The cold and menthol receptor, TRPM8, also designated CMR1, is a member of the transient receptor potential (TRP) family of excitatory ion channels. TRPM8 is a channel activated by cold temperatures, voltage, and menthol. In this study, we characterize the coldand voltage-induced activation of TRPM8 channel in an attempt to identify the temperature-and voltage-dependent components involved in channel activation. Under equilibrium conditions, decreasing temperature has two effects. Since the breakthrough identification of the vanilloid receptor, TRPV1, as the hot-capsaicin receptor (1), six temperature-dependent channels have been cloned during the last 7 years (1-8). Interestingly, all of the cloned channels belong to the extended transient receptor potential (TRP) family of excitatory channels (9, 10). TRP channels are membrane proteins constituted by subunits containing six-transmembrane domains, which assemble into tetramers forming conducting pores (11, 12). Heat-dependent channels belong to the vanilloid receptor TRPV subfamily and the cold-dependent channel TRPM8 belongs to the TRPM subfamily (13). Thermo-TRP channels have different temperature thresholds for activation, and their activity is modulated by several different agonists, allowing us to sense and differentiate a large spectrum of temperatures, from below 0°C to 50°C (14). Among those temperature-activated channels, TRPM8 was the first found to sense a cold stimulus (4, 7). TRPM8 is a nonselective outwardly rectifying channel. The channel opens at Ϸ28°C, and its activity increases as the temperature diminishes and saturates at Ϸ10°C (4). The channel is also able to respond to several agonists, including menthol (15).Channel gating is known to be affected by temperature. Q 10 value is used to estimate the temperature dependence of a given system. Channels with a Q 10 Ն 2 are considered highly temperature-dependent (16). Thermo-TRP receptor channels present exceptionally high Q 10 values (Ͼ20) (13).Several studies have addressed capsaicin binding (17, 18), pH dependencies (19), physiological relevance (13), and, recently, thermodynamics (20) of TRPV1, one of the molecular transducers of heat sensation. Large values for transitional entropy, enthalpy, and Q 10 were found, (20) indicating large rearrangements in the channel structure during activation. In contrast, little is known about regulation of its counterpart, TRPM8.Here we show that the large changes in TRPM8 channel gating induced by temperature are mainly due to modifications of the maximum probability of opening and to a shift along the voltage axis of the conductance-voltage curves. Moreover, the results can be fully explained by using an allosteric model in which temperature has only a moderate effect on the voltage sensors (Q 10 Ϸ 3) when channels are closed. Thus, temperature and voltage sensor activation act almost independently to promote channel opening. MethodsTemperature Control and Ramps. The recording chamber consisted of an electrically isolated bronze block with a hole of the...

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...

No abstract

TRPM1 (melastatin), which encodes the founding member of the TRPM family of transient receptor potential (TRP) ion channels, was first identified by its reduced expression in a highly metastatic mouse melanoma cell line. Clinically, TRPM1 is used as a predictor of melanoma progression in humans because of its reduced abundance in more aggressive forms of melanoma. Although TRPM1 is found primarily in melanin-producing cells and has the molecular architecture of an ion channel, its function is unknown. Here we describe an endogenous current in primary human neonatal epidermal melanocytes and mouse melanoma cells that was abrogated by expression of microRNA directed against TRPM1. Messenger RNA analysis showed that at least five human ion channel-forming isoforms of TRPM1 could be present in melanocytes, melanoma, brain, and retina. Two of these isoforms are encoded by highly conserved splice variants that are generated by previously uncharacterized exons. Expression of these two splice variants in human melanoma cells generated an ionic current similar to endogenous TRPM1 current. In melanoma cells, TRPM1 is prevalent in highly dynamic intracellular vesicular structures. Plasma membrane TRPM1 currents are small, raising the possibility that their primary function is intracellular, or restricted to specific regions of the plasma membrane. In neonatal human epidermal melanocytes, TRPM1 expression correlates with melanin content. We propose that TRPM1 is an ion channel whose function is critical to normal melanocyte pigmentation and is thus a potential target for pigmentation disorders.

Although a unifying characteristic common to all transient receptor potential (TRP) channel functions remains elusive, they could be described as tetramers formed by subunits with six transmembrane domains and containing cation-selective pores, which in several cases show high calcium permeability. TRP channels constitute a large superfamily of ion channels, and can be grouped into seven subfamilies based on their amino acid sequence homology: the canonical or classic TRPs, the vanilloid receptor TRPs, the melastatin or long TRPs, ankyrin (whose only member is the transmembrane protein 1 [TRPA1]), TRPN after the nonmechanoreceptor potential C (nonpC), and the more distant cousins, the polycystins and mucolipins. Because of their role as cellular sensors, polymodal activation and gating properties, many TRP channels are activated by a variety of different stimuli and function as signal integrators. Thus, how TRP channels function and how function relates to given structural determinants contained in the channel-forming protein has attracted the attention of biophysicists as well as molecular and cell biologists. The main purpose of this review is to summarize our present knowledge on the structure of channels of the TRP ion channel family. In the absence of crystal structure information for a complete TRP channel, we will describe important protein domains present in TRP channels, structure-function mutagenesis studies, the few crystal structures available for some TRP channel modules, and the recent determination of some TRP channel structures using electron microscopy.

Large conductance Ca 2+ -activated K + (BK) channels belong to the S4 superfamily of K + channels that include voltage-dependent K + (Kv) channels characterized by having six (S1-S6) transmembrane domains and a positively charged S4 domain. As Kv channels, BK channels contain a S4 domain, but they have an extra (S0) transmembrane domain that leads to an external NH 2 -terminus. The BK channel is activated by internal Ca 2+ , and using chimeric channels and mutagenesis, three distinct Ca 2+ -dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca 2+ -binding domains activate the BK channel when cytoplasmic Ca 2+ reaches micromolar concentrations, and a low Ca 2+ affinity mechanism may be involved in the physiological regulation by Mg 2+ . The presence in the BK channel of multiple Ca 2+ -binding sites explains the huge Ca 2+ concentration range (0.1 μM-100 μM) in which the divalent cation influences channel gating. BK channels are also voltage-dependent, and all the experimental evidence points toward the S4 domain as the domain in charge of sensing the voltage. Calcium can open BK channels when all the voltage sensors are in their resting configuration, and voltage is able to activate channels in the complete absence of Ca 2+ . Therefore, Ca 2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism.

In this review, we discuss a novel function of ascorbic acid in brain energetics. It has been proposed that during glutamatergic synaptic activity neurons preferably consume lactate released from glia. The key to this energetic coupling is the metabolic activation that occurs in astrocytes by glutamate and an increase in extracellular [K+]. Neurons are cells well equipped to consume glucose because they express glucose transporters and glycolytic and tricarboxylic acid cycle enzymes. Moreover, neuronal cells express monocarboxylate transporters and lactate dehydrogenase isoenzyme 1, which is inhibited by pyruvate. As glycolysis produces an increase in pyruvate concentration and a decrease in NAD+/NADH, lactate and glucose consumption are not viable at the same time. In this context, we discuss ascorbic acid participation as a metabolic switch modulating neuronal metabolism between rest and activation periods. Ascorbic acid is highly concentrated in CNS. Glutamate stimulates ascorbic acid release from astrocytes. Ascorbic acid entry into neurons and within the cell can inhibit glucose consumption and stimulate lactate transport. For this switch to occur, an ascorbic acid flow is necessary between astrocytes and neurons, which is driven by neural activity and is part of vitamin C recycling. Here, we review the role of glucose and lactate as metabolic substrates and the modulation of neuronal metabolism by ascorbic acid.