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.

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.

Ca_V1.3 channels regulate excitability in many neurons. As is the case for all voltage-gated channels, it is widely assumed that individual Ca_V1.3 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 (Ca_V1.3_S), but not the long (Ca_V1.3_L), associates in functional clusters of two or more channels that open cooperatively, facilitating Ca²⁺ influx. Ca_V1.3_S channels are coupled via a C-terminus-to-C-terminus interaction that requires binding of the incoming Ca²⁺ to calmodulin (CaM) and subsequent binding of CaM to the pre-IQ domain of the channels. Physically-coupled channels facilitate Ca²⁺ currents as a consequence of their higher open probabilities, leading to increased firing rates in rat hippocampal neurons. We propose that cooperative gating of Ca_V1.3_S channels represents a mechanism for the regulation of Ca²⁺ signaling and electrical activity.

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. 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.

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 Ca_V1.2 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 Ca_V1.2, Ca_V1.3, 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.

Key points: A hallmark of essential hypertension is an anomalous vascular tone due to the increase in intracellular Ca²⁺ ([Ca²⁺]_i) and/or Ca²⁺ sensitivity in vascular smooth muscle. Down‐regulation of K⁺ channels together with increased Ca_V1.2 channel function has been proposed as one pathogenic mechanism. Using a mouse model of essential hypertension (BPH line), we found a decrease in the global smooth muscle Ca²⁺ influx due to fewer Ca_V1.2 channels. However, these Ca_V1.2 channels are hyperactive, allowing a larger local Ca²⁺ influx at rest that triggers an increased Ca²⁺ release from intracellular stores (sparks). Large conductance, Ca²⁺‐activated K⁺ (BK) channels of BPH myocytes show reduced Ca²⁺ sensitivity, so that its activation by the increased [Ca²⁺]_i is impaired. Our results suggest that changes in the molecular composition of both Ca_V1.2 and BK channels could explain vascular dysfunction during hypertension in BPH mice. Abstract  Hypertension is a clinical syndrome characterized by increased arterial tone. Although the mechanisms are varied, the generally accepted view is that increased Ca_V1.2 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 Ca_V1.2 currents (I_Ca) were lower in hypertensive (BPH line) than normotensive (BPN line) myocytes. However, local Ca_V1.2 sparklet activity was higher in BPH cells, suggesting that the relatively low I_Ca in these cells was produced by a few hyperactive Ca_V1.2 channels. Furthermore, our data suggest that while the lower expression of the pore‐forming α_1c subunit of Ca_V1.2 currents underlies the lower I_Ca in BPH myocytes, the increased sparklet activity was due to a different composition in the auxiliary subunits of the Ca_V1.2 complexes. I_Ca currents in BPN cells were produced by channels composed of α_1c/α₂δ/β₃ subunits, while in BPH myocytes currents were probably generated by the opening of channels formed by α_1c/α₂δ/β₂ subunits. In addition, Ca²⁺ sparks evoked large conductance, Ca²⁺‐activated K⁺ (BK) currents of lower magnitude in BPH than in BPN myocytes, because BK channels were less sensitive to Ca²⁺. Our data are consistent with a model in which a decrease in the global number of Ca_V1.2 currents coexist with the existence of a subpopulation of highly active channels that dominate the resting Ca²⁺ influx. The decrease in BK channel activity makes the hyperpolarizing brake ineffective and leads BPH myocytes to a more contracted resting state.