The transient receptor potential channels TRPML2 and TRPML3 (MCOLN2 and MCOLN3) are nonselective cation channels. They are widely expressed in mammals. However, little is known about their physiological function(s) and activation mechanism(s). TRPML3 can be activated or rather de-inhibited by exposing it first to sodium-free extracellular solution and subsequently to high extracellular sodium. TRPML3 can also be activated by a variety of small chemical compounds identified in a high throughput screen and is inhibited by low pH. Furthermore, it was found that TRPML3 is constitutively active in low or no sodium-containing extracellular solution. This constitutive activity is independent of the intracellular presence of sodium, and whole-cell current densities are similar with pipette solutions containing cesium, potassium, or sodium. Here, we present mutagenesis data generated based on the hypothesis that negatively charged amino acids in the extracellular loops of TRPML3 may interfere with the observed sodium inhibition. We systematically mutated negatively charged amino acids in the first and second extracellular loops and found that mutating Glu-361 in the second loop has a significant impact on the sodium-mediated block of TRPML3. We further demonstrate that the TRPML3-related cation channel TRPML2 is also activated by lowering the extracellular sodium concentration as well as by a subset of small chemical compounds that were previously identified as activators of TRPML3, thus confirming the functional activity of TRPML2 at the plasma membrane and suggesting similar gating mechanisms for both TRPML channels.
"These compounds are synthetic and it is unclear whether similar compounds exist in living organisms that might act as natural agonists of TRPML2 and TRPML3. Another stimulus shown to trigger currents in cells heterologously expressing wild-type TRPML2 is the transient lowering of extracellular sodium followed by the replenishment of sodium or other permeant cations (Grimm et al. 2012), a maneuver that was previously found to activate TRPML3 (Grimm et al. 2010; Kim et al. 2007). However, it is unclear whether these ionic changes occur in a natural setting and thus whether this form of channel activation bears physiological relevance. "
[Show abstract][Hide abstract] ABSTRACT: The TRPML2 protein, encoded by the Mcoln2 gene, is one of the three mucolipins (TRPML1-3), a subset of the TRP superfamily of ion channels. Although there are no thorough studies on the cellular distribution of TRPML2, its mRNA appears to be largely restricted to lymphocytes and other immune cells. This contrasts with the ubiquitous expression of TRPML1 and the limited but diverse expression of TRPML3 and clearly suggests a specialized role for TRPML2 in immunity. Localization studies indicate that TRPML2 is present in lysosomes (including the specialized lysosome-related organelle that B-lymphocytes use for processing of the antigen-bound B-cell receptor), late endosomes, recycling endosomes, and, at a much lower level, the plasma membrane. Heterologously expressed TRPML2, like TRPML1 and/or TRPML3, forms ion channels that can be activated by a gain-of-function mutation (alanine to proline in the fifth transmembrane domain, close to the pore) that favors the open state, by a transient reduction of extracellular sodium followed by sodium replenishment, by small chemicals related to sulfonamides, and by PI(3,5)P2, a rare phosphoinositide that naturally accumulates in the membranes of endosomes and lysosomes and thus could act as a physiologically relevant agonist. TRPML2 channels are inwardly rectifying and permeable to Ca(2+), Na(+), and Fe(2+). When heterologously co-expressed, TRPML2 can form heteromultimers with TRPML1 and TRPML3. In B-lymphocytes, TRPML2 and TRPML1 may play redundant roles in the function of their specialized lysosome. Although the specific subcellular function of TRPML2 is unknown, distribution and channel properties suggest roles in calcium release from endolysosomes, perhaps to regulate vesicle fusion and/or subsequent scission or to release calcium from intracellular acidic stores for signaling in the cytosol. Alternatively, TRPML2 could function in the plasma membrane, and its abundance in vesicles of the endocytic pathway could simply be due to regulation by endocytosis and exocytosis. The Mcoln2 gene is closely downstream from and in the same orientation as Mcoln3 in the genomes of most jawed vertebrates (from humans to sharks) with the exception of pigs, Xenopus tropicalis, and ray-finned fishes. The close homology of TRPML2 and 3 (closer to each other than to TRPML1) suggests that Mcoln2 and Mcoln3 arose from unequal crossing over that duplicated a common ancestor and placed both gene copies in tandem. These genes would have come apart subsequently in pigs, Xenopus, and the ancestor to ray-finned fishes. All jawed vertebrates for which we have thorough genomic knowledge have distinct Mcoln1, 2, and 3 genes (except ray-finned fishes which, probably due to the whole-genome duplication in their common ancestor, have two Mcoln1-like genes and two Mcoln3-like genes, although only one Mcoln2 gene). However, the available genomes of invertebrate deuterostomes (a sea urchin, lancelet, and two tunicates) contain a single mucolipin gene that is equally distant from the three vertebrate mucolipins. Hence, vertebrate mucolipins arose through two rounds of gene duplication (the first one likely producing Mcoln1 and the ancestor to Mcoln2 and 3) at some time between the onset of craniates and that of jawed vertebrates. This is also the evolutionary period during which adaptive immunity appeared. Given the restricted expression of TRPML2 in immune cells, this evolutionary history suggests a functional role in the adaptive immunity characteristic of vertebrates.
Handbook of experimental pharmacology 04/2014; 222:647-58. DOI:10.1007/978-3-642-54215-2_25
"Condition 2 contained K+ as the major cation (150 mM K+), a low concentration of Na+ (2 mM), and 1.5 mM Ca2+. As expected, TRPV5 was inactive in this condition and TRPML3 displayed inwardly rectifying currents as shown previously ,  with a current density of −0.10±0.006 nA/pF (n = 12) at −150 mV. "
[Show abstract][Hide abstract] ABSTRACT: TRPML3 and TRPV5 are members of the mucolipin (TRPML) and TRPV subfamilies of transient receptor potential (TRP) cation channels. Based on sequence similarities of the pore forming regions and on structure-function evidence, we hypothesized that the pore forming domains of TRPML and TRPV5/TRPV6 channels have similarities that indicate possible functional interactions between these TRP channel subfamilies. Here we show that TRPML3 and TRPV5 associate to form a novel heteromeric ion channel. This novel conductance is detectable under conditions that do not activate either TRPML3 or TRPV5. It has pharmacological similarity with TRPML3 and requires functional TRPML3 as well as functional TRPV5. Single channel analyses revealed that TRPML3 and TRPV5 heteromers have different features than the respective homomers, and furthermore, that they occur in potentially distinct stoichiometric configurations. Based on overlapping expression of TRPML3 and TRPV5 in the kidney and the inner ear, we propose that TRPML3 and TRPV5 heteromers could have a biological function in these organs.
PLoS ONE 02/2013; 8(2):e58174. DOI:10.1371/journal.pone.0058174 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Survival in the wild requires organismal adaptations to the availability of nutrients. Endosomes and lysosomes are key intracellular organelles that couple nutrition and metabolic status to cellular responses, but how they detect cytosolic ATP levels is not well understood. Here, we identify an endolysosomal ATP-sensitive Na(+) channel (lysoNa(ATP)). The channel is a complex formed by two-pore channels (TPC1 and TPC2), ion channels previously thought to be gated by nicotinic acid adenine dinucleotide phosphate (NAADP), and the mammalian target of rapamycin (mTOR). The channel complex detects nutrient status, becomes constitutively open upon nutrient removal and mTOR translocation off the lysosomal membrane, and controls the lysosome's membrane potential, pH stability, and amino acid homeostasis. Mutant mice lacking lysoNa(ATP) have much reduced exercise endurance after fasting. Thus, TPCs make up an ion channel family that couples the cell's metabolic state to endolysosomal function and are crucial for physical endurance during food restriction.
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