Figure 8.

Inhibition of the outwardly rectifying cation current by intracellular Mg²⁺. (A) I–V relationships for cells patch clamped with ATP- and GTP-free pipette solutions containing 0–6 mM MgCl₂. Chloride concentrations in the solutions were maintained constant by addition of 0–12 mM NMDGCl. EGTA was replaced with 10 mM BAPTA to buffer intracellular Ca²⁺ at 14 nM. Note that the Mg²⁺ concentrations indicated on the figure are those added to the pipette solution. Therefore, 0 Mg²⁺ should be considered nominally Mg²⁺ free. (B) Dose–response relationship for inhibition of the outwardly rectifying current by intracellular free Mg²⁺. Data were fit using the equation I = 1/1 + ([Mg²⁺]/K_1/2)ⁿ. K_1/2 and n are 692 μM and 0.8, respectively. Open circle is inhibition observed when free Mg²⁺ concentration is elevated by addition of 6 mM MgATP. (C) Effect of Mg²⁺ nucleotides on the outwardly rectifying cation current. Whole-cell current is inhibited ∼60% by 6 mM MgATP. Calculated concentration of free Mg²⁺ in the pipette solution containing 6 mM MgATP is 700 μM. The degree of inhibition is similar to that observed when free Mg²⁺ is elevated by addition of MgCl₂ (see B). *, P < 0.05 (compared with 6 mM TrisATP). Values are means ± SEM (n = 5–9). Currents were measured in standard bath medium containing both Ca²⁺ and Mg²⁺ 3–4 min after obtaining whole-cell access when activation was complete. Voltage clamp protocol was the same as described in Fig. 2.