Introduction: Analyses of ligand-gated ion channel receptors (LGIC) have demonstrated that possible sites of anesthetic action exist within their transmembrane domains. We have previously used molecular modeling techniques combined with experimental data to visualize a plausible model of an anesthetic binding site within a LGIC complex.1 We have also previously shown an approximate computational mechanism, based on an elastic network model, by which these ion channels may open and close and postulated how this motion may be affected by the presence of anesthetics.2 The difficulties with these approximation methods, however, lay in their inability to account for the modest effects of a separate ligand or a small mutation on ion channel motion. Here we show the successful application of a formal molecular mechanics force field for the normal mode calculation of protein motions.Methods: Coordinates of the homomeric GABARa1 pentamer complex composed of both an extracellular ligand binding domain and a transmembrane domain came from our previous work.3 Using this structure as a template, we built a model of the glyRa1 homomer using the homology modeling tools within the InsightII 2005 software package (Accelrys, San Diego, CA). This model then underwent a series of restrained optimizations within the GROMACS modeling package using the OPLS force field and no distance cutoffs on electrostatic and van der Waals interactions. After final unrestrained optimization, normal mode analysis was performed with a sparse matrix implementation.Results: As we previously reported for the approximate elastic network technique2, analysis of the entire glyRa1 complex demonstrated a clear iris-like motion of the protein about the central axis of the ion pore as the first (highest amplitude-lowest frequency) normal mode. In this mode, the rotation of the ligand binding domain occurred in the opposite direction to that of the transmembrane domain, producing a “wringing” like motion of the entire protein complex as it traversed its gating cycle. However, unlike the elastic network calculation of normal modes, which could only report relative frequencies of vibration, the GROMACS-based normal mode analysis allows for the calculation of real vibrational frequencies on the order of 321 GHz or around 3.1 ps per cycle. Likewise, while elastic network calculations could be completed in a few hours, the GROMACS calculations took approximately a week to complete on a Dell Workstation with dual 3GHz Xeon processors and a 64 bit software implementation.Conclusions: Despite these proteins containing upwards of 26,000 atoms, our new methods have made it possible to derive normal modes via the full implementation of a formal force field calculation. Despite their length and markedly increased complexity, these calculations still demonstrate that the harmonic motion of LGIC complexes is consistent with the direction of channel opening and closing. Such calculations should now allow the elucidation of the subtle effects on ion channel motion that are due to anesthetic binding.
The 2006 Annual Meeting of the American Society of Anesthesiologists. Chicago, Illinois. October 14-18 2006