Temperature transduction in mammals is possible because of the presence of a set of temperature-dependent transient receptor potential (TRP) channels in dorsal root ganglia neurons and skin cells. Six thermo-TRP channels, all characterized by their unusually high temperature sensitivity (Q 10 Ͼ 10), have been cloned: TRPV1-4 are heat activated, whereas TRPM8 and TRPA1 are activated by cold. Because of the lack of structural information, the molecular basis for regulation by temperature remains unknown. In this study, we assessed the role of the C-terminal domain of thermo-TRPs and its involvement in thermal activation by using chimeras between the heat receptor TRPV1 and the cold receptor TRPM8, in which the entire C-terminal domain was switched. Here, we demonstrate that the C-terminal domain is modular and confers the channel phenotype regarding temperature sensitivity, channel gating kinetics, and PIP 2 (phosphatidylinositol-4,5-bisphophate) modulation. Thus, thermo-TRP channels contain an interchangeable specific region, different from the voltage sensor, which allows them to sense temperature stimuli.

Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a central role in the activation of several transient receptor potential (TRP) channels. The role of PIP2 on temperature gating of thermoTRP channels has not been explored in detail, and the process of temperature activation is largely unexplained. In this work, we have exchanged different segments of the C-terminal region between coldsensitive (TRPM8) and heat-sensitive (TRPV1) channels, trying to understand the role of the segment in PIP2 and temperature activation. A chimera in which the proximal part of the C-terminal of TRPV1 replaces an equivalent section of TRPM8 C-terminal is activated by PIP2 and confers the phenotype of heat activation. PIP2, but not temperature sensitivity, disappears when positively charged residues contained in the exchanged region are neutralized. Shortening the exchanged segment to a length of 11 aa produces voltage-dependent and temperature-insensitive channels. Our findings suggest the existence of different activation domains for temperature, PIP2, and voltage. We provide an interpretation for channel-PIP2 interaction using a full-atom molecular model of TRPV1 and PIP2 docking analysis. chimera ͉ temperature activation ͉ C-terminal domain ͉ molecular model P hosphatidylinositol 4,5-bisphosphate (PIP 2 ) acts as a second messenger phospholipid and is the source of another three lipidic-derived messengers (DAG, IP 3 , PIP 3 ). Although the amount of PIP 2 in the membrane is very low, it is able to regulate the activity of ion channels transporters and enzymes (1-3). Several TRP channels reveal some degree of PIP 2 dependence. PIP 2 depletion inhibits TRPM7, TRPM5, TRPM8, TRPV5, and TRPM4 currents (4-9). In the case of TRPM8, some key positively charged residues present in a well conserved sequence contained in the C-terminal region of TRP channels, the TRP domain, were found to be crucial in determining the apparent affinity of PIP 2 activation (7). Residues K995, R998, and R1008 in the TRP box and TRP domain are critically involved in the activation of TRPM8 by PIP 2 . The hydrolysis of PIP 2 also constitutes an important mechanism for the Ca 2ϩ -dependent desensitization of TRPM8 (6, 7). Because of the high sequence similarity among TRP channels in the TRP domain region, it has been proposed that the family of TRP channels possesses a common PIP 2 -binding site located on its proximal C terminus (7, 10, 11). Different from its counterparts, TRPV1 shows a PLC/ NGF-dependent inhibition (12), where binding of NGF to trkA is coupled to PLC activation that leads to PIP 2 hydrolysis. Mutagenesis experiments suggested the presence of a PIP 2 -dependent inhibitory domain (13). In this model, the sensitization observed in TRPV1 is explained on the basis of PIP 2 hydrolysis as it acts as a tonical inhibitor. An alternative model has been proposed for the inhibition based on NGF-dependent phosphorylation of the TRPV1 C-terminal domain and a subsequent increase in membrane expression (14). These observations, together with the finding that...

Serum creatine kinase (CK) activity, calcium (Ca) and magnesium (Mg) contents of skeletal muscle and isolated mitochondria, as well as oxidative phosphorylation of X-linked muscular dystrophic (mdx) mice were compared with normal control animals at ages 5, 10, and 23 weeks. Serum CK is elevated in mdx mice at all ages, with highest activities at 5 weeks. The Ca content of dystrophic skeletal muscle is increased at all ages, whereas no clearly abnormal trend in muscle Mg levels was observed. Noncollagen protein (NCP), which was used as a reference base, is significantly diminished in muscle from 10- and 23-week-old mdx animals. Isolated mitochondria from mdx mice have elevated calcium content and decreased respiratory control ratios with NAD-linked substrates pyruvate/malate. The findings are distinct from those in dystrophic mice, strain 129/ReJ, but similar to observations in dystrophic hamsters and Duchenne muscular dystrophy and reflect the occurrence of overt muscle cell necrosis.

In voltage-dependent channels, positive charges contained within the S4 domain are the voltage-sensing elements. The ''voltagesensor paddle'' gating mechanism proposed for the KvAP K ؉ channel has been the subject of intense discussion regarding its general applicability to the family of voltage-gated channels. In this model, the voltage sensor composed of the S3b and the S4 segment shuttles across the lipid bilayer during channel activation. Guided by this mechanism, we assessed here the accessibility of residues in the S3 segment of the Shaker K ؉ channel by using cysteine-scanning mutagenesis. Mutants expressed robust K ؉ currents in Xenopus oocytes and reacted with methanethiosulfonate ethyltrimethylammonium in both closed and open conformations of the channel. Because Shaker has a long S3-S4 linker segment, we generated a deletion mutant with only three residues to emulate the KvAP structure. In this short linker mutant, all of the tested residues in the S3b were accessible to methanethiosulfonate ethyltrimethylammonium in both closed and open conformations. Because the S3b moves together with the S4 domain in the paddle model, we tested the effects of deleting two negative charges or adding a positive charge to this region of the channel. We found that altering the S3b net charge does not modify the total gating charge involved in channel activation. We conclude that the S3b segment is always exposed to the external milieu of the Shaker K ؉ channel. Our results are incompatible with any model involving a large membrane displacement of segment S3b. methanethiosulfonate ethyltrimethylammonium accessibility ͉ paddle model ͉ S3-S4 linker ͉ gating charge