Whole cell and outside-out recording configurations are similar with respect to the position of the cell membrane. In both cases the extracellular face of the plasma membrane is exposed to the bath solution and the intracellular face is continuous with the recording pipette solution. These example solutions are for recording Ca channel activity and can be used with either whole-cell or outside-out patches.
A. RECORDING PIPETTE SOLUTIONS
1. Minimize Ca Channel Run-Down (Slesinger and Lansman, 1991a). In mM:120 Aspartic acid, 20 NaCl, 5 EGTA, 10 HEPES, 15 glucose. Titrate with CsOH to pH 7.4 then add 1 MgCl2, 2 Mg-ATP, 1 cAMP. Note: added divalent salts will slightly lower the pH.
2. ATP Regenerating System (Kohr and Mody, 1991; Regan et al. 1991). In mM: 105 Methanesulfonic acid, 10 EGTA, 10 HEPES. Titrate with CsOH to pH 7.4 then add 5 MgCl2, 2 Mg-ATP, 14 - 25 creatine phosphate disodium salt, 50 U creatine kinase.
3. Pipette Solution to Record Ca Channel Currents. In mM: 150 CsOH, 10 HEPES, 10 EGTA, 10 TEA-Cl. Titrate with methanesulfonic acid to pH 7.4. Then add 2 MgCl2, 4 Mg-ATP, 1 cAMP, 0.5 Na2-GTP. OPTIONAL: Addition of protease inhibitors slow Ca channel rundown (Doerner and Alger, 1988; Pflüg. Arch. 1991): 0.1 - 1.0 Leupeptin hemisulfate, 0.05 pepstatin A.
B. BATH SOLUTIONS
1. Ba Solution for Whole Cell Ca Channel Currents (adapted from Slesinger and Lansman, 1991a). In mM: 155 TEA-Cl, 10 HEPES, 10 glucose. Titrate with TEA-OH to pH 7.4. Then add 20 BaCl2, 1 MgCl2, 1µM TTX. Note: the concentration of TEA-Cl was empirically determined to give a final osmolarity of 315 mOsm.