A Non-canonical Voltage-Sensing Mechanism Controls Gating in K2P K(+) Channels.

Two-pore domain (K2P) K(+) channels are major regulators of excitability that endow cells with an outwardly rectifying background "leak" conductance. In some K2P channels, strong voltage-dependent activation has been observed, but the mechanism remains unresolved because they lack a canonical voltage-sensing domain. Here, we show voltage-dependent gating is common to most K2P channels and that this voltage sensitivity originates from the movement of three to four ions into the high electric field of an inactive selectivity filter. Overall, this ion-flux gating mechanism generates a one-way "check valve" within the filter because outward movement of K(+) induces filter opening, whereas inward movement promotes inactivation. Furthermore, many physiological stimuli switch off this flux gating mode to convert K2P channels into a leak conductance. These findings provide insight into the functional plasticity of a K(+)-selective filter and also refine our understanding of K2P channels and the mechanisms by which ion channels can sense voltage.

An otopetrin family proton channel promotes cellular acid efflux critical for biomineralization in a marine calcifier.

Otopetrins comprise a family of proton-selective channels that are critically important for the mineralization of otoliths and statoconia in vertebrates but whose underlying cellular mechanisms remain largely unknown. Here, we demonstrate that otopetrins are critically involved in the calcification process by providing an exit route for protons liberated by the formation of CaCO3 Using the sea urchin larva, we examined the otopetrin ortholog otop2l, which is exclusively expressed in the calcifying primary mesenchymal cells (PMCs) that generate the calcitic larval skeleton. otop2l expression is stimulated during skeletogenesis, and knockdown of otop2l impairs spicule formation. Intracellular pH measurements demonstrated Zn2+-sensitive H+ fluxes in PMCs that regulate intracellular pH in a Na+/HCO3--independent manner, while Otop2l knockdown reduced membrane proton permeability. Furthermore, Otop2l displays unique features, including strong activation by high extracellular pH (>8.0) and check-valve-like outwardly rectifying H+ flux properties, making it into a cellular proton extrusion machine adapted to oceanic living conditions. Our results provide evidence that otopetrin family proton channels are a central component of the cellular pH regulatory machinery in biomineralizing cells. Their ubiquitous occurrence in calcifying systems across the animal kingdom suggest a conserved physiological function by mediating pH at the site of mineralization. This important role of otopetrin family proton channels has strong implications for our view on the cellular mechanisms of biomineralization and their response to changes in oceanic pH.

The versatile regulation of K2P channels by polyanionic lipids of the phosphoinositide and fatty acid metabolism.

Work over the past three decades has greatly advanced our understanding of the regulation of Kir 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 K2P channel family by phosphoinositides and by long-chain fatty acid-CoA esters, such as oleoyl-CoA. We screened 12 mammalian K2P 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 K2P 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 K2P channel activity.

A pharmacological master key mechanism that unlocks the selectivity filter gate in K+ channels.

Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+ channels gated at their selectivity filter (SF), including many two-pore domain K+ (K2P) channels, voltage-gated hERG (human ether-à-go-go-related gene) channels and calcium (Ca2+)-activated big-conductance potassium (BK)-type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+ occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+ channel activators and highlight a filter gating machinery that is conserved across different families of K+ channels with implications for rational drug design.