Mechanosensitive ion channels (MSC) were first observed in chick skeletal muscle in 1984. Recently, a family of Eukaryotic MSCs, called Piezo, was identified and cloned. These channels sense mechanical forces in a variety of tissues and allow cells to adapt to mechanical stressors. The channels are abundant in the skin (Piezo1) and are found in cartilage (Piezo1 and 2). Their functional presence in joints is related to osteoarthritis. The hereditary disease Arthrogryposis arises from mutations to Piezo2 producing congenital joint contractures. We can analyze Piezo channels with high precision in the patch clamp or by whole cell recording but these measurements do not reflect the in situ channel environment and we have shown that the channel?s response to stress depends upon the local environment. We propose to modify the Piezo1 channel so that its activity is controlled optically and this will allow us to study a channel?s contribution to a particular physiological response. The Piezo1 protein can accommodate large insertions within the channel (position 1591) and remain functional, but in some instances the insertion inhibits channel function. The protein can be split at position 1591, and if the segments are co-expressed, they can reassemble into functional channels although neither half alone is functional. We hypothesize that this region sits at junction involved in protein movement which will us to control channel function optically. We plan to use the well-known LOV domain in a folded conformation to prevent a transition to the open state. Then with light stimuli, the LOV domain unfolds and activates the channel. This reaction is reversible.
Specific Aim 1 describes how we intend to introduce the LOV protein into PIEZO1 to serve as a photoactivatable switch and the ways that we will test channel function using patch clamp and fluid shear stress stimuli. A second approach to creating a photoactivated Piezo is described in Specific Aim2. We will determine what sequences at the split sight (1591) are necessary for formation of the active channel when coexpressed and remove them. To restore channel function, we will encode protein LOV domain on one channel subunit and a protein called Zdrk on the other. The LOV-Zdrk proteins interact and the channel is assembled in the dark (active) and can be dissociated in light (inactivated). We will characterize the property of channel dimerization in situ and measure channel function. The overall goal is to develop optically controlled mechanosensitive ion channels thus providing a new tool to study the physiological response of cells to mechanical stress in situ as well as in vivo and in vitro.
Eukaryotic cells are able to sense and adapt to environmental forces, and a key player in the cell?s ability to respond to mechanical stress is a family of ion channels called Piezo. They are integral to how our vascular system develops and cell homeostasis of epithelial cells. We will control PIEZO1 channels optically and use these probes to study the cell?s response to mechanical forces.
