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Predictions and experimental tests of a new biophysical model of the mammalian respiratory oscillator.

ABSTRACT: Previously our computational modeling studies (Phillips et al., 2019) proposed that neuronal persistent sodium current (I_NaP) and calcium-activated non-selective cation current (I_CAN) are key biophysical factors that, respectively, generate inspiratory rhythm and burst pattern in the mammalian preBötzinger complex (preBötC) respiratory oscillator isolated in vitro. Here, we experimentally tested and confirmed three predictions of the model from new simulations concerning the roles of I_NaP and I_CAN: (1) I_NaP and I_CAN blockade have opposite effects on the relationship between network excitability and preBötC rhythmic activity; (2) I_NaP is essential for preBötC rhythmogenesis; and (3) I_CAN is essential for generating the amplitude of rhythmic output but not rhythm generation. These predictions were confirmed via optogenetic manipulations of preBötC network excitability during graded I_NaP or I_CAN blockade by pharmacological manipulations in slices in vitro containing the rhythmically active preBötC from the medulla oblongata of neonatal mice. Our results support and advance the hypothesis that I_NaP and I_CAN mechanistically underlie rhythm and inspiratory burst pattern generation, respectively, in the isolated preBötC.

SUBMITTER: Phillips RS

PROVIDER: S-EPMC9262387 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Molecular docking computationally screens thousands to millions of organic molecules against protein structures, looking for those with complementary fits. Many approximations are made, often resulting in low "hit rates." A strategy to overcome these approximations is to rescore top-ranked docked molecules using a better but slower method. One such is afforded by molecular mechanics-generalized Born surface area (MM-GBSA) techniques. These more physically realistic methods have improved models for solvation and electrostatic interactions and conformational change compared to most docking programs. To investigate MM-GBSA rescoring, we re-ranked docking hit lists in three small buried sites: a hydrophobic cavity that binds apolar ligands, a slightly polar cavity that binds aryl and hydrogen-bonding ligands, and an anionic cavity that binds cationic ligands. These sites are simple; consequently, incorrect predictions can be attributed to particular errors in the method, and many likely ligands may actually be tested. In retrospective calculations, MM-GBSA techniques with binding-site minimization better distinguished the known ligands for each cavity from the known decoys compared to the docking calculation alone. This encouraged us to test rescoring prospectively on molecules that ranked poorly by docking but that ranked well when rescored by MM-GBSA. A total of 33 molecules highly ranked by MM-GBSA for the three cavities were tested experimentally. Of these, 23 were observed to bind--these are docking false negatives rescued by rescoring. The 10 remaining molecules are true negatives by docking and false positives by MM-GBSA. X-ray crystal structures were determined for 21 of these 23 molecules. In many cases, the geometry prediction by MM-GBSA improved the initial docking pose and more closely resembled the crystallographic result; yet in several cases, the rescored geometry failed to capture large conformational changes in the protein. Intriguingly, rescoring not only rescued docking false positives, but also introduced several new false positives into the top-ranking molecules. We consider the origins of the successes and failures in MM-GBSA rescoring in these model cavity sites and the prospects for rescoring in biologically relevant targets.

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Predictions and experimental tests of a new biophysical model of the mammalian respiratory oscillator.

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