Pannexins (Panx1-3) comprise a unique family of membrane channels that mediate a variety of immune responses including apoptotic cell clearance, cytokine secretion, and T-cell activation. Pannexins also play crucial roles in modulating synaptic activity and plasticity; for instance, Panx1 knockout mice display diminished excitatory postsynaptic potentials, resulting in impaired object recognition and spatial learning. Also, Panx1 promotes aberrant postsynaptic activity after repetitive NMDA receptor stimulation, suggesting that Panx1 plays important roles in epileptic seizures. These studies highlight the vital roles of pannexins throughout the body, however, little is understood about what activates pannexin channels, how they open and close, and what cellular events accompany these changes. Our long-term goal is to uncover the mechanisms underlying pannexin gating, regulation, and downstream signaling using structural and functional approaches.
The specific aims of this proposal are approached through the following lines of investigation: 1) Solve the first crystal structure of a pannexin. We have identified a pannexin species that expresses at a high level, assembles into a stable and monodisperse oligomer in solution, and forms crystals that diffract to ~9. We will continue optimizing crystallization conditions and determine the firt crystal structure of a pannexin. We expect the atomic resolution structure of a pannexin will serve as a solid foundation for determining what constitutes the channel pore and how the opening and closing are controlled. 2) Dissect the mechanism underlying Panx1 channel gating. Our preliminary experiments suggest that Panx1--the most studied subtype thus far--most likely harbors an intrinsic voltage sensor. We also discovered that carbenoxolone, the most commonly used nonspecific antagonist of Panx1, allosterically inhibits the voltage-gated Panx1 channel activity. Taking advantage of this drug and electrophysiology, we will identify the key residues that govern voltage gating, defining their position relative to the membrane field, and probing how they move in response to voltage. Our proposed research is innovative not only because it will uncover the first atomic resolution structure of a pannexin, but also because it wil provide the first detailed molecular mechanism of this unique class of a voltage-gated membrane channel. Moreover, this is the first detailed investigation for the mechanism of action of any known pannexin inhibitors. We expect that our studies will open new avenues for developing long-awaited Panx1 specific agonists or antagonists through structure-based drug design or in vitro screening based on the conformational changes associated with Panx1 channel opening. Once such molecular tools become available, we hope to clarify why pannexin misexpression results in widespread dysfunctions in vivo, and in the long term, potential new strategies for treating devastating conditions such as chronic pain and epilepsy.
Pannexins are membrane channels widely distributed throughout the human body and play important roles in inflammatory response and neurotransmission. Our research objective is to understand mechanisms of how pannexins function at a molecular level, which may lead to the design of novel pharmacological agents to treat inflammatory and neurological diseases.
| Michalski, Kevin; Henze, Erik; Nguyen, Phillip et al. (2018) The weak voltage dependence of pannexin 1 channels can be tuned by N-terminal modifications. J Gen Physiol 150:1758-1768 |
| Karasawa, Akira; Kawate, Toshimitsu (2017) Expression and Purification of a Mammalian P2X7 Receptor from Sf9 Insect Cells. Bio Protoc 7: |
| Kawate, Toshimitsu (2017) P2X Receptor Activation. Adv Exp Med Biol 1051:55-69 |
| Karasawa, Akira; Michalski, Kevin; Mikhelzon, Polina et al. (2017) The P2X7 receptor forms a dye-permeable pore independent of its intracellular domain but dependent on membrane lipid composition. Elife 6: |
| Michalski, Kevin; Kawate, Toshimitsu (2016) Carbenoxolone inhibits Pannexin1 channels through interactions in the first extracellular loop. J Gen Physiol 147:165-74 |
| Karasawa, Akira; Kawate, Toshimitsu (2016) Structural basis for subtype-specific inhibition of the P2X7 receptor. Elife 5: |
