Most if not all plasma-membrane ion-channels are influenced by lipid interactions, although few characterized roles for lipid-regulation of ion-channels have been determined. Further, the lipid-binding domains in ion-channels are often cryptic, and not identified by traditional bioinformatics approaches. In this project, studies will be focused on the canonical TRP (TRPC) channels regulate central physiological functions such as neuronal growth cone guidance, vascular tone regulation, and secretion. TRPC channels are activated downstream of G protein coupled receptors and phospholipase C (PLC), yet the exact mechanism of their activation remains obscure. TRPC channels are activated by lipids, yet the mechanisms are unknown. As TRPC channels do not contain conventional lipid binding domains, novel approaches are necessary to identify the structural and functional determinants of TRPC regulation by lipids. The goal of this project is to computationally predict and experimentally verify lipid binding domains within the TRPC family. The core predictive approach that will be utilized within the course of this project is based on structural comparison of functionally related proteins and identifying evolutionary conserved amino acid that dictate the function of the given domain. These predictions will be experimentally validated and assayed for their alteration of (i) channel activity, (ii) channel multimerizaiton, (iii) trafficking, and (iv) protein-protein interactions. The tools and data developed in this project are expected to improve fundamental understanding of ion-channel regulation by lipids.
Broader Impacts: Both Penn State and University of Pittsburgh are dedicated to the education of undergraduate and graduate students. Dr. Patterson, Dr. van Rossum, and Dr. Kiselyov teach cell signaling related undergraduate and graduate courses. The experimental paradigms and principles formulated within this project will be brought into the classrooms to illustrate the development of modern biomedical research.
Dr. Patterson has established a scientific career development graduate course during which the students will directly participate in data analysis, manuscript and grant writing as well as scientific presentations. Dr. Kiselyov and Dr. van Rossum will participate in teaching this class, in particular covering areas of the course, which extend to channel function, plasma-membrane signal transduction, and lyzosomal biology. The present project will provide important experimental and logistical bases for this course. Further, the course will be taught via simulcast to University of Pittsburgh and Howard University and broadcast via YouTube, providing access to anyone interested and incorporated into the University of Pittsburgh-run Supercourse that connects the US educators with students in developing countries. Both lab groups actively recruit undergraduate and graduate students as volunteer and salaried researchers. This project provides an excellent opportunity for undergraduate and graduate student researchers to gain hands-on experience in modern biological research.
Research Strategy and Synergistic Developments- Our NSF grant laid out a two pronged approach (computational analysis and biochemical/cell-biological experimentation) to uncover key determinants of protein-lipid interactions for TRP channels and to derive mechanistic insight into their regulation. Specifically, our computational endeavors included: (a) refining lipid-specific position-specific scoring matrices (PSSMs) for the detection of lipid-binding domains and rational mutagenesis (e.g. Libraries for PIP3-, cardiolipin-, fatty acid-binding etc.); (b) creating methods for the accurate detection of transmembrane domain topology, (c) improving template-based structural modeling of soluble domains, and integral domains such as pore and filter models of ion-channels, (d) performing molecular simulations of ion channel models towards a refined understanding of ion channel gating, and (e) improved methods for domain detection and annotation in general with respect to both speed and accuracy. These types of computational analyses informed our biochemical/cell-biological experiments. The functional analyses largely consisted of mutating key amino acids predicted by modeling, identifying membrane-targeted mutants by in vitro lipid-binding, and analyzing their functional properties using imaging and electrophysiological approaches. Due to the limited scope of this proposal, the analysis was limited to generating the battery of mutants, proving their localization and providing the first tier analysis of their activity using Ca2+ imaging. For example, the Patterson and van Rossum labs rationally engineered over 30 mutants of TRPC1 and TRPC3 channels, which were further analyzed by the Kiselyov group. Sixteen mutants yielded consistently good expression, of which 5 were retained in the ER and 6 showed punctate stain consistent with retainment in the delivery vesicles. The rest of the mutants were found to localize in the plasma membrane, majority showing normal response to cell stimulation. Our continued efforts will be focused on identifying the sites of TRPC retainment, and characterizing plasma membrane localized mutants using electrophysiological techniques. Outside of these experimental aims there were multiple projects that emerged from our interdisciplinary study. For example, the structural analysis initiated by the Patterson and van Rossum groups contributed to a new project in the Kiselyov lab, which is focused on structural determinants of TRPC targeting in polarized cells. Pairwise analysis of TRPC channels revealed basolateral targeting motifs in some of the TRPCs. Interestingly, only the lipid sensitive TRPC channels contain these motifs. Current efforts are focused on the interaction between basolateral targeting and lipid sensing motifs, and on the role of such interaction in TRPC regulation in polarized cells. These data are expected to result in a publication and a grant application within a year. In addition, methodology developed in the course of this project was adapted to functional studies of TRPC relatives, TRPML1 and TRPML3 channels, in the Kiselyov lab. Structural analysis of TRPML channels revealed structure-functional determinants of the block and permeability of these channels to transition metals. These data explain the loss of lysosomal integrity and release of lysosomal caspase under the conditions of TRPML1 loss, recently published by the Kiselyov group in JBC (Loss of the lysosomal ion channel TRPML1 leads to cathepsin B-dependent apoptosis. Colletti GA, Miedel MT, Quinn J, Andharia N, Weisz OA, Kiselyov K. J Biol Chem. 2012 Jan 18. [Epub ahead of print] PMID: 22262857). Two more publications and a grant application are expected to result within a year. Research Accomplishments- Over the course of this project we solidified the collaborative efforts of the Patterson, van Rossum, and Kiselyov labs, outlined the key structural determinants of TRPC regulation by lipids and by membrane traffic, adopted experimental techniques for further investigation of TRPC regulation, and expanded experimental paradigms to a related group of TRP channels and other soluble proteins that interact with lipids. The NSF funding thus laid foundation for numerous peer-reviewed manuscripts, reviews, and a book chapter and was leveraged for grants in preparation (PIs: Patterson and van Rossum) and for full sized funded NIH grants including: NIH RO1 "The TRPML1 Role in Lysosomes." PI: Kirill Kiselyov, Ph.D. and NIH R21 "The role of mucolipin 1 block in transition metal toxicity." PI: Kirill Kiselyov, Ph.D. In addition, we were able to use NSF funds to promote young investigator research. Overall, this was a highly collaborative and productive use of NSF funds. Specifically, through the support of the NSF we were able to make considerable progress in our understanding of: (a) the evolution of potassium, cyclic-nucleotide, and TRP channels with respect to their transmembrane spanning region, (b) the dynamic nature of the TRP channel pore-forming region and the mechanism(s) for their ion selectivity, (c) structural/functional determinants of multiple lipid-binding domains in TRP channels and their interaction with trafficking proteins, and (d) the emergent behaviors of complex signaling systems through the use of Boolean modeling for phospholipase C-mediated cascades.
