Figure 4.

Dependence of CDI on unitary Ca²⁺ influx. (A) (Left, top) Shown here is a schematic of voltage-ramp protocol and average leak current (〈i_leak〉) detected during cell-attached voltage ramps with no channel activity (gray). Given here are single-channel Na⁺-only currents (i_Na) from single sweep (black) and averaged over 70–100 sweeps (red). The averaged current (〈i〉) was recorded at multiple external [Ca²⁺] (0–75 mM). (Bottom) The 〈i〉 was fit with the Jahr and Stevens model (white line) to determine conductance (γ_in), reversal potential (E_rev), and fractional Ca²⁺ current (f_Ca) (28). (Right) Given here are representative stationary unitary currents in cell-attached patches at the indicated external [Ca²⁺]. Unitary current amplitude values (i) C and O indicate closed and open current levels. (B) (Top) Whole-cell currents were recorded with the indicated external [Ca²⁺] (5 mM BAPTA internal). (Bottom) Shown here is CDI measured with increasing external [Ca²⁺] plotted against the equivalent unitary Ca²⁺ current corrected for channel opening, 〈i_Ca〉 (see Table S1). EI₅₀ = 0.024 pA was extracted from the Hill equation fitted to data. (C) (Top) Simulated steady-state Ca²⁺ spatial profile during channel opening is corrected for channel P_o to reflect the time-averaged Ca²⁺ signal driving CDI_EQ in macroscopic recordings plotted for various Ca²⁺ diffusion coefficients. Experimentally derived [Ca²⁺]/flux ratio (EC₅₀/EI₅₀, red horizontal dashed line) predicts r_CaM = 9 nm. When Ca²⁺ diffusion coefficient (D_Ca) is varied across several orders of magnitude, r_CaM likely resides between 2 and 16 nm (vertical pink dashed lines). (C) (Bottom) Systematic error in the model was evaluated as BAPTA kinetic (k_on) and diffusion (D_B) parameters were varied across several orders of magnitude; the estimated distance of r_CaM remains approximately stable for a given value of D_Ca. To see this figure in color, go online.