Intracellular Mg2+ inhibits the IP3-activated I K(Ca) in NG108-15 cells. [Why intracellular citrate can be useful for recording I K(Ca)]

Receptor-mediated formation of inositol 1,4,5-trisphosphate (IP₃) can induce an outward Ca²⁺-activated K⁺ current I _K(Ca)] some neural cells. We have investigated I _K(Ca) activated by intracellular injections of IP₃ in whole-cell patch-clamped neuroblastoma×glioma hybrid cells. The current could only be recorded reliably using citrate as the anion in the pipette, but not using acetate, aspartate, chloride, fluoride, gluconate or methylsulphate. This could be attributed to buffering of intracellular Mg²⁺ by citrate. Theoretical calculations suggested free Mg²⁺] of 1.0 and 0.07 mM respectively in the acetate- and citrate-based recording solutions. Further, IP₃-activated I _K(Ca) could be recorded when the free Mg²⁺ level in the acetate, chloride or methylsulphate solutions was lowered to the range (0.05 mM) calculated for the citrate solution. Thus, raised Mg²⁺] blocks I _K(Ca). This appeared to be due to inhibition of the response to released Ca²⁺, since high Mg²⁺] also blocked the response to intracellular injections of Ca²⁺ ions. Mean Mg²⁺ levels in intact neuroblastoma×glioma hybrid cells measured by Mag-Indo-1/AM fluorescence were estimated to be less than 0.14 mM. We therefore conclude that IP₃-induced I _K(Ca) is expressed under normal conditions, but may be subject to regulation by intracellular Mg²⁺.