Project Grant K99GM155547

THE MOLECULAR BASIS OF PIEZO ION CHANNEL FUNCTION - PROJECT SUMMARY PIEZO1 AND PIEZO2 ARE MECHANOSENSITIVE ION CHANNELS THAT CONVERT FORCE INTO CHEMOELECTRIC SIGNALS. PIEZOS PLAY DIVERSE ROLES IN PHYSIOLOGICAL PROCESSES SUCH AS TOUCH, PROPRIOCEPTION, BREATHING, AND VASCULAR DEVELOPMENT. PATHOGENIC MUTATIONS IN PIEZOS CAN CAUSE EXTENSIVE SENSORY DEFECTS AND DEBILITATING NEUROLOGICAL DISEASES SUCH AS DISTAL ARTHROGRYPOSIS. DESPITE RECENT ADVANCES IN ELUCIDATING THE PHYSIOLOGICAL ROLES OF PIEZO ION CHANNELS, THE UNDERLYING STRUCTURAL CORRELATES OF FUNCTION ARE LARGELY UNKNOWN. PIEZOS ARE HOMOTRIMERIC ION CHANNELS WITH EXTENSIVE BLADES OF TRANSMEMBRANE DOMAINS THAT ARE THOUGHT TO BE THE PRINCIPAL TRANSDUCERS OF MECHANICAL FORCE, BUT STATIC STRUCTURAL SNAPSHOTS AND IN VITRO STUDIES ARE POORLY EQUIPPED TO DESCRIBE WHICH STRUCTURAL REARRANGEMENTS UNDERLIE CHANNEL GATING. TO ADDRESS THESE FUNDAMENTAL GAPS IN KNOWLEDGE, THIS PROPOSAL WILL USE FLUORESCENCE NANOSCOPY TO DETERMINE THE STRUCTURAL MECHANICS OF PIEZO ION CHANNELS IN A CELL AT SINGLE MOLECULE RESOLUTION. THESE STRUCTURAL MECHANICS WILL THEN BE CORRELATED WITH THE FUNCTIONAL OUTPUT OF THE CHANNEL USING ELECTROPHYSIOLOGY. AIM 1 WILL DETERMINE WHICH UNIQUE STRUCTURAL MECHANICS UNDERLIE THE FUNCTIONAL DIFFERENCES BETWEEN PIEZO1 AND PIEZO2. THIS WILL BE ACCOMPLISHED BY EXAMINING HOW THE CELLULAR ENVIRONMENT AND INTRINSIC STRUCTURAL MECHANICS SHAPE THE FUNCTIONAL OUTPUT OF EACH CHANNEL. AIM 2 WILL DEFINE HOW THE INTERPLAY BETWEEN THE PROTEIN AND THE PLASMA MEMBRANE SHAPES THE ACTIVITY OF PIEZOS, ESPECIALLY IN THE CONTEXT OF PATHOGENIC GAIN-OF-FUNCTION MUTATIONS THAT CAUSE DISTAL ARTHROGRYPOSIS. THIS WILL BE ACCOMPLISHED BY MANIPULATING THE LIPID COMPOSITION OF THE PLASMA MEMBRANE AND BY ASSESSING HOW DIVERSE MODULATORS SHAPE THE STRUCTURE OF THE CHANNEL. THIS PROPOSAL WILL ULTIMATELY PROVIDE FUNDAMENTAL INSIGHT INTO HOW PIEZO ION CHANNELS FUNCTION INSIDE OF A CELL AND PROVIDE A FOUNDATION FOR THE STUDY OF OTHER SENSORY ION CHANNELS.