Deviation of the ambient temperature is one of the most ubiquitous stimuli that continuously affect mammals' skin. Although the role of the warmth receptors in epidermal homeostasis (EH) was elucidated in recent years, the mystery of the keratinocyte mild-cold sensor remains unsolved. Here we report the cloning and characterization of a new functional epidermal isoform of the transient receptor potential M8 (TRPM8) mild-cold receptor, dubbed epidermal TRPM8 (eTRPM8), which is localized in the keratinocyte endoplasmic reticulum membrane and controls mitochondrial Ca 2+ concentration ([Ca 2+ ] m ). In turn, [Ca 2+ ] m modulates ATP and superoxide (O •−2 ) synthesis in a cold-dependent manner. We report that this fine tuning of ATP and O •−2 levels by cooling controls the balance between keratinocyte proliferation and differentiation. Finally, to ascertain eTRPM8's role in EH in vivo we developed a new functional knockout mouse strain by deleting the pore domain of TRPM8 and demonstrated that eTRPM8 knockout impairs adaptation of the epidermis to low temperatures.he skin epidermis provides a protective barrier that guards the body against an uncongenial environment. Under the influence of a variety of ambient factors the skin epidermis undergoes continuous regeneration through so-called epidermal homeostasis (EH): the fine-tuning of the balance between proliferation, directional migration, differentiation, and death of keratinocytes. EH involves complex molecular and chemical pathways, regulating dynamic and continuous transition of keratinocytes from the proliferating state in the basal layer to the nonproliferating state in the suprabasal layer before the beginning of the differentiation in the stratum spinosum and stratum granulosum. The terminal differentiation step, characterized by keratinocyte death, transforms keratinocytes into corneocytes, which form the waterproof, mechanically resistant sheath of the stratum corneum (1).Deviation of the ambient temperature is one of the most important stimuli that constantly affect mammals' skin. At ambient temperatures from +10°C to +30°C, the unprotected human skin temperature settles at mean steady-state values within the range of +24°C to +33°C, respectively (2). Temperature is perceived by thermoreceptors, the ion channels that belong to the transient receptor potential (TRP) superfamily (for review see ref.3). Of these, TRPV1 and TRPV2 are activated by heat (above 42°C and above 52°C, respectively) (4), whereas TRPM8 and likely TRPA1 are activated by mild (5, 6) and noxious (7-9) cold, respectively. Heatstimulated keratinocytes have been shown to secrete ATP (10) and, taking into account that purinergic receptors are expressed in keratinocytes (11), TRPV3 is involved in a paracrine heat-

Transient receptor potential (TRP) channels are recently identified proteins that form a versatile family of ion channels, the majority of which are calcium permeable and exhibit complex regulatory patterns with sensitivity to multiple environmental factors. While this sensitivity has captured early attention, leading to recognition of TRP channels as environmental and chemical sensors, many later studies concentrated on the regulation of intracellular calcium by TRP channels. Due to mutations, dysregulation of ion channel gating or expression levels, normal spatiotemporal patterns of local Ca(2+) distribution become distorted. This causes deregulation of downstream effectors sensitive to changes in Ca(2+) homeostasis that, in turn, promotes pathophysiological cancer hallmarks, such as enhanced survival, proliferation and invasion. These observations give rise to the appreciation of the important contributions that TRP channels make to many cellular processes controlling cell fate and positioning these channels as important players in cancer regulation. This review discusses the accumulated scientific knowledge focused on TRP channel involvement in regulation of cell fate in various transformed tissues.

The anti-apoptotic Bcl-2 protein is emerging as an efficient inhibitor of IP3R function, contributing to its oncogenic properties. Yet, the underlying molecular mechanisms remain not fully understood. Using mutations or pharmacological inhibition to antagonize Bcl-2's hydrophobic cleft, we excluded this functional domain as responsible for Bcl-2-mediated IP3Rs inhibition. In contrast, the deletion of the C-terminus, containing the trans-membrane domain, which is only present in Bcl-2α, but not in Bcl-2β, led to impaired inhibition of IP3R-mediated Ca2+ release and staurosporine-induced apoptosis. Strikingly, the trans-membrane domain was sufficient for IP3R binding and inhibition. We therefore propose a novel model, in which the Bcl-2's C-terminus serves as a functional anchor, which beyond mere ER-membrane targeting, underlies efficient IP3R inhibition by (i) positioning the BH4 domain in the close proximity of its binding site on IP3R, thus facilitating their interaction; (ii) inhibiting IP3R-channel openings through a direct interaction with the C-terminal region of the channel downstream of the channel-pore. Finally, since the hydrophobic cleft of Bcl-2 was not involved in IP3R suppression, our findings indicate that ABT-199 does not interfere with IP3R regulation by Bcl-2 and its mechanism of action as a cell-death therapeutic in cancer cells likely does not involve Ca2+ signaling.

Intracellular ion channels are involved in multiple signaling processes, including such crucial ones as regulation of cellular motility and fate. With 95% of the cellular membrane belonging to intracellular organelles, it is hard to overestimate the importance of intracellular ion channels. Multiple studies have been performed on these channels over the years, however, a unified approach allowing not only to characterize their activity but also to study their regulation by partner proteins, analogous to the patch clamp “golden standard”, is lacking. Here, we present a universal approach that combines the extraction of intracellular membrane fractions with the preparation of patchable substrates that allows to characterize these channels in endogenous protein environment and to study their regulation by partner proteins. We validate this method by characterizing activity of multiple intracellular ion channels localized to different organelles and by providing detailed electrophysiological characterization of the regulation of IP3R activity by endogenous Bcl-2. Thus, after synthesis and reshaping of the well-established approaches, organelle membrane derived patch clamp provides the means to assess ion channels from arbitrary cellular membranes at the single channel level.

Store-operated calcium entry (SOCE) plays important roles in a multitude of cellular processes, from muscle contraction to cellular proliferation and migration. Dysregulation of SOCE is responsible for the advancement of multiple diseases, ranging from immune diseases, myopathies, to terminal ones like cancer. Naturally, SOCE has been a focus of many studies and review papers which, however, primarily concentrated on the principal players localized to the plasma membrane and responsible for Ca entry into the cell. Much less has been said about other players participating in the entire SOCE event. This review aims to address this shortcoming by discussing the accumulated scientific knowledge focused on the inositol trisphosphate receptors (IPRs), principal player responsible for emptying intracellular Ca stores in a majority of cells, and their involvement in regulation of cell migration and invasion in cancer.

Aim: Inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitous calcium (Ca2+) channel involved in the regulation of cellular fate and motility. Its modulation by anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) plays an important role in cancer progression. Disrupting this interaction could overcome apoptosis avoidance, one of the hallmarks of cancer, and is, thus, of great interest. Earlier reports have shown the involvement of both the Bcl-2 homology 4 (BH4) and the transmembrane domains (TMDs) of Bcl-2 in regulating IP3R activity, while the Bcl-2 hydrophobic cleft was associated primarily with its anti-apoptotic and IP3R-independent action at the mitochondria (Oncotarget. 2016;7:55704–20. doi: 10.18632/oncotarget.11005). The aim of this study was to investigate how targeting the BH3 hydrophobic cleft of Bcl-2 affects IP3R:Bcl-2 interaction. Methods: Organelle membrane-derived (OMD) patch-clamp and circular dichroism (CD) thermal melting experiments were used to elucidate the effects of the ABT-199 (venetoclax) on the IP3R:Bcl-2 interaction. Molecular dynamics (MD) simulations of free and ABT-199 bound Bcl-2 were used to propose a molecular model of such interaction. Results: It was shown that occlusion of Bcl-2’s hydrophobic cleft by the drug ABT-199 finely modulates IP3R gating in the low open probability (Po) regime, characteristic of the basal IP3R activity in non-excited cells. Complementary MD simulations allowed to propose a model of this modulation, involving an allosteric interaction with the BH4 domain on the opposite side of Bcl-2. Conclusions: Bcl-2 is an important regulator of IP3R activity and, thus of Ca2+ release from internal stores and associated processes, including cellular proliferation and death. The presence of multiple regulatory domains in both proteins suggests a complex interaction. Thus, it was found that the occlusion of the hydrophobic cleft of Bcl-2 by ABT-199 disrupts IP3R activity, leading to Bcl-2 rebinding with smaller affinity and lesser inhibitory effect. MDs simulations of free and ABT-199 bound Bcl-2 propose a molecular model of such disruption, involving an allosteric interaction with the BH4 domain on the opposite side of Bcl-2.

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