hit rate and tiny current

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xsmart
xsmart's picture
hit rate and tiny current

I am trying to record Glutamate receptor from HEK cells transfected by CaPO4 method (outside out). My internal solution is 115 NaCl, 10 NaF,0.5 CaCl2, 1 MgCl2, 5 Na4BAPTA, 5 HEPES and 10 Na2ATP, pH 7.3. and external solution is  (in mM)150 NaCl, 0.1MgCl2, 0.1 CaCl2, 5 HEPES, titrated to pH 7.3 with NaOH, My patch ppt is 2-5Mohm. I see very tiny current ( 25pA) and my hit rate of getting current is also very low. I loose them within 5-7 min. In few paper I saw internal solution instead of sodium they use cesium. Can somebody explain me why cesium is used. glutamate unedite receptor allows sodium ion to pass through it. Will using cesium improve my current?

neuro
neuro's picture
the amplitude of the current

the amplitude of the current you record through the channels depends on the driving force of the ions through the channels, which is a function of ion concentrations on both sides of the membrane and also the transmembrane potential that you're clamping the channel at.  Make sure that all concentrations AND the holding potential are correct.  you say you are getting few events.... if this is in a single channel then change the concentration of the agonist... if it is in whole cell mode then you've got poor transfection efficiency.  try changing your method of transfection, or switch to cells that will produce more channels (like TSA 201 cells, which are modified HEK 293 cells that have higher protein expression)

Bluejay
Bluejay's picture
Also, keep in mind that most

Also, keep in mind that most media contains glutamate or glutamine that quickly degrates to glutamate.  This could downregulate your ion channel expression or cause other problems.  Some authors use glutamax or other glutamine substitutes.