Distance constraints on activation of TRPV4 channels by AKAP150-bound PKCα in arterial myocytes.

TRPV4 (transient receptor potential vanilloid 4) channels are Ca2+-permeable channels that play a key role in regulating vascular tone. In arterial myocytes, opening of TRPV4 channels creates local increases in Ca2+ influx, detectable optically as "TRPV4 sparklets." TRPV4 sparklet activity can be enhanced by the action of the vasoconstrictor angiotensin II (AngII). This modulation depends on the activation of subcellular signaling domains that comprise protein kinase C α (PKCα) bound to the anchoring protein AKAP150. Here, we used super-resolution nanoscopy, patch-clamp electrophysiology, Ca2+ imaging, and mathematical modeling approaches to test the hypothesis that AKAP150-dependent modulation of TRPV4 channels is critically dependent on the distance between these two proteins in the sarcolemma of arterial myocytes. Our data show that the distance between AKAP150 and TRPV4 channel clusters varies with sex and arterial bed. Consistent with our hypothesis, we further find that basal and AngII-induced TRPV4 channel activity decays exponentially as the distance between TRPV4 and AKAP150 increases. Our data suggest a maximum radius of action of ∼200 nm for local modulation of TRPV4 channels by AKAP150-associated PKCα

Dynamic L-type CaV1.2 channel trafficking facilitates CaV1.2 clustering and cooperative gating.

L-type CaV1.2 channels are key regulators of gene expression, cell excitability and muscle contraction. CaV1.2 channels organize in clusters throughout the plasma membrane. This channel organization has been suggested to contribute to the concerted activation of adjacent CaV1.2 channels (e.g. cooperative gating). Here, we tested the hypothesis that dynamic intracellular and perimembrane trafficking of CaV1.2 channels is critical for formation and dissolution of functional channel clusters mediating cooperative gating. We found that CaV1.2 moves in vesicular structures of circular and tubular shape with diverse intracellular and submembrane trafficking patterns. Both microtubules and actin filaments are required for dynamic movement of CaV1.2 vesicles. These vesicles undergo constitutive homotypic fusion and fission events that sustain CaV1.2 clustering, channel activity and cooperative gating. Our study suggests that CaV1.2 clusters and activity can be modulated by diverse and unique intracellular and perimembrane vesicular dynamics to fine-tune Ca2+ signals

Kv2.1 channels play opposing roles in regulating membrane potential, Ca2+ channel function, and myogenic tone in arterial smooth muscle.

The accepted role of the protein Kv2.1 in arterial smooth muscle cells is to form K+ channels in the sarcolemma. Opening of Kv2.1 channels causes membrane hyperpolarization, which decreases the activity of L-type CaV1.2 channels, lowering intracellular Ca2+ ([Ca2+]i) and causing smooth muscle relaxation. A limitation of this model is that it is based exclusively on data from male arterial myocytes. Here, we used a combination of electrophysiology as well as imaging approaches to investigate the role of Kv2.1 channels in male and female arterial myocytes. We confirmed that Kv2.1 plays a canonical conductive role but found it also has a structural role in arterial myocytes to enhance clustering of CaV1.2 channels. Less than 1% of Kv2.1 channels are conductive and induce membrane hyperpolarization. Paradoxically, by enhancing the structural clustering and probability of CaV1.2-CaV1.2 interactions within these clusters, Kv2.1 increases Ca2+ influx. These functional impacts of Kv2.1 depend on its level of expression, which varies with sex. In female myocytes, where expression of Kv2.1 protein is higher than in male myocytes, Kv2.1 has conductive and structural roles. Female myocytes have larger CaV1.2 clusters, larger [Ca2+]i, and larger myogenic tone than male myocytes. In contrast, in male myocytes, Kv2.1 channels regulate membrane potential but not CaV1.2 channel clustering. We propose a model in which Kv2.1 function varies with sex: in males, Kv2.1 channels control membrane potential but, in female myocytes, Kv2.1 plays dual electrical and CaV1.2 clustering roles. This contributes to sex-specific regulation of excitability, [Ca2+]i, and myogenic tone in arterial myocytes

High blood pressure associates with the remodelling of inward rectifier K+ channels in mice mesenteric vascular smooth muscle cells

Producción CientíficaThe increased vascular tone that defines essential hypertension is associated with depolarization of vascular smooth muscle cells (VSMCs) and involves a change in the expression profile of ion channels promoting arterial contraction. As a major regulator of VSMC resting membrane potential (VM), K+channel activity is an important determinant of vascular tone and vessel diameter. However, hypertension-associated changes in the expression and/or modulation of K+channels are poorly defined, due to their large molecular diversity and their bed-specific pattern of expression. Moreover, the impact of these changes on the integrated vessel functionand their contribution to the development of altered vascular tone under physiological conditions need to be confirmed. Hypertensive (BPH) and normotensive (BPN) mice strains obtained by phenotypic selection were used to explore whether changes in the functional expression of VSMC inward rectifier K+channels contribute to the more depolarized resting VM and the increased vascular reactivity of hypertensive arteries. We determined the expression levels of inward rectifierK+channel mRNA in several vascular beds from BPN and BPH animals, and their functional contribution to VSMC excitability and vascular tone in mesenteric arteries. We found a decrease in the expression of Kir2.1, Kir4.1, Kir6.x and SUR2 mRNA in BPH VSMCs, and a decreased functional contribution of both KIRand KATP channels in isolated BPH VSMCs. However, only the effect of KATP channel modulators was impaired when exploring vascular tone, suggesting that decreased functional expression of KATP channels may be an important element in the remodelling of VSMCs in essential hypertension.Ministerio de Sanidad, Consumo y Bienestar Social - Instituto de Salud Carlos III (grant R006/009)Ministerio de Ciencia, Innovación y Universidades (grant BFU2010-15898)Junta de Castilla y León (grant VA094A11-2

Kv1.3 channels can modulate cell proliferation during phenotypic switch by an ion-flux independent mechanism

Producción CientíficaObjective: Phenotypic modulation of vascular smooth muscle cells has been associated with a decreased expression of all voltage-dependent potassium channel (Kv)1 channel encoding genes but Kcna3 (which encodes Kv1.3 channels). In fact, upregulation of Kv1.3 currents seems to be important to modulate proliferation of mice femoral vascular smooth muscle cells in culture. This study was designed to explore if these changes in Kv1 expression pattern constituted a landmark of phenotypic modulation across vascular beds and to investigate the mechanisms involved in the proproliferative function of Kv1.3 channels. Methods and Results: Changes in Kv1.3 and Kv1.5 channel expression were reproduced in mesenteric and aortic vascular smooth muscle cells, and their correlate with protein expression was electrophysiologicaly confirmed using selective blockers. Heterologous expression of Kv1.3 and Kv1.5 channels in HEK cells has opposite effects on the proliferation rate. The proproliferative effect of Kv1.3 channels was reproduced by “poreless” mutants but disappeared when voltagedependence of gating was suppressed. Conclusion: These findings suggest that the signaling cascade linking Kv1.3 functional expression to cell proliferation is activated by the voltage-dependent conformational change of the channels without needing ion conduction. Additionally, the conserved upregulation of Kv1.3 on phenotypic modulation in several vascular beds makes this channel a good target to control unwanted vascular remodeling.Instituto de Salud Carlos III (grant R006/009)Ministerio de Ciencia, Innovación y Universidades (grant BFU2010-15898)Junta de Castilla y León (grant VA094A11-2

TRPA1 channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins

Producción CientíficaGram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation occurs through activation of the Toll-like receptor 4 (TLR4) signalling pathway by lipopolysaccharide (LPS), a toxic by-product of bacterial lysis. Here we show that LPS exerts fast, membrane delimited, excitatory actions via TRPA1, a transient receptor potential cation channel that is critical for transducing environmental irritant stimuli into nociceptor activity. Moreover, we find that pain and acute vascular reactions, including neurogenic inflammation (CGRP release) caused by LPS are primarily dependent on TRPA1 channel activation in nociceptive sensory neurons, and develop independently of TLR4 activation. The identification of TRPA1 as a molecular determinant of direct LPS effects on nociceptors offers new insights into the pathogenesis of pain and neurovascular responses during bacterial infections and opens novel avenues for their treatment.Projects SAF2010-14990 and PROMETEO2010-046. Instituto de Salud Carlos III. CONSOLIDER-INGENIO 2010. ISCIII grants R006/009 (Red Heracles), the Spanish Fundación Marcelino Botín and Belgian Federal Government (IUAP P6/28 and P7/13), the Research Foundation-Flanders and the Research Council of the KU Leuven

Role of Ca2+ and K+ channels in a model of essential hypertension

En este trabajo se han investigado los mecanismos que provocan la disfunción vascular en un modelo de ratón con hipertensión genética. La hipertensión esencial implica un aumento gradual y sostenido de las resistencias periféricas, lo que provoca un tono vascular aumentado. Este cambio se asocia con una despolarización de las células del músculo liso vascular (Vascular Smooth Muscle Cells, VSMCs) y se produce como consecuencia de un cambio en el perfil de expresión de los canales iónicos dependientes de voltaje (principalmente canales de K+ y Ca2+) que promueven la contracción arterial. Nosotros hemos caracterizado la expresión molecular y funcional de los canales de K+ “inwar rectifier” así como de los canales de Ca2+ dependiente de voltaje en miocitos vasculares de animales hipertensión (BPH) y normotensos (BPN) y su contribución a la excitabilidad de las VSMCs y al tono vascular en arterias mesentéricas.Departamento de Bioquímica y Biología Molecular y Fisiologí

Calcium permeable channels in cancer hallmarks

Cancer, the second cause of death worldwide, is characterized by several common criteria, known as the “cancer hallmarks” such as unrestrained cell proliferation, cell death resistance, angiogenesis, invasion and metastasis. Calcium permeable channels are proteins present in external and internal biological membranes, diffusing Ca2+ ions down their electrochemical gradient. Numerous physiological functions are mediated by calcium channels, ranging from intracellular calcium homeostasis to sensory transduction. Consequently, calcium channels play important roles in human physiology and it is not a surprise the increasing number of evidences connecting calcium channels disorders with tumor cells growth, survival and migration. Multiple studies suggest that calcium signals are augmented in various cancer cell types, contributing to cancer hallmarks. This review focuses in the role of calcium permeable channels signaling in cancer with special attention to the mechanisms behind the remodeling of the calcium signals. Transient Receptor Potential (TRP) channels and Store Operated Channels (SOC) are the main extracellular Ca2+ source in the plasma membrane of non-excitable cells, while inositol trisphosphate receptors (IP3R) are the main channels releasing Ca2+ from the endoplasmic reticulum (ER). Alterations in the function and/or expression of these calcium channels, as wells as, the calcium buffering by mitochondria affect intracellular calcium homeostasis and signaling, contributing to the transformation of normal cells into their tumor counterparts. Several compounds reported to counteract several cancer hallmarks also modulate the activity and/or the expression of these channels including non-steroidal anti-inflammatory drugs (NSAIDs) like sulindac and aspirin, and inhibitors of polyamine biosynthesis, like difluoromethylornithine (DFMO). The possible role of the calcium permeable channels targeted by these compounds in cancer and their action mechanism will be discussed also in the review.This work has been supported by competitive grants RTI2018- 099298-B-100 from Ministerio de Ciencia, Innovación y Universidades, Spain, BFU2015-70131 from Ministerio de Economia y Competitividad, Spain and VA294P18 from Junta de Castilla y León, Spain.Peer reviewe

Total Internal Reflection Fluorescence Microscopy in Vascular Smooth Muscle

This chapter provides an overview of the basic concepts behind Total Internal Reflection Fluorescence Microscopy (TIRFM), and its application to the study of the function and regulation of plasmalemmal Ca2+-permeable channels in vascular smooth muscle. TIRFM utilizes an evanescent wave to selectively excite fluorophores in regions of a sample directly adjacent to the glass coverslip-buffer interface. The principles at the heart of TIRFM are based on the laws of refraction of light and properties of the refractive media. TIRFM relies on the ability to introduce light at angles exceeding a critical angle. A particular innovative use of TIRFM has been on the recording of Ca2+ signals produced by the opening of Ca2+-permeable channels at the plasma membrane of a cell, including vascular smooth muscle cells. The quality of vascular smooth muscle cells is critical for successful recording of sparklets using TIRFM. Single vascular smooth muscle cells can be obtained by enzymatic digestion of freshly dissected arteries from different vascular beds