Alicia de la Cruz

Effects of n-3 Polyunsaturated Fatty Acids on Cardiac Ion Channels.

Dietary n−3 polyunsaturated fatty acids (PUFAs) have been reported to exhibit antiarrhythmic properties, and these effects have been attributed to their capability to modulate ion channels. In the present review, we will focus on the effects of PUFAs on a cardiac sodium channel (Nav1.5) and two potassium channels involved in cardiac atrial and ventricular repolarization (Kv) (Kv1.5 and Kv11.1). n−3 PUFAs of marine (docosahexaenoic, DHA and eicosapentaenoic acid, EPA) and plant origin (alpha-linolenic acid, ALA) block Kv1.5 and Kv11.1 channels at physiological concentrations. Moreover, DHA and EPA decrease the expression levels of Kv1.5, whereas ALA does not. DHA and EPA also decrease the magnitude of the currents elicited by the activation of Nav1.5 and calcium channels. These effects on sodium and calcium channels should theoretically shorten the cardiac action potential duration (APD), whereas the blocking actions of n−3 PUFAs on Kv channels would be expected to produce a lengthening of cardiac action potential. Indeed, the effects of n−3 PUFAs on the cardiac APD and, therefore, on cardiac arrhythmias vary depending on the method of application, the animal model, and the underlying cardiac pathology.

A new KCNQ1 mutation at the S5 segment that impairs its association with KCNE1 is responsible for short QT syndrome

AIMS KCNQ1 and KCNE1 encode Kv7.1 and KCNE1, respectively, the pore-forming and the accessory subunits of the slow delayed rectifier potassium current, IKs. KCNQ1 mutations are associated with long and short QT syndrome. The aim of this study was to characterize the biophysical and cellular phenotype of a KCNQ1 missense mutation, F279I, found in a 23-year-old man with a corrected QT interval (QTc) of 356 ms and a family history of sudden cardiac death. METHODS AND RESULTS Experiments were performed using perforated patch-clamp, western blot, co-immunoprecipitation, biotinylation, and immunocytochemistry techniques in HEK293, COS7 cells and in cardiomyocytes transfected with WT Kv7.1/KCNE1 or F279I Kv7.1/KCNE1 channels. In the absence of KCNE1, F279I Kv7.1 current exhibited a lesser degree of inactivation than WT Kv7.1. Also, functional analysis of F279I Kv7.1 in the presence of KCNE1 revealed a negative shift in the activation curve and an acceleration of the activation kinetics leading to a gain of function in IKs. The co-assembly between F279I Kv7.1 channels and KCNE1 was markedly decreased compared with WT Kv7.1 channels, as revealed by co-immunoprecipitation and Föster Resonance Energy Transfer experiments. All these effects contribute to the increase of IKs when channels incorporate F279I Kv7.1 subunits, as shown by a computer model simulation of these data that predicts a shortening of the action potential (AP) consistent with the patient phenotype. CONCLUSION The F279I mutation induces a gain of function of IKs due to an impaired gating modulation of Kv7.1 induced by KCNE1, leading to a shortening of the cardiac AP.

Modulation of Voltage-Dependent and Inward Rectifier Potassium Channels by 15-Epi-Lipoxin-A4 in Activated Murine Macrophages: Implications in Innate Immunity

Potassium channels modulate macrophage physiology. Blockade of voltage-dependent potassium channels (Kv) by specific antagonists decreases macrophage cytokine production and inhibits proliferation. In the presence of aspirin, acetylated cyclooxygenase-2 loses the activity required to synthesize PGs but maintains the oxygenase activity to produce 15R-HETE from arachidonate. This intermediate product is transformed via 5-LOX into epimeric lipoxins, termed 15-epi-lipoxins (15-epi-lipoxin A4 [e-LXA4]). Kv have been proposed as anti-inflammatory targets. Therefore, we studied the effects of e-LXA4 on signaling and on Kv and inward rectifier potassium channels (Kir) in mice bone marrow–derived macrophages (BMDM). Electrophysiological recordings were performed in these cells by the whole-cell patch-clamp technique. Treatment of BMDM with e-LXA4 inhibited LPS-dependent activation of NF-κB and IκB kinase β activity, protected against LPS activation–dependent apoptosis, and enhanced the accumulation of the Nrf-2 transcription factor. Moreover, treatment of LPS-stimulated BMDM with e-LXA4 resulted in a rapid decrease of Kv currents, compatible with attenuation of the inflammatory response. Long-term treatment of LPS-stimulated BMDM with e-LXA4 significantly reverted LPS effects on Kv and Kir currents. Under these conditions, e-LXA4 decreased the calcium influx versus that observed in LPS-stimulated BMDM. These effects were partially mediated via the lipoxin receptor (ALX), because they were significantly reverted by a selective ALX receptor antagonist. We provide evidence for a new mechanism by which e-LXA4 contributes to inflammation resolution, consisting of the reversion of LPS effects on Kv and Kir currents in macrophages.

Marine n-3 PUFAs modulate IKs gating, channel expression, and location in membrane microdomains

AIMS Polyunsaturated fatty n-3 acids (PUFAs) have been reported to exhibit antiarrhythmic properties. However, the mechanisms of action remain unclear. We studied the electrophysiological effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on IKs, and on the expression and location of Kv7.1 and KCNE1. METHODS AND RESULTS Experiments were performed using patch-clamp, western blot, and sucrose gradient techniques in COS7 cells transfected with Kv7.1/KCNE1 channels. Acute perfusion with both PUFAs increased Kv7.1/KCNE1 current, this effect being greater for DHA than for EPA. Similar results were found in guinea pig cardiomyocytes. Acute perfusion of either PUFA slowed the activation kinetics and EPA shifted the activation curve to the left. Conversely, chronic EPA did not modify Kv7.1/KCNE1 current magnitude and shifted the activation curve to the right. Chronic PUFAs decreased the expression of Kv7.1, but not of KCNE1, and induced spatial redistribution of Kv7.1 over the cell membrane. Cholesterol depletion with methyl-β-cyclodextrin increased Kv7.1/KCNE1 current magnitude. Under these conditions, acute EPA produced similar effects than those induced in non-cholesterol-depleted cells. A ventricular action potential computational model suggested antiarrhythmic efficacy of acute PUFA application under IKr block. CONCLUSIONS We provide evidence that acute application of PUFAs increases Kv7.1/KCNE1 through a probably direct effect, and shows antiarrhythmic efficacy under IKr block. Conversely, chronic EPA application modifies the channel activity through a change in the Kv7.1/KCNE1 voltage-dependence, correlated with a redistribution of Kv7.1 over the cell membrane. This loss of function may be pro-arrhythmic. This shed light on the controversial effects of PUFAs regarding arrhythmias.

Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease

Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD.

Functional Assembly of Kv7.1/Kv7.5 Channels With Emerging Properties on Vascular Muscle Physiology

Objective—Voltage-dependent K+ (Kv) channels from the Kv7 family are expressed in blood vessels and contribute to cardiovascular physiology. Although Kv7 channel blockers trigger muscle contractions, Kv7 activators act as vasorelaxants. Kv7.1 and Kv7.5 are expressed in many vessels. Kv7.1 is under intense investigation because Kv7.1 blockers fail to modulate smooth muscle reactivity. In this study, we analyzed whether Kv7.1 and Kv7.5 may form functional heterotetrameric channels increasing the channel diversity in vascular smooth muscles. Approach and Results—Kv7.1 and Kv7.5 currents elicited in arterial myocytes, oocyte, and mammalian expression systems suggest the formation of heterotetrameric complexes. Kv7.1/Kv7.5 heteromers, exhibiting different pharmacological characteristics, participate in the arterial tone. Kv7.1/Kv7.5 associations were confirmed by coimmunoprecipitation, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching experiments. Kv7.1/Kv7.5 heterotetramers were highly retained at the endoplasmic reticulum. Studies in HEK-293 cells, heart, brain, and smooth and skeletal muscles demonstrated that the predominant presence of Kv7.5 stimulates release of Kv7.1/Kv7.5 oligomers out of lipid raft microdomains. Electrophysiological studies supported that KCNE1 and KCNE3 regulatory subunits further increased the channel diversity. Finally, the analysis of rat isolated myocytes and human blood vessels demonstrated that Kv7.1 and Kv7.5 exhibited a differential expression, which may lead to channel diversity. Conclusions—Kv7.1 and Kv7.5 form heterotetrameric channels increasing the diversity of structures which fine-tune blood vessel reactivity. Because the lipid raft localization of ion channels is crucial for cardiovascular physiology, Kv7.1/Kv7.5 heteromers provide efficient spatial and temporal regulation of smooth muscle function. Our results shed light on the debate about the contribution of Kv7 channels to vasoconstriction and hypertension.

Polyunsaturated Fatty Acids Modify the Gating of Kv Channels

Polyunsaturated fatty acids (PUFAs) have been reported to exhibit antiarrhythmic properties, which are attributed to their capability to modulate ion channels. This PUFAs ability has been reported to be due to their effects on the gating properties of ion channels. In the present review, we will focus on the role of PUFAs on the gating of two Kv channels, Kv1.5 and Kv11.1. Kv1.5 channels are blocked by n−3 PUFAs of marine [docosahexaenoic acid (DHA) and eicosapentaenoic acid] and plant origin (alpha-linolenic acid, ALA) at physiological concentrations. The blockade of Kv1.5 channels by PUFAs steeply increased in the range of membrane potentials coinciding with those of Kv1.5 channel activation, suggesting that PUFAs-channel binding may derive a significant fraction of its voltage sensitivity through the coupling to channel gating. A similar shift in the activation voltage was noted for the effects of n–6 arachidonic acid (AA) and DHA on Kv1.1, Kv1.2, and Kv11.1 channels. PUFAs-Kv1.5 channel interaction is time-dependent, producing a fast decay of the current upon depolarization. Thus, Kv1.5 channel opening is a prerequisite for the PUFA-channel interaction. Similar to the Kv1.5 channels, the blockade of Kv11.1 channels by AA and DHA steeply increased in the range of membrane potentials that coincided with the range of Kv11.1 channel activation, suggesting that the PUFAs-Kv channel interactions are also coupled to channel gating. Furthermore, AA regulates the inactivation process in other Kv channels, introducing a fast voltage-dependent inactivation in non-inactivating Kv channels. These results have been explained within the framework that AA closes voltage-dependent potassium channels by inducing conformational changes in the selectivity filter, suggesting that Kv channel gating is lipid dependent.

Stereoselective interactions between local anesthetics and ion channels

Local anesthetics are useful probes of ion channel function and structure. Stereoselective interactions are especially interesting because they can reveal three-dimensional relationships between drugs and channels with otherwise identical biophysical and physicochemical properties. Furthermore, stereoselectivity suggests direct and specific receptor-mediated action, and identification of such stereospecific interactions may have important clinical consequences. The fact that drug targets are able to discriminate between the enantiomers present in a racemic drug is the consequence of the ordered asymmetric macromolecular units that form living cells. However, almost 25% of the drugs used in the clinical practice are racemic mixtures, and their individual enantiomers frequently differ in both their pharmacodynamic and pharmacokinetic profiles. Moreover, their effects can be similar to or different from the pharmacological effect of the drug and may contribute to the undesired effects of the drug. In other cases, the pharmacological effects induced by the two enantiomers on the molecular target are opposite. In the present manuscript, we will review the stereoselective effects of bupivacaine-like local anesthetics on cardiac sodium and potassium channels.

In-depth study of the interaction, sensitivity, and gating modulation by PUFAs on K+ channels; interaction and new targets

Voltage gated potassium channels (KV) are membrane proteins that allow selective flow of K+ ions in a voltage-dependent manner. These channels play an important role in several excitable cells as neurons, cardiomyocytes, and vascular smooth muscle. Over the last 20 years, it has been shown that omega-3 polyunsaturated fatty acids (PUFAs) enhance or decrease the activity of several cardiac KV channels. PUFAs-dependent modulation of potassium ion channels has been reported to be cardioprotective. However, the precise cellular mechanism underlying the cardiovascular benefits remained unclear in part because new PUFAs targets and signaling pathways continue being discovered. In this review, we will focus on recent data available concerning the following aspects of the KV channel modulation by PUFAs: (i) the exact residues involved in PUFAs-KV channels interaction; (ii) the structural PUFAs determinants important for their effects on KV channels; (iii) the mechanism of the gating modulation of KV channels and, finally, (iv) the PUFAs modulation of a few new targets present in smooth muscle cells (SMC), KCa1.1, K2P, and KATP channels, involved in vascular relaxation.

D242N, a KV7.1 LQTS mutation uncovers a key residue for IKs voltage dependence

KV7.1 and KCNE1 co-assemble to give rise to the IKs current, one of the most important repolarizing currents of the cardiac action potential. Its relevance is underscored by the identification of >500 mutations in KV7.1 and, at least, 36 in KCNE1, that cause Long QT Syndrome (LQTS). The aim of this study was to characterize the biophysical and cellular consequences of the D242N KV7.1 mutation associated with the LQTS. The mutation is located in the S4 transmembrane segment, within the voltage sensor of the KV7.1 channel, disrupting the conserved charge balance of this region. Perforated patch-clamp experiments show that, unexpectedly, the mutation did not disrupt the voltage-dependent activation but it removed the inactivation and slowed the activation kinetics of D242N KV7.1 channels. Biotinylation of cell-surface protein and co-immunoprecipitation experiments revealed that neither plasma membrane targeting nor co-assembly between KV7.1 and KCNE1 was altered by the mutation. However, the association of D242N KV7.1 with KCNE1 strongly shifted the voltage dependence of activation to more depolarized potentials (+50mV), hindering IKs current at physiologically relevant membrane potentials. Both functional and computational analysis suggest that the clinical phenotype of the LQTS patients carrying the D242N mutation is due to impaired action potential adaptation to exercise and, in particular, to increase in heart rate. Moreover, our data identify D242 aminoacidic position as a potential residue involved in the KCNE1-mediated regulation of the voltage dependence of activation of the KV7.1 channel.