Laura Solé

KCNE4 suppresses Kv1.3 currents by modulating trafficking, surface expression and channel gating

Voltage-dependent potassium channels (Kv) play a crucial role in the activation and proliferation of leukocytes. Kv channels are either homo- or hetero-oligomers. This composition modulates their surface expression and serves as a mechanism for regulating channel activity. Kv channel interaction with accessory subunits provides mechanisms for channels to respond to stimuli beyond changes in membrane potential. Here, we demonstrate that KCNE4 (potassium voltage-gated channel subfamily E member 4), but not KCNE2, functions as an inhibitory Kv1.3 partner in leukocytes. Kv1.3 trafficking, targeting and activity are altered by the presence of KCNE4. KCNE4 decreases current density, slows activation, accelerates inactivation, increases cumulative inactivation, retains Kv1.3 in the ER and impairs channel targeting to lipid raft microdomains. KCNE4 associates with Kv1.3 in the ER and decreases the number of Kv1.3 channels at the cell surface, which diminishes cell excitability. Kv1.3 and KCNE4 are differentially regulated upon activation or immunosuppression in macrophages. Thus, lipopolysaccharide-induced activation increases Kv1.3 and KCNE4 mRNA, whereas dexamethasone triggers a decrease in Kv1.3 with no changes in KCNE4. The channelosome composition determines the activity and affects surface expression and membrane localization. Therefore, KCNE4 association might play a crucial role in controlling immunological responses. Our results indicate that KCNE ancillary subunits could be new targets for immunomodulation.

Skeletal muscle Kv7 (KCNQ) channels in myoblast differentiation and proliferation

Voltage-dependent K(+) channels (Kv) are involved in myocyte proliferation and differentiation by triggering changes in membrane potential and regulating cell volume. Since Kv7 channels may participate in these events, the purpose of this study was to investigate whether skeletal muscle Kv7.1 and Kv7.5 were involved during proliferation and myogenesis. Here we report that, while myotube formation did not regulate Kv7 channels, Kv7.5 was up-regulated during cell cycle progression. Although, Kv7.1 mRNA also increased during the G(1)-phase, pharmacological evidence mainly involves Kv7.5 in myoblast growth. Our results indicate that the cell cycle-dependent expression of Kv7.5 is involved in skeletal muscle cell proliferation.

Multiple Kv1.5 targeting to membrane surface microdomains.

Surface expression of voltage‐dependent K+ channels (Kv) has a pivotal role in leukocyte physiology. Although little is known about the physiological role of lipid rafts, these microdomains concentrate signaling molecules and their ion channel substrates. Kv1.3 associates with Kv1.5 to form functional channels in macrophages. Different isoform stoichiometries lead to distinct heteromeric channels which may be further modulated by targeting the complex to different membrane surface microdomains. Kv1.3 targets to lipid rafts, whereas Kv1.5 localization is under debate. With this in mind, we wanted to study whether heterotetrameric Kv1.5‐containing channels target to lipid rafts. While in transfected HEK‐293 cells, homo‐ and heterotetrameric channels targeted to rafts, Kv1.5 did not target to rafts in macrophages. Therefore, Kv1.3/Kv1.5 hybrid channels are mostly concentrated in non‐raft microdomains. However, LPS‐induced activation, which increases the Kv1.3/Kv1.5 ratio and caveolin, targeted Kv1.5 back to lipid rafts. Moreover, Kv1.5 did not localize to low‐buoyancy fractions in L6E9 skeletal myoblasts, which also coexpress both channels, heart membranes or cardiomyocyes. Coexpression of a Cav3DGV‐mutant confined Kv1.5 to Cav3DGV‐vesicles of HEK cells. Contrarily, coexpression of Kvβ2.1 impaired the Kv1.5 targeting to raft microdomains in HEK cells. Our results indicate that Kv1.5 partnership interactions are underlying mechanisms governing channel targeting to lipid rafts. J. Cell. Physiol. 217: 667–673, 2008.

Preferential Targeting of Nav1.6 Voltage-Gated Na+ Channels to the Axon Initial Segment during Development

During axonal maturation, voltage-gated sodium (Nav) channels accumulate at the axon initial segment (AIS) at high concentrations. This localization is necessary for the efficient initiation of action potentials. The mechanisms underlying channel trafficking to the AIS during axonal development have remained elusive due to a lack of Nav reagents suitable for high resolution imaging of channels located specifically on the cell surface. Using an optical pulse-chase approach in combination with a novel Nav1.6 construct containing an extracellular biotinylation domain we demonstrate that Nav1.6 channels are preferentially inserted into the AIS membrane during neuronal development via direct vesicular trafficking. Single-molecule tracking illustrates that axonal channels are immediately immobilized following delivery, while channels delivered to the soma are often mobile. Neither a Nav1.6 channel lacking the ankyrin-binding motif nor a chimeric Kv2.1 channel containing the Nav ankyrinG-binding domain show preferential AIS insertion. Together these data support a model where ankyrinG-binding is required for preferential Nav1.6 insertion into the AIS plasma membrane. In contrast, ankyrinG-binding alone does not confer the preferential delivery of proteins to the AIS.

Impact of KCNE subunits on KCNQ1 (Kv7.1) channel membrane surface targeting

The KCNQ1 (Kv7.1) channel plays an important role in cardiovascular physiology. Cardiomyocytes co‐express KCNQ1 with KCNE1‐5 proteins. KCNQ1 may co‐associate with multiple KCNE regulatory subunits to generate different biophysically and pharmacologically distinct channels. Increasing evidence indicates that the location and targeting of channels are important determinants of their function. In this context, the presence of K+ channels in sphingolipid–cholesterol‐enriched membrane microdomains (lipid rafts) is under investigation. Lipid rafts are important for cardiovascular functioning. We aimed to determine whether KCNE subunits modify the localization and targeting of KCNQ1 channels in lipid rafts microdomains. HEK‐293 cells were transiently transfected with KCNQ1 and KCNE1–5, and their traffic and presence in lipid rafts were analyzed. Only KCNQ1 and KCNE3, when expressed alone, co‐localized in raft fractions. In addition, while KCNE2 and KCNE5 notably stained the cell surface, KCNQ1 and the rest of the KCNEs showed strong intracellular retention. KCNQ1 targets multiple membrane surface microdomains upon association with KCNE peptides. Thus, while KCNQ1/KCNE1 and KCNQ1/KCNE2 channels target lipid rafts, KCNQ1 associated with KCNE3–5 did not. Channel membrane dynamics, analyzed by fluorescence recovery after photobleaching (FRAP) experiments, further supported these results. In conclusion, the trafficking and targeting pattern of KCNQ1 can be influenced by its association with KCNEs. Since KCNQ1 is crucial for cardiovascular physiology, the temporal and spatial regulations that different KCNE subunits may confer to the channels could have a dramatic impact on membrane electrical activity and putative endocrine regulation. J. Cell. Physiol. 225: 692–700, 2010.

Functional implications of KCNE subunit expression for the Kv7.5 (KCNQ5) channel.

Kv7 (KCNQ) proteins form a family of voltage-gated potassium channels that is comprised of five members, Kv7.1-Kv7.5. While Kv7.1 is crucial in the heart, the Kv7.2, Kv7.3, Kv7.4 and Kv7.5 channels contribute to the M-current in the nervous system. In addition to the brain, Kv7.5 is expressed in skeletal and smooth muscle, where its physiological role is currently under evaluation. Kv7 associations with KCNE accessory subunits (KCNE1-5) enhance channel diversity and their interaction provides mechanisms to respond to a variety of stimuli. KCNE peptides control the surface expression, voltage-dependence, kinetics of gating, unitary conductance, ion selectivity and pharmacology of several channels. KCNE subunits have been primarily studied in the heart; however, their activity in the brain and in many other tissues is being increasingly recognized. Here, we found that Kv7.5 and KCNE subunits are present in myoblasts. Therefore, oligomeric associations may underlie some Kv7.5 functional diversity in skeletal muscle. An extensive study in Xenopus oocytes and HEK-293 cells demonstrates that KCNE1 and KCNE3, but none of the other KCNE subunits, affect Kv7.5 currents. While KCNE1 slows activation and suppresses inward rectification, KCNE3 drastically inhibits Kv7.5 currents. In addition, KCNE1 increases Kv7.5 currents in HEK cells. Changes in gating and amplitude indicate functional interactions. Our results have physiological relevance since Kv7.5 is abundant in skeletal and smooth muscle and its association with KCNE peptides may fine-tune cellular responses.

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.

Ergodicity breaking on the neuronal surface emerges from random switching between diffusive states

Stochastic motion on the surface of living cells is critical to promote molecular encounters that are necessary for multiple cellular processes. Often the complexity of the cell membranes leads to anomalous diffusion, which under certain conditions it is accompanied by non-ergodic dynamics. Here, we unravel two manifestations of ergodicity breaking in the dynamics of membrane proteins in the somatic surface of hippocampal neurons. Three different tagged molecules are studied on the surface of the soma: the voltage-gated potassium and sodium channels Kv1.4 and Nav1.6 and the glycoprotein CD4. In these three molecules ergodicity breaking is unveiled by the confidence interval of the mean square displacement and by the dynamical functional estimator. Ergodicity breaking is found to take place due to transient confinement effects since the molecules alternate between free diffusion and confined motion.

Does a physiological role for KCNE subunits exist in the immune system

The study of channel modulation by regulatory subunits has attracted considerable attention. Evidence indicates a pivotal role for accessory proteins in the channelosome. For instance, these regulatory subunits are necessary to recapitulate in vivo ion currents and to further understand the physiological role of ion channels. KCNEs are a family of regulatory subunits that interact with a wide range of channels. We have described for the first time a molecular interaction between KCNE4 and the voltage-dependent potassium channel Kv1.3. The association of KCNE4, which alters the biophysical properties, trafficking and membrane localization of Kv1.3, functions as an endogenous dominant-negative mechanism. Since both proteins are expressed in the immune system, Kv1.3/KCNE4 channels may contribute to the fine-tuning of the immune response. Therefore, our results point to KCNE4 as a novel target for immunomodulation. KCNE4 is not the only KCNE which is expressed in leukocytes. All KCNEs (KCNE1-5) are present, and some members demonstrate modulation during proliferation and cancer. In summary, regulatory KCNE subunits are expressed in the immune system. In addition, several voltage-dependent K+ channels, which could interact with KCNEs, are also detected. Therefore, KCNE subunits may play a yet undiscovered role in the physiology of the immune system.

Elucidating distinct ion channel populations on the surface of hippocampal neurons via single-particle tracking recurrence analysis

Protein and lipid nanodomains are prevalent on the surface of mammalian cells. In particular, it has been recently recognized that ion channels assemble into surface nanoclusters in the soma of cultured neurons. However, the interactions of these molecules with surface nanodomains display a considerable degree of heterogeneity. Here, we investigate this heterogeneity and develop statistical tools based on the recurrence of individual trajectories to identify subpopulations within ion channels in the neuronal surface. We specifically study the dynamics of the K^{+} channel Kv1.4 and the Na^{+} channel Nav1.6 on the surface of cultured hippocampal neurons at the single-molecule level. We find that both these molecules are expressed in two different forms with distinct kinetics with regards to surface interactions, emphasizing the complex proteomic landscape of the neuronal surface. Further, the tools presented in this work provide new methods for the analysis of membrane nanodomains, transient confinement, and identification of populations within single-particle trajectories.