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Ca²⁺ influx via the L-type Ca²⁺ channel during tail current and above current reversal potential in ferret ventricular myocytes

Zhuan Zhou * and Donald M. Bers

1. Current through L-type Ca²⁺ channels (I_Ca) was measured electrophysiologically at the same time as Ca²⁺ influx was measured by trapping entering Ca²⁺ with a high concentration of indo-1 (> 1 mM) in ferret ventricular myocytes.

2. Na⁺-free conditions prevented Na⁺-Ca²⁺ exchange and K⁺ currents were blocked by Cs⁺ and TEA. Thapsigargin (5 µM) prevented Ca²⁺ uptake and release by the sarcoplasmic reticulum. I_Ca was pre-activated by brief pulses to +120 mV (the equilibrium potential for Ca²⁺, E_Ca), followed by steps to different membrane potentials (E_m, -80 to +100 mV), in some cases in the presence of the Ca²⁺ channel agonist FPL-64176.

3. Integrated I_Ca (I_Ca) was linearly related to the change in the concentration of Ca²⁺ bound to indo-1, which was assessed by the fluorescence difference signal F_d (F_d = F₅₀₀ - F₄₀₀). This created an internal calibration of F_d as a measure of Ca²⁺ influx.

4. The F_d/I_Cadt relationship was virtually unchanged at all measurable inward I_Ca (at E_m from -80 to +50 mV). This indicates that the fractional current carried by Ca²⁺ and channel selectivity are unchanged over this E_m range, and also that the selectivity for Ca²⁺ is very high.

5. Ca²⁺ influx was readily detected by F_d beyond the I_Ca reversal potential (+65 to +100 mV) and was not abolished until E_m was +120 mV (i.e. E_Ca). This is explained by the fact that inward Ca²⁺ flux at the I_Ca reversal potential is exactly balanced by outward Cs⁺ current through the Ca²⁺ channels and can be described by classic Goldman flux analysis with a Ca²⁺/Cs⁺ selectivity of the order of 5000.

6. This result also emphasizes that net Ca²⁺ influx via Ca²⁺ channels occurs over a voltage range where the net channel current is outward.

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