The effect of external group IIB divalent cations on the Y1618H, W1716H, and Y1618H plus W1716H mutant channels. (A) The representative currents in the absence (solid black lines) and presence of 100 μM Cd²⁺ (green lines) and 300 μM Zn²⁺ (red lines) on the same oocyte. The oocyte was held at −120 mV and stepped to −10 mV to elicit Na⁺ currents. The pulse protocol was repeated every 3 s until a steady-state effect of Cd²⁺ or Zn²⁺ was reached. The effects of both Cd²⁺ and Zn²⁺ can be readily washed out with the control ND-96 solution, and the observed effect remains the same regardless of the order of Cd²⁺ and Zn²⁺ application. The dotted lines mark the zero current level. (B) The activation curves in the absence (open circles and solid black lines) and presence of 100 μM Cd²⁺ (top panels; closed circles and green lines) or 300 μM Zn²⁺ (bottom panels; closed circles and red lines) were done by the protocols described in Materials and methods. The lines are fits with a Boltzmann function with V_1/2 values (control vs. Cd²⁺) in mV of −17.2 versus −14.2, −19.7 versus −10.6, and −18.6 versus −19.5, and k values (control vs. Cd²⁺) of 4.8 versus 5.0, 4.9 versus 5.3, and 3.9 versus 3.8, and V_1/2 values (control vs. Zn²⁺) in mV of −16.5 versus −12.4, −19.5 versus −4.7, and −18.6 versus −16.0, and k values (control vs. Zn²⁺) of 5.9 versus 5.8, 3.7 versus 4.2, and 3.9 versus 4.0 for the W1716H, the Y1618H plus W1716H, and the Y1618H mutant channels, respectively. The averaged shift of the V_1/2 of the activation curve by Cd²⁺ is 0.8 ± 0.2, 1.9 ± 1.0, and 8.2 ± 0.9 mV, and the averaged V_1/2 shift by Zn²⁺ is 2.6 ± 1.1, 3.7 ± 2.0, and 10.7 ± 2.6 mV for the Y1618H, the W1716H, and the Y1618H plus W1716H mutant channels, respectively (n = 3–4).