PIP2 regulation of TRPV4 channels: Binding sites and dynamic coupling

Transient Receptor Potential subfamily V4 (TRPV4) is a nonselective cation channel that plays important roles in thermo-sensing, osmoregulation, nociception, and bone homeostasis. The activities of TRV4 channels are known to be regulated by phosphatidylinositol 4,5-bisphosphate (PIP2), even though its molecular basis remains poorly understood at present. Existing studies reveal great uncertainty or even controversy on the binding sites as well as functional effects of PIP2 on TRPV4. Analysis of available cryo-EM structures suggests that the previously proposed sites on the N-terminal domain and the ankyrin repeat domain are too distal from the membrane interface and thus unlikely to be the primary sites for PIP2 regulation. Instead, we have identified two possible PIP2 binding sites near the cytosolic membrane interface using structural analysis and molecular docking. Atomistic simulations and free energy analysis reveal that these two sites belong to a single broad binding groove, where PIP2 binding is dynamic and can sample multiple configurations of interactions with positively charged side chains within the groove. These local free energy minima are separated by small free energy barriers and offer ∼4 kcal/mol stability with respect to the membrane bulk. Furthermore, dynamic network analysis suggests that PIP2 binding in the predicted groove can modulate the dynamic coupling between various domains of TRPV4, potentially priming the channel for responding to various stimuli. Together, these results provide important new insights on the possible molecular basis of PIP2 binding and regulation of TRPV4 activities.