The subjective feeling of cold is mediated by the activation of TRPM8 channels in thermoreceptive neurons by cold or by cooling agents such as menthol. Here, we demonstrate a central role for phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) in the activation of recombinant TRPM8 channels by both cold and menthol. Moreover, we show that Ca(2+) influx through these channels activates a Ca(2+)-sensitive phospholipase C and that the subsequent depletion of PI(4,5)P(2) limits channel activity, serving as a unique mechanism for desensitization of TRPM8 channels. Finally, we find that mutation of conserved positive residues in the highly conserved proximal C-terminal TRP domain of TRPM8 and two other family members, TRPM5 and TRPV5, reduces the sensitivity of the channels for PI(4,5)P(2) and increases inhibition by PI(4,5)P(2) depletion. These data suggest that the TRP domain of these channels may serve as a PI(4,5)P(2)-interacting site and that regulation by PI(4,5)P(2) is a common feature of members of the TRP channel family.
Summary Cyclic nucleotide-gated (CNG) channels are essential for vision and olfaction. They belong to the voltage-gated ion channel superfamily but their activities are controlled by intracellular cyclic nucleotides instead of transmembrane voltage. Here we report a 3.5 Å-resolution single-particle electron cryomicroscopy structure of a CNG channel from C. elegans in the cGMP-bound open state. The channel has an unusual voltage-sensor-like domain (VSLD), accounting for its deficient voltage dependence. A C-terminal linker connecting S6 and the cyclic nucleotide-binding domain interacts directly with both the VSLD and pore domain, forming a gating ring that couples conformational changes triggered by cyclic nucleotide binding to the gate. The selectivity filter is lined by the carboxylate side chains of a functionally important glutamate and three rings of backbone carbonyls. This structure provides a new framework for understanding mechanisms of ion permeation, gating and channelopathy of CNG channels and cyclic nucleotide modulation of related channels.
Organellar ion channels are essential for cell physiology. Their activities are often regulated by Ca2+ and H+, which are concentrated in many organelles. Here we report a novel structural element critical for Ca2+/pH dual regulation of TRPML1, a Ca2+ release channel crucial for endolysosomal functions. TRPML1 mutations cause mucolipidosis type IV (MLIV), a severe lysosomal storage disorder characterized by neurodegeneration, mental retardation and blindness. We obtained high-resolution crystal structures of a 213-amino acid luminal domain of human TRPML1 that harbors three missense MLIV-causing mutations. This domain forms a tetramer with a highly electronegative central pore formed by a novel luminal pore-loop. Cysteine crosslinking and cryo-EM confirm this structure in the full-length channel. Structure-function studies demonstrate that Ca2+ and H+ interact with the luminal pore to exert physiologically important regulation. The MLIV-causing mutations disrupt the luminal domain structure and cause TRPML1 mislocalization. Our study provides a structural underpinning for TRPML1's regulation, assembly and pathogenesis.
Regulation of the abundance of NMDA receptors (NMDARs) at excitatory synapses is critical during changes in synaptic efficacy underlying learning and memory as well as during synapse formation throughout neural development. However, the molecular signals that govern NMDAR delivery, maintenance, and internalization remain unclear. In this study, we identify a conserved family of membraneproximal endocytic signals, two within the NMDAR type 1 (NR1) subunit and one within the NR2A and NR2B subunits, necessary and sufficient to drive the internalization of NMDARs. These endocytic motifs reside in the region of NMDAR subunits immediately after the fourth membrane segment, a region implicated in use-dependent rundown and NMDA channel inactivation. Although endocytosis driven by the distal C-terminal domain of NR2B is followed by rapid recycling, internalization mediated by membrane-proximal motifs selectively targets receptors to late endosomes and accelerates degradation. These results define a novel conserved signature of NMDARs regulating internalization and postendocytic trafficking.
Phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2 or PIP2] is a direct modulator of a diverse array of proteins in eukaryotic cells. The functional integrity of transmembrane proteins, such as ion channels and transporters, is critically dependent on specific interactions with PIP2 and other phosphoinositides. Here, we report a novel requirement for PIP2 in the activation of the epidermal growth factor receptor (EGFR). Down-regulation of PIP2 levels either via pharmacological inhibition of PI kinase activity, or via manipulation of the levels of the lipid kinase PIP5K1α and the lipid phosphatase synaptojanin, reduced EGFR tyrosine phosphorylation, whereas up-regulation of PIP2 levels via overexpression of PIP5K1α had the opposite effect. A cluster of positively charged residues in the juxtamembrane domain (basic JD) of EGFR is likely to mediate binding of EGFR to PIP2 and PIP2-dependent regulation of EGFR activation. A peptide mimicking the EGFR juxtamembrane domain that was assayed by surface plasmon resonance displayed strong binding to PIP2. Neutralization of positively charged amino acids abolished EGFR/PIP2 interaction in the context of this peptide and down-regulated epidermal growth factor (EGF)-induced EGFR autophosphorylation and EGF-induced EGFR signaling to ion channels in the context of the full-length receptor. These results suggest that EGFR activation and downstream signaling depend on interactions of EGFR with PIP2 and point to the basic JD’s critical involvement in these interactions. The addition of this very different class of membrane proteins to ion channels and transporters suggests that PIP2 may serve as a general modulator of the activity of many diverse eukaryotic transmembrane proteins through their basic JDs.
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