Taxol-treatment alters endogenous TRPV1 expression and mitochondrial membrane potential in mesenchymal stem cells: Relevant in chemotherapy-induced pathophysiology.

Microtubule-based chemotherapeutics, primarily Taxane-derived agents are still used as the major live-saving agents, yet have several side effects including serious loss of immune cells, bone density etc. which lowers the quality of life. This imposes the need to understand the effects of these agents on Mesenchymal Stem Cells (MSCs) in details. In this work we demonstrate that Taxol and Nocodazole affects the endogenous expression of TRPV1, a non-selective cation channel in MSCs. These agents also affect the status of polymerized Actin as well as Tyrosinated-tubulin, basal cytosolic Ca2+ and mitochondrial membrane potential (ΔΨm). Notably, pharmacological modulation of TRPV1 by Capsaicin or Capsazepine can also alter the above-mentioned parameters in a context-dependent manner. We suggest that endogenous expression of TRPV1 and pharmacological modulation of TRPV1 can be utilized to rescue some of these parameters effectively. These findings may have significance in the treatments and strategies with Microtubule-based chemotherapeutics and stem-cell based therapy.

Ratio of Hydrophobic-Hydrophilic and Positive-Negative Residues at Lipid-Water-Interface Influences Surface Expression and Channel Gating of TRPV1.

During evolution, TRPV1 has lost, retained or selected certain residues at Lipid-Water-Interface (LWI) and formed specific patterns there. The ratio of "hydrophobic-hydrophilic" and "positive-negative-charged" residues at the inner LWI remains conserved throughout vertebrate evolution and plays important role in regulating TRPV1 trafficking and localization. Arg575 is an important residue as Arg575Asp mutant has reduced surface expression, co-localization with lipid raft markers, cell area and increased cell lethality. This lethality is most likely due to the disruption of the ratio between positive-negative charges caused by the mutation. Such lethality can be rescued by either using TRPV1-specfic inhibitor 5'-IRTX or by restoring the positive-negative charge ratio at that position, i.e. by introducing Asp576Arg mutation in Arg575Asp backbone. We propose that Arg575Asp mutation confers TRPV1 in a "constitutive-open-like" condition. These findings have broader implication in understanding the molecular evolution of thermo-sensitive ion channels and the micro-environments involved in processes that goes erratic in different diseases. The segment of TRPV1 that is present at the inner lipid-water-interface (LWI) has a specific pattern of amino acid combinations. The overall ratio of +ve charge /-ve charge and the ratio of hydrophobicity/hydrophilicity remain constant throughout the vertebrate evolution (ca 450 million years). This specific pattern is not observed in the outer LWI region of TRPV1.

Capsaicin-insensitivity of TRPV1-R575D mutant located at the lipid-water-interface region can be rescued by either extracellular Ca2+-chelation or cholesterol reduction.

TRPV1 acts as a unique polymodal ion channel having distinct structure and gating properties. In this context, TRPV1-R575D represents a special mutant located at the inner lipid-water-interface (LWI) region that has less possibility of interaction with membrane cholesterol. In control conditions, this lab-generated mutant of TRPV1 shows no "ligand-sensitivity", reduced surface expression, reduced localization in the lipid rafts, yet induces high cellular lethality. Notably, the cellular lethality induced by TRPV1-R575D expression can be rescued by adding 5'I-RTX (a specific inhibitor of TRPV1) or by introducing another mutation in the next position, i.e. in TRPV1-R575D/D576R. In this work we characterized TRPV1-R575D and TRPV1-R575D/D576R mutants in different cellular conditions and compared with the TRPV1-WT. We report that the "ligand-insensitivity" of TRPV1-R575D can be rescued in certain conditions, such as by chelation of extracellular Ca2+, or by reduction of the membrane cholesterol. Here we show that Ca2+ plays an important role in the channel gating of TRPV1-WT as well as LWI mutants (TRPV1-R575D, TRPV1-R575D/D576R). However, chelation of intracellular Ca2+ or depletion of ER Ca2+ did not have a significant effect on the TRPV1-R575D. Certain properties related to channel gating of mutant TRPV1-R575D/D576R can be rescued partially or fully in a context -dependent manner. Cholesterol depletion also alters these properties. Our data suggests that lower intracellular basal Ca2+ acts as a pre-requisite for further opening of TRPV1-R575D. These findings enable better understanding of the structure-function relationship of TRPV1 and may be critical in comprehending the channelopathies induced by other homologous thermosensitive TRPVs.

Human skeletal dysplasia causing L596P-mutant alters the conserved amino acid pattern at the lipid-water-Interface of TRPV4.

TRPV4 is a polymodal and non-selective cation channel that is activated by multiple physical and chemical stimuli. >50 naturally occurring point-mutation of TRPV4 have been identified in human, most of which induce different diseases commonly termed as channelopathies. While, these mutations are either "gain-of-function" or "loss-of-function" in nature, the exact molecular and cellular mechanisms behind such diverse channelopathies are largely unknown. In this work, we analyze the evolutionary conservation of individual amino acids present in the lipid-water-interface (LWI) regions and the relationship of TRPV4 with membrane cholesterol. Our data suggests that the positive-negative charges and hydrophobic-hydrophilic amino acids form "specific patterns" in the LWI region which remain conserved throughout the vertebrate evolution and thus suggesting for the specific microenvironment where TRPV4 remain functional. Notably, Spondylometaphyseal Dysplasia, Kozlowski (SMDK) disease causing L596P mutation disrupts this pattern significantly at the LWI region. L596P mutant also sequesters Caveolin-1 differently, especially in partial cholesterol-depleted (~40 % reduction) conditions. L596P shows altered localization in membrane and enhanced Ca2+-influx properties in cell as well as in filopodia-like structures. We propose that conserved pattern of amino acids is an important parameter for proper localization and functions of TRPV4 in physiological conditions. These findings also offer a new paradigm to analyze the channelopathies caused by mutations in LWI regions of other channels as well.