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.

BIN1 Induces the Formation of T-Tubules and Adult-Like Ca2+ Release Units in Developing Cardiomyocytes.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are at the center of new cell-based therapies for cardiac disease, but may also serve as a useful in vitro model for cardiac cell development. An intriguing feature of hESC-CMs is that although they express contractile proteins and have sarcomeres, they do not develop transverse-tubules (T-tubules) with adult-like Ca2+ release units (CRUs). We tested the hypothesis that expression of the protein BIN1 in hESC-CMs promotes T-tubules formation, facilitates CaV 1.2 channel clustering along the tubules, and results in the development of stable CRUs. Using electrophysiology, [Ca2+ ]i imaging, and super resolution microscopy, we found that BIN1 expression induced T-tubule development in hESC-CMs, while increasing differentiation toward a more ventricular-like phenotype. Voltage-gated CaV 1.2 channels clustered along the surface sarcolemma and T-tubules of hESC-CM. The length and width of the T-tubules as well as the expression and size of CaV 1.2 clusters grew, as BIN1 expression increased and cells matured. BIN1 expression increased CaV 1.2 channel activity and the probability of coupled gating within channel clusters. Interestingly, BIN1 clusters also served as sites for sarcoplasmic reticulum (SR) anchoring and stabilization. Accordingly, BIN1-expressing cells had more CaV 1.2-ryanodine receptor junctions than control cells. This was associated with larger [Ca2+ ]i transients during excitation-contraction coupling. Our data support the view that BIN1 is a key regulator of T-tubule formation and CaV 1.2 channel delivery. By studying the role of BIN1 during the differentiation of hESC-CMs, we show that BIN1 is also important for CaV 1.2 channel clustering, junctional SR organization, and the establishment of excitation-contraction coupling. Stem Cells 2019;37:54-64.

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.

Cinnamaldehyde inhibits L-type calcium channels in mouse ventricular cardiomyocytes and vascular smooth muscle cells.

Cinnamaldehyde (CA), a major component of cinnamon, is known to have important actions in the cardiovascular system, including vasorelaxation and decrease in blood pressure. Although CA-induced activation of the chemosensory cation channel TRPA1 seems to be involved in these phenomena, it has been shown that genetic ablation of Trpa1 is insufficient to abolish CA effects. Here, we confirm that CA relaxes rat aortic rings and report that it has negative inotropic and chronotropic effects on isolated mouse hearts. Considering the major role of L-type Ca(2+) channels in the control of the vascular tone and cardiac contraction, we used whole-cell patch-clamp to test whether CA affects L-type Ca(2+) currents in mouse ventricular cardiomyocytes (VCM, with Ca(2+) as charge carrier) and in mesenteric artery smooth muscle cells (VSMC, with Ba(2+) as charge carrier). We found that CA inhibited L-type currents in both cell types in a concentration-dependent manner, with little voltage-dependent effects. However, CA was more potent in VCM than in VSMC and caused opposite effects on the rate of inactivation. We found these divergences to be at least in part due to the use of different charge carriers. We conclude that CA inhibits L-type Ca(2+) channels and that this effect may contribute to its vasorelaxing action. Importantly, our results demonstrate that TRPA1 is not a specific target of CA and indicate that the inhibition of voltage-gated Ca(2+) channels should be taken into account when using CA to probe the pathophysiological roles of TRPA1.

Down-regulation of CaV1.2 channels during hypertension: how fewer CaV1.2 channels allow more Ca(2+) into hypertensive arterial smooth muscle.

Hypertension is a clinical syndrome characterized by increased arterial tone. Although the mechanisms are varied, the generally accepted view is that increased CaV1.2 channel function is a common feature of this pathological condition. Here, we investigated the mechanisms underlying vascular dysfunction in a mouse model of genetic hypertension. Contrary to expectation, we found that whole-cell CaV1.2 currents (ICa) were lower in hypertensive (BPH line) than normotensive (BPN line) myocytes. However, local CaV1.2 sparklet activity was higher in BPH cells, suggesting that the relatively low ICa in these cells was produced by a few hyperactive CaV1.2 channels. Furthermore, our data suggest that while the lower expression of the pore-forming α1c subunit of CaV1.2 currents underlies the lower ICa in BPH myocytes, the increased sparklet activity was due to a different composition in the auxiliary subunits of the CaV1.2 complexes. ICa currents in BPN cells were produced by channels composed of α1c/α2δ/ß3 subunits, while in BPH myocytes currents were probably generated by the opening of channels formed by α1c/α2δ/ß2 subunits. In addition, Ca(2+) sparks evoked large conductance, Ca(2+)-activated K(+) (BK) currents of lower magnitude in BPH than in BPN myocytes, because BK channels were less sensitive to Ca(2+). Our data are consistent with a model in which a decrease in the global number of CaV1.2 currents coexist with the existence of a subpopulation of highly active channels that dominate the resting Ca(2+) influx. The decrease in BK channel activity makes the hyperpolarizing brake ineffective and leads BPH myocytes to a more contracted resting state.

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

OBJECTIVE: 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 voltage-dependence of gating was suppressed. CONCLUSIONS: 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.

Transcriptional Basis of Ca2+ Remodeling Reversal Induced by Polyamine Synthesis Inhibition in Colorectal Cancer Cells.

Colorectal cancer (CRC) is associated with mutations in APC/Wnt leading to c-myc activation and the overexpression of ODC1, the limiting step in polyamine synthesis. CRC cells also display a remodeling of intracellular Ca2+ homeostasis that contributes to cancer hallmarks. As polyamines may modulate Ca2+ homeostasis during epithelial tissue repair, we investigated whether polyamine synthesis inhibition may reverse Ca2+ remodeling in CRC cells and, if so, the molecular basis for this reversal. To this end, we used calcium imaging and transcriptomic analysis in normal and CRC cells treated with DFMO, an ODC1 suicide inhibitor. We found that polyamine synthesis inhibition partially reversed changes in Ca2+ homeostasis associated with CRC, including a decrease in resting Ca2+ and SOCE along with an increased Ca2+ store content. We also found that polyamine synthesis inhibition reversed transcriptomic changes in CRC cells without affecting normal cells. Specifically, DFMO treatment enhanced the transcription of SOCE modulators CRACR2A; ORMDL3; and SEPTINS 6, 7, 8, 9, and 11, whereas it decreased SPCA2, involved in store-independent Orai1 activation. Therefore, DFMO treatment probably decreased store-independent Ca2+ entry and enhanced SOCE control. Conversely, DFMO treatment decreased the transcription of the TRP channels TRPC1 and 5, TRPV6, and TRPP1 while increasing TRPP2, thus probably decreasing Ca2+ entry through TRP channels. Finally, DFMO treatment enhanced the transcription of the PMCA4 Ca2+ pump and mitochondrial channels MCU and VDAC3 for enhanced Ca2+ extrusion through the plasma membrane and mitochondria. Collectively, these findings suggested the critical role of polyamines in Ca2+ remodeling in colorectal cancer.

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

The 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 (V(M)), 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 function and 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 V(M) and the increased vascular reactivity of hypertensive arteries. We determined the expression levels of inward rectifier K(+) 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 K(IR) and K(ATP) channels in isolated BPH VSMCs. However, only the effect of K(ATP) channel modulators was impaired when exploring vascular tone, suggesting that decreased functional expression of K(ATP) channels may be an important element in the remodelling of VSMCs in essential hypertension.

Genetically engineered mice for combinatorial cardiovascular optobiology.

Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage-specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca2+ sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP3 (optoα1AR) and cAMP (optoß2AR), or Ca2+-permeant membrane channels (CatCh2) in smooth muscle (Acta2) and endothelium (Cdh5). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca2+ imaging in Hcn4BAC-CatCh2/Myh6-GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.

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.

A stochastic model of ion channel cluster formation in the plasma membrane.

Ion channels are often found arranged into dense clusters in the plasma membranes of excitable cells, but the mechanisms underlying the formation and maintenance of these functional aggregates are unknown. Here, we tested the hypothesis that channel clustering is the consequence of a stochastic self-assembly process and propose a model by which channel clusters are formed and regulated in size. Our hypothesis is based on statistical analyses of the size distributions of the channel clusters we measured in neurons, ventricular myocytes, arterial smooth muscle, and heterologous cells, which in all cases were described by exponential functions, indicative of a Poisson process (i.e., clusters form in a continuous, independent, and memory-less fashion). We were able to reproduce the observed cluster distributions of five different types of channels in the membrane of excitable and tsA-201 cells in simulations using a computer model in which channels are "delivered" to the membrane at randomly assigned locations. The model's three parameters represent channel cluster nucleation, growth, and removal probabilities, the values of which were estimated based on our experimental measurements. We also determined the time course of cluster formation and membrane dwell time for CaV1.2 and TRPV4 channels expressed in tsA-201 cells to constrain our model. In addition, we elaborated a more complex version of our model that incorporated a self-regulating feedback mechanism to shape channel cluster formation. The strong inference we make from our results is that CaV1.2, CaV1.3, BK, and TRPV4 proteins are all randomly inserted into the plasma membranes of excitable cells and that they form homogeneous clusters that increase in size until they reach a steady state. Further, it appears likely that cluster size for a diverse set of membrane-bound proteins and a wide range of cell types is regulated by a common feedback mechanism.

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.

Ca(2+) entry into neurons is facilitated by cooperative gating of clustered CaV1.3 channels.

CaV1.3 channels regulate excitability in many neurons. As is the case for all voltage-gated channels, it is widely assumed that individual CaV1.3 channels behave independently with respect to voltage-activation, open probability, and facilitation. Here, we report the results of super-resolution imaging, optogenetic, and electrophysiological measurements that refute this long-held view. We found that the short channel isoform (CaV1.3S), but not the long (CaV1.3L), associates in functional clusters of two or more channels that open cooperatively, facilitating Ca(2+) influx. CaV1.3S channels are coupled via a C-terminus-to-C-terminus interaction that requires binding of the incoming Ca(2+) to calmodulin (CaM) and subsequent binding of CaM to the pre-IQ domain of the channels. Physically-coupled channels facilitate Ca(2+) currents as a consequence of their higher open probabilities, leading to increased firing rates in rat hippocampal neurons. We propose that cooperative gating of CaV1.3S channels represents a mechanism for the regulation of Ca(2+) signaling and electrical activity.

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

Gram-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.