Nanodiscs that hold a lipid bilayer surrounded by a boundary of scaffold proteins have emerged as a powerful tool for membrane protein solubilization and analysis. By combining nanodiscs and cell-free expression technologies, even completely detergent-free membrane protein characterization protocols can be designed. Nanodiscs are compatible with various techniques, and due to their bilayer environment and increased stability, they are often superior to detergent micelles or liposomes for membrane protein solubilization. However, transport assays in nanodiscs have not been conducted so far, due to limitations of the two-dimensional nature of nanodisc membranes that offers no compartmentalization. Here, we study Krokinobacter eikastus rhodopsin-2 (KR2), a microbial light-driven sodium or proton pump, with noncovalent mass-spectrometric, electrophysiological, and flash photolysis measurements after its cotranslational insertion into nanodiscs. We demonstrate the feasibility of adsorbing nanodiscs containing KR2 to an artificial bilayer. This allows us to record light-induced capacitive currents that reflect KR2's ion transport activity. The solid-supported membrane assay with nanodisc samples provides reliable control over the ionic condition and information of the relative ion activity of this promiscuous pump. Our strategy is complemented with flash photolysis data, where the lifetimes of different photointermediates were determined at different ionic conditions. The advantage of using identical samples to three complementary approaches allows for a comprehensive comparability. The cell-free synthesis in combination with nanodiscs provides a defined hydrophobic lipid environment minimizing the detergent dependence often seen in assays with membrane proteins. KR2 is a promising tool for optogenetics, thus directed engineering to modify ion selectivity can be highly beneficial. Our approach, using the fast generation of functional ion pumps incorporated into nanodiscs and their subsequent analysis by several biophysical techniques, can serve as a versatile screening and engineering platform. This may open new avenues for the study of ion pumps and similar electrogenic targets.

We investigated the temperature-dependent kinetics of the light-driven Na þ pump Krokinobacter rhodopsin 2 (KR2) at Na þ -pumping conditions. The recorded microsecond flash photolysis data were subjected to detailed global target analysis, employing Eyring constraints and spectral decomposition. The analysis resulted in the kinetic rates, the composition of the different photocycle equilibria, and the spectra of the involved photointermediates. Our results show that with the temperature increase (from 10 to 40 C), the overall photocycle duration is accelerated by a factor of 6, with the L-to-M transition exhibiting an impressive 40-fold increase. It follows from the analysis that in KR2 the chromophore and the protein scaffold are more kinetically decoupled than in other microbial rhodopsins. We link this effect to the rigidity of the retinal protein environment. This kinetic decoupling should be considered in future studies and could potentially be exploited for fine-tuning biotechnological applications.

Sleep apnea is a common disorder that represents a global public health burden. KCNK3 encodes TASK-1, a K+ channel implicated in the control of breathing, but its link with sleep apnea remains poorly understood. Here we describe a new developmental disorder with associated sleep apnea (developmental delay with sleep apnea, or DDSA) caused by rare de novo gain-of-function mutations in KCNK3. The mutations cluster around the ‘X-gate’, a gating motif that controls channel opening, and produce overactive channels that no longer respond to inhibition by G-protein-coupled receptor pathways. However, despite their defective X-gating, these mutant channels can still be inhibited by a range of known TASK channel inhibitors. These results not only highlight an important new role for TASK-1 K+ channels and their link with sleep apnea but also identify possible therapeutic strategies.

Here we applied target analysis to a temperature dependent flash photolysis dataset of the light-driven sodium ion pump Krokinobacter rhodopsin 2 (KR2) at sodium pumping conditions. With an increase in temperature from 10 – 40 °C, the overall photocycle duration was accelerated by a factor of six, while single transitions like the L to M transition increased by a factor of 40. Using kinetic modeling with the Eyring constraint as well as spectral corrections on the datasets the spectral position as well as the equilibria of the different photointermediates could be resolved. The results provide further insight into KR2s photocycle and energetics.STATEMENT OF SIGNIFICANCEKR2 is the most prominent member of the new class of non-proton cation pumps, as it represents an interesting new optogenetic tool. Despite widespread biophysical investigations, the molecular mechanisms of light-induced sodium pumping in KR2 are still not sufficiently understood. Therefore, an expanded set of thermodynamic parameters is essential for a complete picture. Our study of the KR2 photocycle shows that different steps in the photocycle are affected differently by temperature changes. Rigorous data analysis provides strong evidence that the transient states observed in time-resolved experiments represent rather equilibria between the different photocycle intermediates than pure intermediates. Gaining access to the dynamics and energetics of KR2 helps to answer long standing open questions concerning the molecular mechanism of cation pumping.

Sleep apnea is a common disorder that represents a global public health burden. KCNK3 encodes TASK-1, a K+ channel implicated in the control of breathing, but its reported link with sleep apnea remains poorly understood. Here we describe a novel developmental disorder with sleep apnea caused by rare de novo gain-of-function mutations in KCNK3. The mutations cluster around the X-gate, a gating motif which controls channel opening, and produce overactive channels that no longer respond to inhibition by G-protein coupled receptor pathways but which can be inhibited by several clinically relevant drugs. These findings demonstrate a clear role for TASK-1 in sleep apnea and identify possible therapeutic strategies.