Marianne Musinszki

Two crystallographic states of mechanosensitive TREK/TRAAK K2P channels - a low-activity down-state and a high-activity up-state - have been proposed to underlie gating, but the origin of the low activity remains debated. Competing models suggest either lipid-mediated pore block or selectivity filter (SF) inactivation. Using systematic mutagenesis of M2/M4 helices, we identified 16 highly active mutants and assessed their activation mechanisms via free-energy calculations, molecular dynamics simulations, and a state-dependent pharmacological probe. The computational approaches reliably predicted mutation-induced shifts in the down-up equilibrium. We further show that intracellular acidification and regulatory lipids primarily stabilize the up-state, consistent with stretch, temperature, and dephosphorylation. These findings support the down-up transition as the principal physiological activation pathway and suggest that mechanosensitivity arises from the larger membrane footprint of the up-state. Our data argue against a physiological role of a lipid-blocked pore and instead support gating via conformational control of the SF in TREK/TRAAK channels.

Polyphenolic compounds are widely explored for health benefits, including hypertension, but their active ingredients, molecular targets, and mechanisms remain poorly defined. We identify the xanthone Mangostin from Garcinia mangostana as a potent modulator of several potassium channels, with large-conductance K ⁺ (BK) channels as its primary target for vasorelaxation. Mangostin-activated BK channels as α subunits alone, in complexes with vascular β1 subunits, and in reconstituted BKα/β1–Ca _v nanodomains. It shifted BK voltage activation to more negative potentials by antagonizing channel closure and promoting channel opening without markedly altering Ca² ⁺ sensitivity. Docking, competition, single-channel analysis, and mutagenesis localized the binding site in the pore cavity below the SF, involving gating-critical S6 residues I308, L312, and A316, and suggest that Mangostin stays bound in closed and open states. These findings establish BK channel activation as the core molecular mechanism driving Mangostin’s vascular effects and define its structural mode of action, informing nutraceutical safety assessment and BK-targeted drug design.

Polyphenolic compounds are widely explored for health benefits, including hypertension, but their active ingredients, molecular targets, and mechanisms remain poorly defined. We identify the xanthone Mangostin from Garcinia mangostana as a potent modulator of several potassium channels, with large-conductance K+ (BK) channels as its primary target for vasorelaxation. Mangostin activated BK channels as α subunits alone, in complexes with vascular β1 subunits, and in reconstituted BKα/β1–Cav nanodomains. It shifted BK voltage activation to more negative potentials by antagonizing channel closure and promoting channel opening without markedly altering Ca²⁺ sensitivity. Docking, competition, single channel analysis and mutagenesis localized the binding site in the pore cavity below the SF, involving gating-critical S6 residues I308, L312, and A316, and suggest that Mangostin stays bound in closed and open states. These findings establish BK channel activation as the core molecular mechanism driving Mangostin’s vascular effects and define its structural mode of action, informing nutraceutical safety assessment and BK-targeted drug design.

Biomineralization shapes the geology of our planet and is an integral part of the global carbon cycle. Many calcifying organisms generate biomineral precursors within their cells but the mechanisms controlling carbonate chemistry in the calcifying vesicles remain unknown. Using the sea urchin embryo that is a prime model for biomineralization by intracellular amorphous calcium carbonate (ACC) formation this work demonstrated a high proton permeability of the calcifying cells and their vesicles. Intra-vesicular pH and Ca ²⁺ recordings demonstrate highly alkaline conditions in ACC forming endocytotic Ca ²⁺ -rich vesicles, that are enriched during the mineralization process. Using confocal live-cell imaging we observe how vesicles exocytose their Ca ²⁺ -rich and alkaline contents to the calcification front. We identify the proton channel Otop2l as a master regulator for membrane proton conductance in calcifying cells, using the membrane potential to control pH conditions at the site of mineral precursor formation. Electrophysiological investigations demonstrate that Otop2l is activated by alkaline conditions as well as Mg ²⁺ and Ca ²⁺ ions. We provide evidence for high intracellular proton conductance as mechanism to generate alkaline pH conditions in the calcification vesicles. This deep mechanistic knowledge can help explain sensitivities of marine calcifiers to rapid changes in seawater pH in past and future marine habitats.

Polyphenolic compounds are widely explored for health benefits, including hypertension, but their active ingredients, molecular targets, and mechanisms remain poorly defined. We identify the xanthone Mangostin from Garcinia mangostana as a potent modulator of several potassium channels, with large-conductance K ⁺ (BK) channels as its primary target for vasorelaxation. Mangostin activated BK channels as α subunits alone, in complexes with vascular β1 subunits, and in reconstituted BKα/β1–Ca _v nanodomains. It shifted BK voltage activation to more negative potentials by antagonizing channel closure and promoting channel opening without markedly altering Ca ²⁺ sensitivity. Docking, competition, single channel analysis and mutagenesis localized the binding site in the pore cavity below the SF, involving gating-critical S6 residues I308, L312, and A316, and suggest that Mangostin stays bound in closed and open states. These findings establish BK channel activation as the core molecular mechanism driving Mangostin’s vascular effects and define its structural mode of action, informing nutraceutical safety assessment and BK-targeted drug design.

Temperature sensing is an essential component of animal perception and enables individuals to avoid painful or lethal temperatures. Many temperature sensors in central and peripheral neurons are ion channels. Here, we focus on the thermosensitive TREK/TRAAK subfamily of K2P channels—the only known K⁺ selective thermosensitive channels. The C-terminal domain is essential for the temperature activation of TREK channels, but the mechanism of temperature sensation and the nature of the temperature sensor are unknown. We studied the thermosensitivity of representatives of all K2P channel subfamilies and identified TREK-1 and TREK-2 as the only thermosensitive K2P channels, while TRAAK, the third member of the mechano-gated subfamily, showed no temperature dependence. We transferred the thermosensitivity of TREK-1 to TRAAK channels by exchanging the C-termini, demonstrating that the C-terminal domain is sufficient to confer thermosensitivity. By gradually truncating the C-terminus, we isolated a specific temperature responsive element (TRE) consisting of 18 amino acids that constitutes a unique feature in mammalian thermosensitive channels. Within this TRE lie both the binding domain for microtubule associated protein 2 (MAP2) and the PKA phosphorylation site. Pharmacological disruption of the microtubular network as well as the loss of the MAP2 binding site suppressed the temperature response, and PKA activation completely abolished temperature sensitivity. Thus, the connection to the microtubular network enables the thermosensitivity of TREK channels, which is not intrinsic to the channel itself, while the PKA-mediated phosphorylation status acts as a switch that determines if TREK channels are thermosensitive at all.

Temperature sensing is an essential component of animal perception and enables individuals to avoid painful or lethal temperatures. Many temperature sensors in central and peripheral neurons are ion channels. Here, we focus on the thermosensitive TREK/TRAAK subfamily of K2P channels – the only known K ⁺ selective thermosensitive channels. The C-terminal domain is essential for the temperature activation of TREK channels, but the mechanism of temperature sensation and the nature of the temperature sensor are unknown. We studied the thermosensitivity of representatives of all K2P channel subfamilies and identified TREK-1 and TREK-2 as the only thermosensitive K2P channels, while TRAAK, the third member of the mechano-gated subfamily, showed no temperature dependence. We transferred the thermosensitivity of TREK-1 to TRAAK channels by exchanging the C-termini, demonstrating that the C-terminal domain is sufficient to confer thermosensitivity. By gradually truncating the C-terminus, we isolated a specific temperature responsive element (TRE) consisting of 18 amino acids that constitutes a unique feature in mammalian thermosensitive channels. Within this TRE lie both the binding domain for microtubule associated protein 2 (MAP2) and the PKA phosphorylation site. Pharmacological disruption of the microtubular network as well as the loss of the MAP2 binding site suppressed the temperature response, and PKA activation completely abolished temperature sensitivity. Thus, the connection to the microtubular network enables the thermosensitivity of TREK channels, which is not intrinsic to the channel itself, while the PKA-mediated phosphorylation status acts as a switch that determines if TREK channels are thermosensitive at all.

Work over the past three decades has greatly advanced our understanding of the regulation of K_ir K⁺ channels by polyanionic lipids of the phosphoinositide (e.g., PIP₂) and fatty acid metabolism (e.g., oleoyl-CoA). However, comparatively little is known regarding the regulation of the K_2P channel family by phosphoinositides and by long-chain fatty acid–CoA esters, such as oleoyl-CoA. We screened 12 mammalian K_2P channels and report effects of polyanionic lipids on all tested channels. We observed activation of members of the TREK, TALK, and THIK subfamilies, with the strongest activation by PIP₂ for TRAAK and the strongest activation by oleoyl-CoA for TALK-2. By contrast, we observed inhibition for members of the TASK and TRESK subfamilies. Our results reveal that TASK-2 channels have both activatory and inhibitory PIP₂ sites with different affinities. Finally, we provided evidence that PIP₂ inhibition of TASK-1 and TASK-3 channels is mediated by closure of the recently identified lower X-gate as critical mutations within the gate (i.e., L244A, R245A) prevent PIP₂-induced inhibition. Our findings establish that K⁺ channels of the K_2P family are highly sensitive to polyanionic lipids, extending our knowledge of the mechanisms of lipid regulation and implicating the metabolism of these lipids as possible effector pathways to regulate K_2P channel activity.

Significance: Otopetrins comprise a family of proton channels that are required for the development of calcified structures including otoliths and statoconia in vertebrates. To date, it remains unknown how otopetrins contribute to the calcification process. Using the sea urchin larva, we could demonstrate that the otopetrin ortholog otop2l encodes a proton channel that is essential for the formation of the CaCO ₃ skeleton. otop2l is exclusively expressed by the calcifying primary mesenchyme cells, where it promotes the exit of protons liberated by the mineralization process. Given the deep phylogenetic origin of otopetrins in animals, our work identified a key mechanism in the mineralization process with relevance for many calcifying species and their responses to changes in environmental pH.

Work of the past three decades provided tremendous insight into the regulation of K ⁺ channels - in particular K _ir channels - by polyanionic lipids of the phosphoinositide (e.g. PIP ₂ ) and fatty acid metabolism (e.g. oleoyl-CoA). However, comparatively little is known regarding the phosphoinositide regulation in the K _2P channel family and the effects of long-chain fatty acid CoA esters (LC-CoA, e.g. oleoyl-CoA) are so far unexplored. By screening most mammalian K _2P channels (12 in total), we report strong effects of polyanionic lipids (activation and inhibition) for all tested K _2P channels. In most cases the effects of PIP ₂ and oleoyl-CoA were similar causing either activation or inhibition depending on the respective subgroup. Activation was observed for members of the TREK, TALK and THIK subfamily with the strongest activation by PIP ₂ seen for TRAAK (~110-fold) and by oleoyl-CoA for TALK-2 (~90-fold). In contrast, inhibition was observed for members of the TASK and TRESK subfamilies up to ~85 %. In TASK-2 channels our results indicated an activatory as well as an inhibitory PIP ₂ site with different affinities. Finally, we provided evidence that PIP ₂ inhibition in TASK-1 and TASK-3 channels is mediated by closure of the recently identified lower X-gate as critical mutations within the gate (i.e. L244A, R245A) prevent PIP ₂ induced inhibition. Our results disclosed K _2P channels as a family of ion channels highly sensitive to polyanionic lipids (PIP ₂ and LC-CoA), extended our knowledge on the mechanisms of lipid regulation and implicate the metabolisms of these lipids as possible effector pathways to regulate K _2P channel activity.