Debapriya Ghosh

Inhibition of the cation channel TRPV4 improves bladder function in mice and rats with cyclophosphamide-induced cystitis

Reduced functional bladder capacity and concomitant increased micturition frequency (pollakisuria) are common lower urinary tract symptoms associated with conditions such as cystitis, prostatic hyperplasia, neurological disease, and overactive bladder syndrome. These symptoms can profoundly affect the quality of life of afflicted individuals, but available pharmacological treatments are often unsatisfactory. Recent work has demonstrated that the cation channel TRPV4 is highly expressed in urothelial cells and plays a role in sensing the normal filling state of the bladder. In this article, we show that the development of cystitis-induced bladder dysfunction is strongly impaired in Trpv4−/− mice. Moreover, we describe HC-067047, a previously uncharacterized, potent, and selective TRPV4 antagonist that increases functional bladder capacity and reduces micturition frequency in WT mice and rats with cystitis. HC-067047 did not affect bladder function in Trpv4−/− mice, demonstrating that its in vivo effects are on target. These results indicate that TRPV4 antagonists may provide a promising means of treating bladder dysfunction.

Cholesterol loss during glutamate-mediated excitotoxicity

The deregulation of brain cholesterol metabolism is typical in acute neuronal injury (such as stroke, brain trauma and epileptic seizures) and chronic neurodegenerative diseases (Alzheimers disease). Since both conditions are characterized by excessive stimulation of glutamate receptors, we have here investigated to which extent excitatory neurotransmission plays a role in brain cholesterol homeostasis. We show that a short (30 min) stimulation of glutamatergic neurotransmission induces a small but significant loss of membrane cholesterol, which is paralleled by release to the extracellular milieu of the metabolite 24S‐hydroxycholesterol. Consistent with a cause–effect relationship, knockdown of the enzyme cholesterol 24‐hydroxylase (CYP46A1) prevented glutamate‐mediated cholesterol loss. Functionally, the loss of cholesterol modulates the magnitude of the depolarization‐evoked calcium response. Mechanistically, glutamate‐induced cholesterol loss requires high levels of intracellular Ca2+, a functional stromal interaction molecule 2 (STIM2) and mobilization of CYP46A1 towards the plasma membrane. This study underscores the key role of excitatory neurotransmission in the control of membrane lipid composition, and consequently in neuronal membrane organization and function.

Regulation of the transient receptor potential channel TRPM3 by phosphoinositides

TRPM3 is dynamically regulated by plasma membrane PI(4,5)P2 and related PIPs.

VAMP7 regulates constitutive membrane incorporation of the cold-activated channel TRPM8

The cation channel TRPM8 plays a central role in the somatosensory system, as a key sensor of innocuously cold temperatures and cooling agents. Although increased functional expression of TRPM8 has been implicated in various forms of pathological cold hypersensitivity, little is known about the cellular and molecular mechanisms that determine TRPM8 abundance at the plasma membrane. Here we demonstrate constitutive transport of TRPM8 towards the plasma membrane in atypical, non-acidic transport vesicles that contain lysosomal-associated membrane protein 1 (LAMP1), and provide evidence that vesicle-associated membrane protein 7 (VAMP7) mediates fusion of these vesicles with the plasma membrane. In line herewith, VAMP7-deficient mice exhibit reduced functional expression of TRPM8 in sensory neurons and concomitant deficits in cold avoidance and icilin-induced cold hypersensitivity. Our results uncover a cellular pathway that controls functional plasma membrane incorporation of a temperature-sensitive TRP channel, and thus regulates thermosensitivity in vivo.

TRPV4 participates in the establishment of trailing adhesions and directional persistence of migrating cells.

Calcium signaling participates in different cellular processes leading to cell migration. TRPV4, a non-selective cation channel that responds to mechano-osmotic stimulation and heat, is also involved in cell migration. However, the mechanistic involvement of TRPV4 in cell migration is currently unknown. We now report that expression of the mutant channel TRPV4-121AAWAA (lacking the phosphoinositide-binding site 121KRWRK125 and the response to physiological stimuli) altered HEK293 cell migration. Altered migration patterns included periods of fast and persistent motion followed by periods of stalling and turning, and the extension of multiple long cellular protrusions. TRPV4-WT overexpressing cells showed almost complete loss of directionality with frequent turns, no progression, and absence of long protrusions. Traction microscopy revealed higher tractions forces in the tail of TRPV4-121AAWAA than in TRPV4-WT expressing cells. These results are consistent with a defective and augmented tail retraction in TRPV4-121AAWAA- and TRPV4-WT-expressing cells, respectively. The activity of calpain, a protease implicated in focal adhesion (FA) disassembly, was decreased in TRPV4-121AAWAA compared with TRPV4-WT-expressing cells. Consistently, larger focal adhesions were seen in TRPV4-121AAWAA compared with TRPV4-WT-expressing HEK293 cells, a result that was also reproduced in T47D and U87 cells. Similarly, overexpression of the pore-dead mutant TRPV4-M680D resumed the TRPV4-121AAWAA phenotype presenting larger FA. The migratory phenotype obtained in HEK293 cells overexpressing TRPV4-121AAWAA was mimicked by knocking-down TRPC1, a cationic channel that participates in cell migration. Together, our results point to the participation of TRPV4 in the dynamics of trailing adhesions, a function that may require the interplay of TRPV4 with other cation channels or proteins present at the FA sites.

Distinct modes of perimembrane TRP channel turnover revealed by TIR-FRAP

Transient Receptor Potential (TRP) channels form a broadly expressed and functionally diverse family of cation channels involved in various (patho)physiological processes. Whereas the mechanisms that control opening of TRP channels have been extensively studied, little is known about the transport processes of TRP channels to and within the plasma membrane. Here we used Total Internal Reflection - Fluorescence Recovery after Photobleaching (TIR-FRAP) to selectively visualize and bleach the fluorescently labeled TRP channels TRPV2 and TRPM4 in close proximity of the glass-plasma membrane interface, allowing detailed analysis of their perimembrane dynamics. We show that recovery of TRPM4 occurs via 200-nm diameter transport vesicles, and demonstrate the full fusion of such vesicles with the plasma membrane. In contrast, TRPV2 recovery proceeded mainly via lateral diffusion from non-bleached areas of the plasma membrane. Analysis of the two-dimensional channel diffusion kinetics yielded 2D diffusion coefficients ranging between 0.1 and 0.3 μm2/s, suggesting that these TRP channels move relatively unrestricted within the plasma membrane. These data demonstrate distinct modes of TRP channel turnover at the plasma membrane and illustrate the usefulness of TIR-FRAP to monitor these processes with high resolution.

Definition of two agonist types at the mammalian cold-activated channel TRPM8

Various TRP channels act as polymodal sensors of thermal and chemical stimuli, but the mechanisms whereby chemical ligands impact on TRP channel gating are poorly understood. Here we show that AITC (allyl isothiocyanate; mustard oil) and menthol represent two distinct types of ligands at the mammalian cold sensor TRPM8. Kinetic analysis of channel gating revealed that AITC acts by destabilizing the closed channel, whereas menthol stabilizes the open channel, relative to the transition state. Based on these differences, we classify agonists as either type I (menthol-like) or type II (AITC-like), and provide a kinetic model that faithfully reproduces their differential effects. We further demonstrate that type I and type II agonists have a distinct impact on TRPM8 currents and TRPM8-mediated calcium signals in excitable cells. These findings provide a theoretical framework for understanding the differential actions of TRP channel ligands, with important ramifications for TRP channel structure-function analysis and pharmacology. DOI: http://dx.doi.org/10.7554/eLife.17240.001

Calcium Channels in Vascular Smooth Muscle

Calcium (Ca2+) plays a central role in excitation, contraction, transcription, and proliferation of vascular smooth muscle cells (VSMs). Precise regulation of intracellular Ca2+ concentration ([Ca2+]i) is crucial for proper physiological VSM function. Studies over the last several decades have revealed that VSMs express a variety of Ca2+-permeable channels that orchestrate a dynamic, yet finely tuned regulation of [Ca2+]i. In this review, we discuss the major Ca2+-permeable channels expressed in VSM and their contribution to vascular physiology and pathology.

Dynamic L-type Ca V 1.2 channel trafficking facilitates Ca V 1.2 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.

Journey of a cold sensor - VAMP7-dependent transport of TRPM8.

TRPM8, a member of the transient receptor potential (TRP) superfamily of cation channels, is expressed in a subset of somatosensory neurons, where it plays a central role in thermosensation, as the principal sensor of innocuous cold temperatures and cooling agents such as menthol or icilin. Pharmacological inhibition or genetic deletion of TRPM8 causes strong deficits in the sensation of innocuously cold temperatures, whereas hyperactivation causes pathological cold hypersensitivity. The (patho)physiological functions of TRPM8 depend on the total ionic current through active channels in the plasma membrane, which in turn is governed by its gating mechanisms, ion permeability and the total number of channels at the site of action. Earlier work has greatly advanced our knowledge about the gating and ion permeability properties of TRPM8, including its regulation by cellular signaling pathways. However, very little insight was available about the intracellular trafficking mechanism of TRPM8 to the plasma membrane (PM), as is true for most other TRP channels. Nevertheless, modulation of the number of active TRP channels at the cell surface represents an important regulatory mechanism under normal and pathophysiological conditions. Earlier studies had provided evidence that TRPM8 may reside in lipid rafts at the PM, indicated that TRPM8 may exhibit various modes of near membrane trafficking, and suggested that channel stimulation may stimulate incorporation of additional channels at the PM via exocytosis. However, in these earlier studies, information regarding cellular structures and molecular determinants that governed the intracellular trafficking of TRPM8 to the cell surface were not elucidated. In the article “VAMP7 regulates constitutive membrane incorporation of the cold-activated channel TRPM8” we have elucidated important aspects of the pathway carrying TRPM8 channels to the PM. In the first part of the work, we used Total-Internal-Reflection-Fluorescence–microscopy (TIRFM) to observe near membrane events in live cells. Using this technique, we showed that fluorescently tagged TRPM8 resides in very dynamic vesicles (speed 0.58 mm/s), which exhibit constitutive movement, mainly via microtubules. However, we were unable to measure an increased TRPM8 trafficking to the PM following agonist treatment, in contrast to a previous study. Next, we characterized the nature of these TRPM8-transporting vesicles, by determining which of a list of known markers of intracellular compartments are co-transported within these vesicles. To achieve this, we used dual color TIRFM to simultaneously monitor the movement of TRPM8-mCherry along with GFP-tagged marker proteins. Thus, TRPM8 was considered to dynamically colocalize with specific marker proteins when we observed a vesicular structure exhibiting both GFP and mCherry fluorescence moving alongside. Using this approach, we observed that TRPM8 is primarily found in LAMP1and Rab7carrying vesicles. Although Rab7 and LAMP1 are classically known to as marker of endo-lysosomes, we observed that the TRPM8-containing vesicles were non-acidic in nature, and did not function as protein breakdown organelles.