The lipid bilayer is a critical determinant of ion channel activity; however, efforts to define the lipid dependence of channel function have generally been limited to cellular expression systems in which the membrane composition cannot be fully controlled. We reconstituted purified human Kir2.1 and Kir2.2 channels into liposomes of defined composition to study their phospholipid dependence of activity using (86)Rb(+) flux and patch-clamp assays. Our results demonstrate that Kir2.1 and Kir2.2 have two distinct lipid requirements for activity: a specific requirement for phosphatidylinositol 4,5-bisphosphate (PIP(2)) and a nonspecific requirement for anionic phospholipids. Whereas we previously showed that PIP(2) increases the channel open probability, in this work we find that activation by POPG increases both the open probability and unitary conductance. Oleoyl CoA potently inhibits Kir2.1 by antagonizing the specific requirement for PIP(2), and EPC appears to antagonize activation by the nonspecific anionic requirement. Phosphatidylinositol phosphates can act on both lipid requirements, yielding variable and even opposite effects on Kir2.1 activity depending on the lipid background. Mutagenesis experiments point to the role of intracellular residues in activation by both PIP(2) and anionic phospholipids. In conclusion, we utilized purified proteins in defined lipid membranes to quantitatively determine the phospholipid requirements for human Kir channel activity.

Structures of the prokaryotic K þ channel, KcsA, highlight the role of the selectivity filter carbonyls from the GYG signature sequence in determining a highly selective pore, but channels displaying this sequence vary widely in their cation selectivity. Furthermore, variable selectivity can be found within the same channel during a process called C-type inactivation. We investigated the mechanism for changes in selectivity associated with inactivation in a model K þ channel, KcsA. We found that E71A, a noninactivating KcsA mutant in which a hydrogen-bond behind the selectivity filter is disrupted, also displays decreased K þ selectivity. In E71A channels, Na þ permeates at higher rates as seen with 86 Rb þ and 22 Na þ flux measurements and analysis of intracellular Na þ block. Crystal structures of E71A reveal that the selectivity filter no longer assumes the "collapsed," presumed inactivated, conformation in low K þ , but a "flipped" conformation, that is also observed in high K þ , high Na þ , and even Na þ only conditions. The data reveal the importance of the E71-D80 interaction in both favoring inactivation and maintaining high K þ selectivity. We propose a molecular mechanism by which inactivation and K þ selectivity are linked, a mechanism that may also be at work in other channels containing the canonical GYG signature sequence. P otassium (K þ ) channels exhibit the remarkable feature of catalyzing rapid ion conduction while maintaining strong selectivity for K þ over Na þ . The extensive K þ channel superfamily contains many members that have different selectivities, ranging from nonselective cation channels to highly selective K þ channels, yet containing the same canonical GYG sequence. These residues form the narrow selectivity filter, in which the backbone carbonyls are positioned to coordinate dehydrated potassium ions (1). These carbonyls are constrained by the surrounding protein structure, which forms an intricate network of hydrogen bonds and salt bridges. In inward rectifying potassium (Kir) channels, a key structural feature of this network is a salt bridge that forms a molecular "bowstring" (2) bridging the top and bottom of the selectivity filter loop (2, 3) (Fig. 1). Alterations in this salt bridge are known to disrupt selectivity (2, 4, 5); several mutations have dramatic effects on permeation, rendering the channel essentially nonselective and highly permeable to Na þ . It has also been proposed that members of one subgroup of this family, the HCN channels, are less K þ selective because they lack this network of molecular restraints (6), but the relevance of this structural network for selectivity in channels other than inward rectifiers, as well as the mechanism responsible for variable selectivity, remains to be seen.Furthermore, alterations in the equivalent salt bridge residues that affect selectivity in the Kir channel family also lead to the induction of a phenomenon similar to C-type inactivation in voltage-gated K þ channels (7). There is also a correlation between C-type inac...

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1β3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and β3 (labeled residue β3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β3-L294 and G308) in the interface between the β3(+) and α1(−) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and β3-α1 intersubunit sites are critical for neurosteroid action.

Many ion channels are modulated by phosphatidylinositol 4,5-bisphosphate (PIP 2 ), but studies examining the PIP 2 dependence of channel activity have been limited to cell expression systems, which present difficulties for controlling membrane composition. We have characterized the PIP 2 dependence of purified human Kir2.1 and Kir2.2 activity using 86 Rb ؉ flux and patch clamp assays in liposomes of defined composition. We definitively show that these channels are directly activated by PIP 2 and that PIP 2 is absolutely required in the membrane for channel activity. The results provide the first quantitative description of the dependence of eukaryotic Kir channel function on PIP 2 levels in the membrane; Kir2.1 shows measureable activity in as little as 0.01% PIP 2 , and open probability increases to ϳ0.4 at 1% PIP 2 . Activation of Kir2.1 by phosphatidylinositol phosphates is also highly selective for PIP 2 ; PI, PI(4)P, and PI(5)P do not activate channels, and PI(3,4,5)P 3 causes minimal activity. The PIP 2 dependence of eukaryotic Kir activity is almost exactly opposite that of KirBac1.1, which shows marked inhibition by PIP 2 . This raises the interesting hypothesis that PIP 2 activation of eukaryotic channels reflects an evolutionary adaptation of the channel to the appearance of PIP 2 in the eukaryotic cell membrane.

Nitrous oxide at 50% inhaled concentration has been shown to improve depressive symptoms in patients with treatment-resistant major depression (TRMD). Whether a lower concentration of 25% nitrous oxide provides similar efficacy and persistence of antidepressant effects while reducing the risk of adverse side effects is unknown. In this phase 2 clinical trial (NCT03283670), 24 patients with severe TRMD were randomly assigned in a crossover fashion to three treatments consisting of a single 1-hour inhalation with (i) 50% nitrous oxide, (ii) 25% nitrous oxide, or (iii) placebo (air/oxygen). The primary outcome was the change on the Hamilton Depression Rating Scale (HDRS-21). Whereas nitrous oxide significantly improved depressive symptoms versus placebo (P = 0.01), there was no difference between 25 and 50% nitrous oxide (P = 0.58). The estimated differences between 25% and placebo were −0.75 points on the HDRS-21 at 2 hours (P = 0.73), −1.41 points at 24 hours (P = 0.52), −4.35 points at week 1 (P = 0.05), and −5.19 points at week 2 (P = 0.02), and the estimated differences between 50% and placebo were −0.87 points at 2 hours (P = 0.69), −1.93 points at 24 hours (P = 0.37), −2.44 points at week 1 (P = 0.25), and −7.00 points at week 2 (P = 0.001). Adverse events declined substantially with dose (P < 0.001). These results suggest that 25% nitrous oxide has comparable efficacy to 50% nitrous oxide in improving TRMD but with a markedly lower rate of adverse effects.