Voltage-gatedcalciumchannels(VGCC)regulatethefunctionofcellsrangingfromspermtoinsulin-producing pancreaticcellstoneurons.Thereisgrowingrecognitionthatspecificlipids,sterolsandthegangliosideGM1, regulateVGCCactivityinhealthanddiseaseincludingduringfertilization,regulationofbloodsugar,andpain sensation.However,determiningmechanismsbywhichlipidsregulateVGCCremainsamajorchallengeinthe field.Thisproposaladdressestwoquestionsofbroadimportancetobiology&medicine:Atamolecularlevel, howcanmembranelipidsregulate1)VGCCactivityand2)cellfunction?Spermareanoutstandingmodelfor theseissues,becausetheirabilitytofertilizeaneggiswell-knowntoberegulatedbylipiddynamics.However, thereistremendouscontroversyregardingspermcalcium(Ca2+)channels,withelectrophysiologicalrecordings (atroomtemperature)onlyidentifyingtheactivityofasingle,non-VGCCchannel(CatSper).Incontrast,data fromseveralgroupsusingapproachesofcellbiology,geneticsandpharmacology,showtheactivityofdifferent channelsincludingVGCC.Ofimportance,wefoundthatsterolsandGM1regulatespermVGCCactivityinways thattypicalelectrophysiologypracticescan?tdetect.Thisproposalrepresentsaconsortiumoftwolaboratories, onewithexpertiseinspermelectrophysiology,whohavepioneerednewmethodstobeabletodetectchannels regulatedbylipids.ThesecondlaboratorywasfirsttoidentifymembranelipidregulationofspermVGCC,and hasexpertiseinmembranelipidorganizationandfunction.Together,weproposetousenewtechnologiesand approachestoaddressthiscontroversy,whichiscentraltoourunderstandingofspermfunction&fertilization. Thisknowledgewillempowerclinicianstobetterunderstandthecausesofmaleinfertility,halfofwhicharedue tospermfunctiondefectsandarenotdetectedbytraditionalsemenanalysis.Basedonexcitingpreliminary datathatclearlyshowevidenceofmorethanonetypeofCa2+channel(usingspermfrommicenullfor CatSper),aswellaspublicationsfrombothlaboratories,weproposeaseriesofexperimentstoinvestigate howlipidsregulatemouseandhumanspermVGCCandpossiblyotherCa2+channels(Aim1).Thesestudies willutilizestate-of-the-artmicroscopy,pharmacology,mousegeneticmodelsandelectrophysiologyunder conditionsthatallowspermmembranelipidstobehaveastheydointhefemalereproductivetract.Wenext proposetodeterminetheprecisemolecularmechanismsbywhichsterolsandGM1regulateVGCC(Aim2).To dothis,we?llexpressdifferentVGCCsubunitsinspecificcelllines,aswellasartificialmembranesystems,in whichwecancontrolbothchannelexpressionandthelipidcomposition.InbothAims,wewillutilizeinnovative mousestrainseitherexpressinggeneticallyencodedCa2+indicatorsorlackingspecificchannelsubunits. Together,thesestudieswillprovidebroadmechanisticinsightintotheregulationofVGCCbymembranelipids, amatterofcriticalimportanceinbothnormalphysiologyaswellasimportantdiseasestates.

Public Health Relevance

SterolsandGM1,lipidsinthespermplasmamembrane,regulatethevoltage-gatedcalciumchannel,CaV2.3?a criticalregulatorofexocytosisandfunctionincellsrangingfromspermtopancreaticbetacellstoneurons. Resultsshowthatthisformofregulationiscriticalforspermtobeabletofertilize;?however,theseresultshave beenmadecontroversialbycommonlyusedelectrophysiologyapproachesthatfailtoidentifyanycalcium channelactivityotherthanthatoftheCatSperchannel.Thiscollaborativeproposalusescutting-edge electrophysiologicalapproachesandnovelmousemodelstoresolvethesecontroversiesandexplorethe molecularmechanismsbywhichtheselipidsregulateCaV2.3andcellfunction,withimportantapplicationsto notonlyinfertility,butalsodiabetes,peripheralneuropathyandpain.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD093827-02
Application #
9894820
Study Section
Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
Program Officer
Moss, Stuart B
Project Start
2019-03-15
Project End
2024-02-29
Budget Start
2020-03-01
Budget End
2021-02-28
Support Year
2
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Cornell University
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850