`
`Generation of the TRPM5 FLIPR Response
`
`Carbachol
`
`M1 GPCR
`
`I [Ca++]
`
`W TRPM5 5W“ I [Na+]
`
`
`Membrane
`Potential
`Dye
`
`1 000
`
`E 500
`
`<
`
`250
`
`0
`
`
` + 314M Carbachol
`
`-:-— 0.3 ”M Carbachol
`+ Na'I've (M1 GPCR only)
`
`
`
`0
`
`V
`
`50
`
`100
`
`150
`
`200
`
`Time (sec)
`
`Carbachol
`
`
`
`FIG. 2(A) and 2(B)
`
`Demonstration of a TRPM5 Ion Channel Response
`in Transiently Transfected HEK293 Cells
`
`Method: HEK293 cells were transfected (Lipofectamine) with TRP M5 plasmid cDNA, or not
`(Controls) and grown for 2 days and transferred to 96 well microtiter plates. They were loaded for 1
`hr. with either a membrane potential dye or a Ca*+flux dye. Cells were treated with 30 uM carbachol
`to stimulate a Ca" response as shown by the increase in fluorescence in 18). Only cells transfected
`with the TRPM5 plasmid gave an increase in fluorescence response indicating activation of the
`TRPM5. Carbachol additions and fluorescence readings were perfo rmed on a FLEXstation
`manufactured by Molecular Devices, Inc. The traces are the instantaneous fluorescent signal from
`representative wells.
`
`
`
`(A)
`
`A W v
`
`33"“
`rb
`1
`
`Cells Loadedwith
`.
`CaICIum Dye
`
`—I— mTrpM5 Plasmid
`—.l.— mTrpM5 Plasmid
`——— No Plasmid Control
`
`—0— No Plasmid Control
`
`
`
`CalcmmDyeResponse(RFU)
`
`20000
`
`a.8.aODOODOOOO
`
`o
`
`50
`
`100
`
`1 50
`
`Time (sec)
`
`30 uM
`Carb,
`i
`
`80000
`
`60000
`
`MembranePotentialDyeResponse
`
`(RFU) AO O OO
`
`Cells Loadedwith
`Membrane Potential Dy
`
`\TrpMS Response
`only in plasmid
`
`
`
`tranfectants
`
`O
`
`50
`
`100
`
`1 50
`
`Time (sec)
`
`--— mTrpM5 Plasmid
`
`—t— mTrpM5 Plasmid
`+ No Plasmid Control
`—o— No Plasmid Control
`
`

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