Grant Number: 1R01GM090123-01

Abstract: DESCRIPTION (provided by applicant): The carefully orchestrated movement of ions across cellular membranes is crucial to virtually every biological process. Ion channels play thereby a central role by allowing the regulated flow of relevant ions through their aqueous pore. In the past few decades, progress made in Molecular Biology and Genomics has revealed the unsuspected variety of ion channels expressed in our membranes. The family of TRP channels with more than two dozens of members in humans that function as biosensors and signal integrators is a good illustration of this development. It includes the only known examples of ionic pores fused to enzymatic regions, so-called "chanzymes". TRPM7 and its close relative TRPM6 both harbor a kinase region at their C-terminus that is capable of phosphorylating protein substrates on Serine/Threonine residues. TRPM7 is widely distributed and has been shown to be an essential and irreplaceable molecule in several genetic models, including a B-lymphocyte cell line called DT40. TRPM7-deficiency in this context results in cell growth arrest and death, unless the extracellular media is supplemented with Mg2+, suggesting a role for TRPM7 in Mg2+- homeostasis regulation. Despite these insights into TRPM7's physiological function, the significance of its kinase region remains unclear. Understanding the role of this unique domain holds the promise to shed some light on novel mechanisms of cellular adjustments to a changing ionic environment. The main goal of this proposal is to investigate the relationship between the channel and kinase portions of TRPM7 by utilizing a combination of biochemistry, genetics and biophysics approaches, allowing for a detailed function-structure relationship study. We plan to determine how altering the ionic selectivity of TRPM7 channels through pore mutations might affect its kinase activity. Furthermore the functional importance of the covalent link between kinase and channel will be assessed by studying liberated domains and through the introduction of linker structures. Finally, we also designed experiments allowing us to define molecular determinants of channel assembly and trafficking to the cell surface. PUBLIC HEALTH RELEVANCE: Ion channels form highly sophisticated pores in biological membranes allowing for the controlled movement of ions between biologically relevant compartments. Most are at the surface of the cell and therefore easily accessible for pharmacological intervention; many are validated molecular targets. Understanding the functional versatility of ion channels is therefore key to providing us with novel potential strategies of interfering with biological processes relevant to human health. The structural uniqueness of the TRPM7 protein as an ion channel/kinase fusion appears to be a reflection of its complex involvement in regulating cellular functions in response to nutrient availability. Understanding the molecular determinants of the interplay between kinase and ion channel function will therefore provide us with novel opportunities to influence processes such as cell proliferation or immune function.

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