPCR functional assays. We demonstrate here the validity of this tool with 3 different GPCRs (M2-muscarinic, β2-adrenergic and oxytocin receptors). The final application of this study would be the integration of this technology in the current crystallographic platforms dedicated to GPCR structure determination or for structure-function studies independent of G protein interaction.

Dynamic Control of IKs Current Amplitude by the ‘Late-Assembly’ Strategy of Channel Subunit Association

Background: Ion channels composed of α (pore-forming) and β (auxiliary) subunits use an ‘early-assembly' strategy (Kir6.x/SUR assembly in ER) to control cell surface expression, or a ‘late-assembly' strategy (α/β2 of rat brain Nav channels, both subunits independently traffic to cell surface) to allow dynamic control of current amplitude/gating kinetics. Slow delayed rectifier (IKs) channel is composed of KCNQ1 (Q1, α) and KCNE1 (E1, β) subunits, and functions as ‘repolarization-reserve' in human heart. It is not clear which of the 2 assembly strategies Q1 and E1 use in forming IKs. Methods: We express Q1 and E1 tagged with fluorescent-protein (Q1-GFP and E1-dsR) or extracellular epitope (AU5-Q1 and HA-E1) in COS-7 and neonatal rat ventricular myocytes (NRVM), and use confocal imaging to track Q1 & E1 movements in cells. Results: In COS-7 cells 3 hr after transfection, Q1 and E1 travel in separate transport intermediates without mixing/fusion. E1 reaches the cell surface before Q1. By 24 hr, Q1 and E1 are colocalized on cell surface. Brefeldin-A (blocking protein export from ER) prevents surface expression of Q1-147C/E1-40C and reduces disulfide formation between the two (as a measure of functional Q1/E1 assembly). In NRVM during in vitro development, Q1-GFP and E1-dsR travel in distinctly different transport intermediates. As NRVM develops into mature-like phenotype, Q1-GFP and E1-dsR are colocalized on the cell surface, with a separate cytosolic Q1-GFP pool colocalized with α-actinin and calnexin (z-line and ER/SR markers, respectively). Conclusions: Q1 and E1 use the ‘late-assembly' strategy to afford dynamic control of IKs current amplitude. This explains why native Q1 and E1 in adult ventricular myocytes are often not well-colocalized. We propose that cardiac myocytes regulate IKs amplitude dynamically by adjusting the degree of Q1/E1 colocalization on cell surface.

Probing the structural basis for differential KCNQ1 modulation by KCNE1 and KCNE2

KCNE1 associates with KCNQ1 to increase its current amplitude and slow the activation gating process, creating the slow delayed rectifier channel that functions as a “repolarization reserve” in human heart. The transmembrane domain (TMD) of KCNE1 plays a key role in modulating KCNQ1 pore conductance and gating kinetics, and the extracellular juxtamembrane (EJM) region plays a modulatory role by interacting with the extracellular surface of KCNQ1. KCNE2 is also expressed in human heart and can associate with KCNQ1 to suppress its current amplitude and slow the deactivation gating process. KCNE1 and KCNE2 share the transmembrane topology and a high degree of sequence homology in TMD and surrounding regions. The structural basis for their distinctly different effects on KCNQ1 is not clear. To address this question, we apply cysteine (Cys) scanning mutagenesis to TMDs and EJMs of KCNE1 and KCNE2. We analyze the patterns of functional perturbation to identify high impact positions, and probe disulfide formation between engineered Cys side chains on KCNE subunits and native Cys on KCNQ1. We also use methanethiosulfonate reagents to probe the relationship between EJMs of KCNE subunits and KCNQ1. Our data suggest that the TMDs of both KCNE subunits are at about the same location but interact differently with KCNQ1. In particular, the much closer contact of KCNE2 TMD with KCNQ1, relative to that of KCNE1, is expected to impact the allosteric modulation of KCNQ1 pore conductance and may explain their differential effects on the KCNQ1 current amplitude. KCNE1 and KCNE2 also differ in the relationship between their EJMs and KCNQ1. Although the EJM of KCNE1 makes intimate contacts with KCNQ1, there appears to be a crevice between KCNQ1 and KCNE2. This putative crevice may perturb the electrical field around the voltage-sensing domain of KCNQ1, contributing to the differential effects of KCNE2 versus KCNE1 on KCNQ1 gating kinetics.

Abstract 19413: Regulation of I Ks Current Amplitude by Differential Trafficking Patterns and Interactions Of KCNQ1 and KCNE Subunits

KCNQ1 (Q1, pore-forming subunit) and KCNE1 (E1, modulatory subunit) associate to form the slow delayed rectifier (I Ks ) channel that functions as ‘repolarization reserve’ in human heart. We have shown that in adult guinea pig (GP) cardiomyocytes Q1 & E1 cluster to different subcellular compartments, and the degree of Q1/E1 colocalization affects the I Ks amplitude. We have also shown that KCNE2 (E2) is expressed in human heart, and can associate with Q1 or Q1/E1 to suppress the I Ks amplitude. Objectives: To study the patterns of trafficking and interactions of Q1, E1 and E2. Methods: Q1, E1 and E2 are labeled with eGFP (Q1-G) or mono-dsR (Ex-R, x = 1 or 2) and expressed in COS-7 cells, neonatal rat (NRVM) and adult GP (AGPV) ventricular myocytes; the latter 2 by adenoviral (Adv) gene transfer. Protein trafficking/interactions are monitored by live cell imaging, in conjunction with patch clamping, immunocytochemistry & immunoblotting. Results: Time courses of Q1-G & Ex-R distribution in COS-7 cells suggest that they are translated, packaged and shipped to the cell surface independent of each other. They meet on the cell surface and form Q1-G/Ex-R complexes. Q1-G/Ex-R complexes are endocytosed and colocalized in a cytosolic tubulo-vesicular system. We identify 2 main effects of Ex-R on Q1-G trafficking: (1) Ex-R accelerates Q1-G recovery after photobleaching, (2) 24-48 hr after transfection, E2-R brings Q1-G with it to lysosomes for increased degradation. The distribution patterns of Q1-G and Ex-R in NRVM are similar to those in COS-7 cells. However, under the same Adv incubation conditions, Q1-G and Ex-R are expressed at a much lower level in AGPV than NRVM. In AGPV, Q1-G is distributed mainly as cytosolic striations while Ex-R mainly on cell surface, similar to the native Q1 and Ex immunofluorescence patterns seen in control myocytes. Conclusions: Assembly of Q1 & E1 into I Ks does not occur early during biogenesis. Q1 & E1 meet after they reach the plasma membrane and travel together thereafter. E1 increases the mobility of Q1, while E2 increases Q1 degradation in lysosomes. In adult cardiomyocytes the expression levels and subcellular distribution patterns of Q1 & Ex are tightly regulated, with the sarcomere structure playing a key role in Q1 localization.

Family-based genome-wide association study of frontal θ oscillations identifies potassium channel gene KCNJ6.

Event-related oscillations (EROs) represent highly heritable neuroelectric correlates of cognitive processes that manifest deficits in alcoholics and in offspring at high risk to develop alcoholism. Theta ERO to targets in the visual oddball task has been shown to be an endophenotype for alcoholism. A family-based genome-wide association study was performed for the frontal theta ERO phenotype using 634 583 autosomal single nucleotide polymorphisms (SNPs) genotyped in 1560 family members from 117 families densely affected by alcohol use disorders, recruited in the Collaborative Study on the Genetics of Alcoholism. Genome-wide significant association was found with several SNPs on chromosome 21 in KCNJ6 (a potassium inward rectifier channel; KIR3.2/GIRK2), with the most significant SNP at P = 4.7 × 10(-10)). The same SNPs were also associated with EROs from central and parietal electrodes, but with less significance, suggesting that the association is frontally focused. One imputed synonymous SNP in exon four, highly correlated with our top three SNPs, was significantly associated with the frontal theta ERO phenotype. These results suggest KCNJ6 or its product GIRK2 account for some of the variations in frontal theta band oscillations. GIRK2 receptor activation contributes to slow inhibitory postsynaptic potentials that modulate neuronal excitability, and therefore influence neuronal networks.

The function and molecular identity of inward rectifier channels in vestibular hair cells of the mouse inner ear

Inner ear hair cells respond to mechanical stimuli with graded receptor potentials. These graded responses are modulated by a host of voltage-dependent currents that flow across the basolateral membrane. Here, we examine the molecular identity and the function of a class of voltage-dependent ion channels that carries the potassium-selective inward rectifier current known as I(K1). I(K1) has been identified in vestibular hair cells of various species, but its molecular composition and functional contributions remain obscure. We used quantitative RT-PCR to show that the inward rectifier gene, Kir2.1, is highly expressed in mouse utricle between embryonic day 15 and adulthood. We confirmed Kir2.1 protein expression in hair cells by immunolocalization. To examine the molecular composition of I(K1), we recorded voltage-dependent currents from type II hair cells in response to 50-ms steps from -124 to -54 in 10-mV increments. Wild-type cells had rapidly activating inward currents with reversal potentials close to the K(+) equilibrium potential and a whole-cell conductance of 4.8 ± 1.5 nS (n = 46). In utricle hair cells from Kir2.1-deficient (Kir2.1(-/-)) mice, I(K1) was absent at all stages examined. To identify the functional contribution of Kir2.1, we recorded membrane responses in current-clamp mode. Hair cells from Kir2.1(-/-) mice had significantly (P < 0.001) more depolarized resting potentials and larger, slower membrane responses than those of wild-type cells. These data suggest that Kir2.1 is required for I(K1) in type II utricle hair cells and contributes to hyperpolarized resting potentials and fast, small amplitude receptor potentials in response to current inputs, such as those evoked by hair bundle deflections.

Are all spinal segments equal: intrinsic membrane properties of superficial dorsal horn neurons in the developing and mature mouse spinal cord

Neurons in the superficial dorsal horn (SDH; laminae I-II) of the spinal cord process nociceptive information from skin, muscle, joints and viscera. Most of what we know about the intrinsic properties of SDH neurons comes from studies in lumbar segments of the cord even though clinical evidence suggests nociceptive signals from viscera and head and neck tissues are processed differently. This ‘lumbar-centric' view of spinal pain processing mechanisms also applies to developing SDH neurons. Here we ask whether the intrinsic membrane properties of SDH neurons differ across spinal cord segments in both the developing and mature spinal cord. Whole cell recordings were made from SDH neurons in slices of upper cervical (C2-4), thoracic (T8-10) and lumbar (L3-5) segments in neonatal (P0-5) and adult (P24-45) mice. Neuronal input resistance (R(IN)), resting membrane potential, AP amplitude, half-width and AHP amplitude were similar across spinal cord regions in both neonates and adults (∼100 neurons for each region and age). In contrast, these intrinsic membrane properties differed dramatically between neonates and adults. Five types of AP discharge were observed during depolarizing current injection. In neonates, single spiking dominated (∼40%) and the proportions of each discharge category did not differ across spinal regions. In adults, initial bursting dominated in each spinal region, but was significantly more prevalent in rostral segments (49% of neurons in C2-4 vs. 29% in L3-5). During development the dominant AP discharge pattern changed from single spiking to initial bursting. The rapid A-type potassium current (I(Ar)) dominated in neonates and adults, but its prevalence decreased (∼80% vs. ∼50% of neurons) in all regions during development. I(Ar) steady state inactivation and activation also changed in upper cervical and lumbar regions during development. Together, our data show the intrinsic properties of SDH neurons are generally conserved in the three spinal cord regions examined in both neonate and adult mice. We propose the conserved intrinsic membrane properties of SDH neurons along the length of the spinal cord cannot explain the marked differences in pain experienced in the limbs, viscera, and head and neck.

Impairment of neurovascular coupling in type 1 diabetes mellitus in rats is linked to PKC modulation of BKCa and Kir channels

We hypothesized that chronic hyperglycemia has a detrimental effect on neurovascular coupling in the brain and that this may be linked to protein kinase C (PKC)-mediated phosphorylation. Therefore, in a rat model of streptozotocin-induced chronic type 1 diabetes mellitus (T1DM), and in nondiabetic (ND) controls, we monitored pial arteriole diameter changes during sciatic nerve stimulation and topical applications of the large-conductance Ca(2+)-operated K(+) channel (BK(Ca)) opener, NS-1619, or the K(+) inward rectifier (Kir) channel agonist, K(+). In the T1DM vs. ND rats, the dilatory response associated with sciatic nerve stimulation was decreased by ∼30%, whereas pial arteriolar dilations to NS-1619 and K(+) were largely suppressed. These responses were completely restored by the acute topical application of a PKC antagonist, calphostin C. Moreover, the suffusion of a PKC activator, phorbol 12,13-dibutyrate, in ND rats was able to reproduce the vascular reactivity impairments found in T1DM rats. Assay of PKC activity in brain samples from T1DM vs. ND rats revealed a significant gain in activity only in specimens harvested from the pial and superficial glia limitans tissue, but not in bulk cortical gray matter. Altogether, these findings suggest that the T1DM-associated impairment of neurovascular coupling may be mechanistically linked to a readily reversible PKC-mediated depression of BK(Ca) and Kir channel activity.

Predicting the Effects of Nonspecific Channel Blocking Drugs has Variable Success Rates Between Different Ventricular Myocyte Action Potential Models

Pro-arrhythmic changes to ventricular action potential (AP) duration (APD) are one of the most frequent causes for the removal or restriction of marketed drugs and are difficult to predict a priori. A common link between all arrhythmogenic drugs is block of the rapid delayed rectifier channel (a.k.a. the hERG channel), but many hERG-blocking drugs also affect additional ion transport pathways. These nonspecific effects can attenuate or exacerbate a drug's arrhythmogenic risk and greatly alter the drug-induced changes to APD. Using mathematical models of ventricular myocytes, we sought to determine whether the effects of a nonspecific drug could be predicted from the effects of pure channel-blocking drugs. In several models, we simulated a hypothetical specific hERG-blocking drug, drugs that specifically blocked every other ion transport pathway (10-20 in each model), and each combination of block of hERG plus an additional pathway. The response to each hypothetical drug was expressed as a dose response curve, with log([drug]) on the abscissa and log(APD/APDnodrug) on the ordinate. In a popular mathematical of the human ventricular myocyte, we found that the response to a nonspecific drug could be predicted with a high degree of accuracy by summing the dose-response curves of the specific drugs. This indicates that the pathways affected by uncharacterized compounds can be deduced from dose-response data. In a common model of the canine ventricular myocyte, however, summing the specific drugs' dose-response curves generally failed to approximate the dose-response curve of the corresponding nonspecific drug. This result points to important differences between models, and has implications for the prediction of nonspecific effects during the drug development process.

Finding Inward Rectifier Channel Inhibitors: Why and How?

GENERAL COMMENTARY article Front. Pharmacol., 09 January 2012Sec. Pharmacology of Ion Channels and Channelopathies volume 2 - 2011 | https://doi.org/10.3389/fphar.2011.00095

N-Terminal Arginines Modulate Plasma-Membrane Localization of Kv7.1/KCNE1 Channel Complexes

Background and ObjectiveThe slow delayed rectifier current (IKs) is important for cardiac action potential termination. The underlying channel is composed of Kv7.1 α-subunits and KCNE1 β-subunits. While most evidence suggests a role of KCNE1 transmembrane domain and C-terminus for the interaction, the N-terminal KCNE1 polymorphism 38G is associated with reduced IKs and atrial fibrillation (a human arrhythmia). Structure-function relationship of the KCNE1 N-terminus for IKs modulation is poorly understood and was subject of this study.MethodsWe studied N-terminal KCNE1 constructs disrupting structurally important positively charged amino-acids (arginines) at positions 32, 33, 36 as well as KCNE1 constructs that modify position 38 including an N-terminal truncation mutation. Experimental procedures included molecular cloning, patch-clamp recording, protein biochemistry, real-time-PCR and confocal microscopy.ResultsAll KCNE1 constructs physically interacted with Kv7.1. IKs resulting from co-expression of Kv7.1 with non-atrial fibrillation ‘38S’ was greater than with any other construct. Ionic currents resulting from co-transfection of a KCNE1 mutant with arginine substitutions (‘38G-3xA’) were comparable to currents evoked from cells transfected with an N-terminally truncated KCNE1-construct (‘Δ1-38’). Western-blots from plasma-membrane preparations and confocal images consistently showed a greater amount of Kv7.1 protein at the plasma-membrane in cells co-transfected with the non-atrial fibrillation KCNE1-38S than with any other construct.ConclusionsThe results of our study indicate that N-terminal arginines in positions 32, 33, 36 of KCNE1 are important for reconstitution of IKs. Furthermore, our results hint towards a role of these N-terminal amino-acids in membrane representation of the delayed rectifier channel complex.

Structural basis of PIP2 activation of the classical inward rectifier K+ channel Kir2.2.

The regulation of ion channel activity by specific lipid molecules is widely recognized as an integral component of electrical signalling in cells. In particular, phosphatidylinositol 4,5-bisphosphate (PIP(2)), a minor yet dynamic phospholipid component of cell membranes, is known to regulate many different ion channels. PIP(2) is the primary agonist for classical inward rectifier (Kir2) channels, through which this lipid can regulate a cell's resting membrane potential. However, the molecular mechanism by which PIP(2) exerts its action is unknown. Here we present the X-ray crystal structure of a Kir2.2 channel in complex with a short-chain (dioctanoyl) derivative of PIP(2). We found that PIP(2) binds at an interface between the transmembrane domain (TMD) and the cytoplasmic domain (CTD). The PIP(2)-binding site consists of a conserved non-specific phospholipid-binding region in the TMD and a specific phosphatidylinositol-binding region in the CTD. On PIP(2) binding, a flexible expansion linker contracts to a compact helical structure, the CTD translates 6 Å and becomes tethered to the TMD and the inner helix gate begins to open. In contrast, the small anionic lipid dioctanoyl glycerol pyrophosphatidic acid (PPA) also binds to the non-specific TMD region, but not to the specific phosphatidylinositol region, and thus fails to engage the CTD or open the channel. Our results show how PIP(2) can control the resting membrane potential through a specific ion-channel-receptor-ligand interaction that brings about a large conformational change, analogous to neurotransmitter activation of ion channels at synapses.

Pancreatic β-cell prosurvival effects of the incretin hormones involve post-translational modification of Kv2.1 delayed rectifier channels

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) are the major incretin hormones that exert insulinotropic and anti-apoptotic actions on pancreatic β-cells. Insulinotropic actions of the incretins involve modulation of voltage-gated potassium (Kv) channels. In multiple cell types, Kv channel activity has been implicated in cell volume changes accompanying initiation of the apoptotic program. Focusing on Kv2.1, we examined whether regulation of Kv channels in β-cells contributes to the prosurvival effects of incretins. Overexpression of Kv2.1 in INS-1 β-cells potentiated apoptosis in response to mitochondrial and ER stress and, conversely, co-stimulation with GIP/GLP-1 uncoupled this potentiation, suppressing apoptosis. In parallel, incretins promoted phosphorylation and acetylation of Kv2.1 via pathways involving protein kinase A (PKA)/mitogen- and stress-activated kinase-1 (MSK-1) and histone acetyltransferase (HAT)/histone deacetylase (HDAC). Further studies demonstrated that acetylation of Kv2.1 was mediated by incretin actions on nuclear/cytoplasmic shuttling of CREB binding protein (CBP) and its interaction with Kv2.1. Regulation of β-cell survival by GIP and GLP-1 therefore involves post-translational modifications (PTMs) of Kv channels by PKA/MSK-1 and HAT/HDAC. This appears to be the first demonstration of modulation of delayed rectifier Kv channels contributing to the β-cell prosurvival effects of incretins and of 7-transmembrane G protein-coupled receptor (GPCR)-stimulated export of a nuclear lysine acetyltransferase that regulates cell surface ion channel function.

Mechanisms for Kir channel inhibition by quinacrine: acute pore block of Kir2.x channels and interference in PIP2 interaction with Kir2.x and Kir6.2 channels

Cardiac inward rectifier potassium currents determine the resting membrane potential and contribute repolarization capacity during phase 3 repolarization. Quinacrine is a cationic amphiphilic drug. In this work, the effects of quinacrine were studied on cardiac Kir channels expressed in HEK 293 cells and on the inward rectifier potassium currents, I(K1) and I(KATP), in cardiac myocytes. We found that quinacrine differentially inhibited Kir channels, Kir6.2 ∼ Kir2.3 > Kir2.1. In addition, we found in cardiac myocytes that quinacrine inhibited I(KATP) > I(K1). We presented evidence that quinacrine displays a double action towards strong inward rectifier Kir2.x channels, i.e., direct pore block and interference in phosphatidylinositol 4,5-bisphosphate, PIP(2)-Kir channel interaction. Pore block is evident in Kir2.1 and 2.3 channels as rapid block; channel block involves residues E224 and E299 facing the cytoplasmic pore of Kir2.1. The interference of the drug with the interaction of Kir2.x and Kir6.2/SUR2A channels and PIP(2) is suggested from four sources of evidence: (1) Slow onset of current block when quinacrine is applied from either the inside or the outside of the channel. (2) Mutation of Kir2.3(I213L) and mutation of Kir6.2(C166S) increase their affinity for PIP(2) and lowers its sensitivity for quinacrine. (3) Mutations of Kir2.1(L222I and K182Q) which decreased its affinity for PIP(2) increased its sensitivity for quinacrine. (4) Co-application of quinacrine with PIP(2) lowers quinacrine-mediated current inhibition. In conclusion, our data demonstrate how an old drug provides insight into a dual a blocking mechanism of Kir carried inward rectifier channels.