Reference number : GC-2017-Q1-01481, amendment number : , agreement type : G, recipient legal name : Duff, Henry (University of Calgary), recipient country : CA, recipient province : nan, recipient city : Alberta, recipient postal code : nan, program name english : Discovery Grants Program - Individual, program name french : Programme de subventions à la découverte - individuelles, program purpose english : hERG is a K channel regulating automaticity, cardiogenesis, vasculogenesis and neuritogenesis. It modulates cancer metastasis. My research program characterizes the structural determinants of hERG modulated by endogenous cellular environmental factors such as pHi, lipophilic substances, isoform variants, and common polymorphisms. Our work will provide insight into why variants of this K channel manifest differential response to endogenous factors. 
 
Aim 1 New Atomistic Model of hERG based on crystalized hEAG: A high-resolution cryo-EM structure of neuronal hEAG channel has been resolved in Aug 2016. Our original in silica model of hERG was based on the Kv1.2 crystal. The hEAG and hERG are very homologous but Kv1.2 is distinctly different. We are creating a new model of hERG based on hEAG. 
 
 Aim 2a Lipophilic Access Paths: We discovered that the M651T mutation shifts the IC50 for ivabradine-induced block of the hERG current from 6 &micro;M/L to 120 &micro;M/L, but did not alter response to dofetilide. Moreover, in silico studies from our lab indicate that M651 is a key structural determinant of lipophilic access path for lipophilic drugs to their binding site. We now propose to assess the impact of this access path to endogenous lipophilic substances including ceramide, sphingosine-1-P, and cholesterol. These agents block the hERG current. Our preliminary data indicate that the specific M651 site determines binding of ceramide. 
 
 Aim 2b Intracellular pH: In Nature&rsquo;s Sci Reports we show the impact of changes in intracellular pH on hERG function in response to dofetilide. We propose to assess the impact of pHi on block of hERG by endogenous substances such as ceramide, sphingosine-1-P, and cholesterol. Ceramide is ionizable at physiologic pH whereas cholesterol is not. 
 
 Aim 3 Impact of Common Polymorphism in Different Isoforms of hERG on Response to Endogenous Substances: Common polymorphisms exist in the KCNH2 gene, which encodes hERG. Common polymorphisms include K897T, R1047L; and Y652A. Responses of WT and polymorphism containing channels will be examined in response to lipophilic endogenous substances. Previous studies indicate that the K897T mutation alters QT response to ischemia and forms the rationale to study pHi. Duff lab first discovered the hERG 1b isoform. The impact of these polymorphisms in the different isoforms has not been explored. Moreover, we see exciting differences in the impact of Y652A in the various isoforms. In theme 3 we also dock the PAS-domain to the intracellular ends of the transmembrane domains of hERG model. Our in silico studies suggest the presence of a receptor for the PAS domain in the S2-S3 and the S4-S5 linkers. 
 
Our program provides insights on why different isoforms or common polymorphic variants of this K channel manifest differential sensitive to common environmental factors., program purpose french : hERG is a K channel regulating automaticity, cardiogenesis, vasculogenesis and neuritogenesis. It modulates cancer metastasis. My research program characterizes the structural determinants of hERG modulated by endogenous cellular environmental factors such as pHi, lipophilic substances, isoform variants, and common polymorphisms. Our work will provide insight into why variants of this K channel manifest differential response to endogenous factors. 
 
Aim 1 New Atomistic Model of hERG based on crystalized hEAG: A high-resolution cryo-EM structure of neuronal hEAG channel has been resolved in Aug 2016. Our original in silica model of hERG was based on the Kv1.2 crystal. The hEAG and hERG are very homologous but Kv1.2 is distinctly different. We are creating a new model of hERG based on hEAG. 
 
 Aim 2a Lipophilic Access Paths: We discovered that the M651T mutation shifts the IC50 for ivabradine-induced block of the hERG current from 6 &micro;M/L to 120 &micro;M/L, but did not alter response to dofetilide. Moreover, in silico studies from our lab indicate that M651 is a key structural determinant of lipophilic access path for lipophilic drugs to their binding site. We now propose to assess the impact of this access path to endogenous lipophilic substances including ceramide, sphingosine-1-P, and cholesterol. These agents block the hERG current. Our preliminary data indicate that the specific M651 site determines binding of ceramide. 
 
 Aim 2b Intracellular pH: In Nature&rsquo;s Sci Reports we show the impact of changes in intracellular pH on hERG function in response to dofetilide. We propose to assess the impact of pHi on block of hERG by endogenous substances such as ceramide, sphingosine-1-P, and cholesterol. Ceramide is ionizable at physiologic pH whereas cholesterol is not. 
 
 Aim 3 Impact of Common Polymorphism in Different Isoforms of hERG on Response to Endogenous Substances: Common polymorphisms exist in the KCNH2 gene, which encodes hERG. Common polymorphisms include K897T, R1047L; and Y652A. Responses of WT and polymorphism containing channels will be examined in response to lipophilic endogenous substances. Previous studies indicate that the K897T mutation alters QT response to ischemia and forms the rationale to study pHi. Duff lab first discovered the hERG 1b isoform. The impact of these polymorphisms in the different isoforms has not been explored. Moreover, we see exciting differences in the impact of Y652A in the various isoforms. In theme 3 we also dock the PAS-domain to the intracellular ends of the transmembrane domains of hERG model. Our in silico studies suggest the presence of a receptor for the PAS domain in the S2-S3 and the S4-S5 linkers. 
 
Our program provides insights on why different isoforms or common polymorphic variants of this K channel manifest differential sensitive to common environmental factors., agreement title english : Modulators of hERG/HCN4 Function and Block, agreement title french : Modulators of hERG/HCN4 Function and Block, agreement number : RGPIN, agreement value : 140000.0, agreement start date : 2017-05-10, agreement end date : nan, description english : nan, description french : nan, expected results english : nan, expected results french : nan, additional information english : Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023), additional information french : Subvention ou bourse octroyée s'appliquant à plus d'un exercice financier. (2017-2018 à 2022-2023), owner organization : nserc-crsng, owner organization title : Natural Sciences and Engineering Research Council of Canada | Conseil de recherches en sciences naturelles et en génie du Canada