Lateral Fenestrations Research Articles

Finding the Route of Entry and Binding Site of Local Anaesthetics in Bacterial Voltage Gated Sodium Channels Using Molecular Dynamics Simulation

Voltage-gated sodium channels are proteins in cell membranes that initiate action potentials in neurons. Dysfunctional sodium channels are implicated in diseases such as chronic pain, epilepsy and cardiac arrhythmia, which are often treated by sodium channel blockers such as the family of local anaesthetics. These blockers can have adverse side-effects by blocking healthy channel subtypes, so researchers have strived to understand their mechanism of action to help design new, subtype-specific drugs. While there is no atomic resolution structural data on human sodium channels, the publication of a number of crystal structures of related, bacterial voltage-gated sodium channel in the last 2 years has offered the opportunity to explore the mechanism of entry and location of binding sodium channel blockers. Here, molecular dynamics simulations were used to determine the binding site of two channel blockers, benzocaine and phenytoin in the bacterial channel NavAb. We find that that binding involves nonspecific hydrophobic interaction in the channel lumen, relate this to measured binding affinities and speculate how sequence differences in this region could alter the affinity for anaesthetics. We also show the feasibility of drug entry from the membrane via the lateral fenestrations. By calculating the free energy for this process we show that these fenestrations are likely to represent the long postulated hydrophobic route of entry leading to tonic block of resting channels. While there are many differences between bacterial and human sodium channels, these results provide a first step in understanding the mechanism of action of local anaesthetics and can aid the rational search for subtype-specific drugs for use in mammalian sodium channels.

Ion access pathway to the transmembrane pore in P2X receptor channels.

P2X receptors are trimeric cation channels that open in response to the binding of adenosine triphosphate (ATP) to a large extracellular domain. The x-ray structure of the P2X4 receptor from zebrafish (zfP2X4) receptor reveals that the extracellular vestibule above the gate opens to the outside through lateral fenestrations, providing a potential pathway for ions to enter and exit the pore. The extracellular region also contains a void at the central axis, providing a second potential pathway. To investigate the energetics of each potential ion permeation pathway, we calculated the electrostatic free energy by solving the Poisson-Boltzmann equation along each of these pathways in the zfP2X4 crystal structure and a homology model of rat P2X2 (rP2X2). We found that the lateral fenestrations are energetically favorable for monovalent cations even in the closed-state structure, whereas the central pathway presents strong electrostatic barriers that would require structural rearrangements to allow for ion accessibility. To probe ion accessibility along these pathways in the rP2X2 receptor, we investigated the modification of introduced Cys residues by methanethiosulfonate (MTS) reagents and constrained structural changes by introducing disulfide bridges. Our results show that MTS reagents can permeate the lateral fenestrations, and that these become larger after ATP binding. Although relatively small MTS reagents can access residues in one of the vestibules within the central pathway, no reactive positions were identified in the upper region of this pathway, and disulfide bridges that constrain movements in that region do not prevent ion conduction. Collectively, these results suggest that ions access the pore using the lateral fenestrations, and that these breathe as the channel opens. The accessibility of ions to one of the chambers in the central pathway likely serves a regulatory function.

Structural Changes in Thoracolumbar Disks Following Lateral Fenestration A Study of the Radiographic, Histologic, and Histochemical Changes in the Chondrodystrophoid Dog

Lateral fenestrations of intervertebral disks T12–13 through L2–3 were performed on 16 1‐year‐old chondrodystrophoid dogs. The disks of five dogs were fenestrated by removing the core of disk material with a 20 gauge spinal needle, six were fenestrated in the same manner with a 14 gauge spinal needle, and five were fenestrated with a 14 gauge spinal needle and a tartar scraper. Forty‐two of 48 disks had radiographic evidence of narrowing following surgery. Microscopic examination of the disks indicated that the nucleus pulposus was nonreactive tissue and that fenestration did not incite an inflammatory response that resulted in dissolution of the nucleus pulposus. Results of histochemical analyses of the fenestrated disks were not different from those of control disks. The only effects observed during a 24 week period following fenestration were disruption of the annulus fibrosus and nucleus pulposus by the fenestration device, removal of varying amounts of the nucleus pulposus, and vascular and fibrous tissue invasion of the lateral annulus fibrosus. These findings suggest that the effectiveness of fenestration is governed by the amount of nucleus pulposus removed.