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. 2020 Apr 1;9:e56054. doi: 10.7554/eLife.56054

Figure 2. α1-adrenergic receptor-dependent inward current is carried by sodium entry.

(A) Membrane resistance (Rm, ΔV −65 to −120 mV) decreased during the α1-AR-EPSC indicating an opening of ion channels, as shown in an example trace (left) and in grouped data (right, p<0.0001, n = 31). (B) Representative trace of membrane noise during the α1-AR-EPSC, brackets denote segments shown below on an expanded scale. (C) Membrane noise (variance, σ2) increased during the α1-AR-EPSC (p<0.0001, n = 22). (D) Plot of α1-AR-EPSC variance versus mean amplitude, linear fit represents mean unitary current (i, r2 = 0.713, p<0.0001). (E) Slow voltage ramps (1 mV/10 ms, analyzed from −120 to −10 mV) were used to determine the current-voltage relationship of the α1-AR-EPSC (subtraction), determined by subtracting current at the peak of the α1-AR-EPSC (stim) from current measured in control conditions just prior to stimulation (basal). Current generated during ramps were truncated for clarity. (F) Current-voltage relationship of the α1-AR-EPSC from grouped data. Shaded area represents mean ± SEM. (G) Plot of reversal potentials (Erev) of the α1-AR-EPSC and INA (p>0.999, n = 26 and 14). (H) Replacing 126 mM NaCl with NMDG eliminated inward INA, shown in a time-course plot (Vhold−65 mV, p<0.0001, n = 14 and 13). (I) Plot of α1-AR-EPSC amplitudes measured at Vhold−65 mV, in 2.5, 6.5, and 10.5 mM [K+]o (p=0.162, n = 17). (J) Plot of α1-AR-EPSC reversal potential (Erev) with varying concentration of external K+ ([K+]o), demonstrating a depolarizing shift in Erev as external K+ was increased (p=0.010, n = 26, 10, and 11). (K) Plot of reversal potentials (Erev) of INA, demonstrating no significant difference between control conditions (ctrl), and after removal of external Ca2+ (0[Ca2+]o, p=0.49, n = 14 and 12) or Mg2+ (0[Mg2+]o, p=0.73, n = 14 and 11). (L) Plot of the amplitude of INA (Vhold−65 mV) demonstrating an augmented INA amplitude in 0[Ca2+]o (p=0.017, n = 14), but not in 0[Mg2+]o, (p>0.9999, n = 11) as compared with control conditions (n = 14). Line and error bars represent mean ± SEM, * denotes statistical significance, ns denotes not significant.

Figure 2—source data 1. Numerical data that were used to generate graphs in Figure 2.

Figure 2.

Figure 2—figure supplement 1. Tail current analysis reveals voltage-dependence of the α1-adrenergic receptor-dependent inward current.

Figure 2—figure supplement 1.

(A) INA was isolated using a two-pulse voltage protocol by subtracting current measured during voltage steps (shown in left inset) at the peak of inward current from noradrenaline application from current measured in control conditions just prior to noradrenaline application. (B) Representative traces of current at Vhold−120 mV following conditioning pre-pulses to −120 mV (black) or to 30 mV (gray) in control conditions (basal) and after noradrenaline. Traces below show membrane noise at Vhold−120 mV on an expanded scale. (C) Conductance (GNA) was measured at peak INA from Vhold−120 mV, calculated using a Erev of −25.1 mV. Plot of GNA versus pre-pulse Vhold, demonstrating a decrease in GNA with depolarizing pre-pulses (linear trend of −0.022 nS/mV, p<0.0001). (D) Plot of unitary current (i, measured at Vhold−120 mV) versus pre-pulse Vhold demonstrating a decrease in i with depolarizing pre-pulses (linear trend of 0.026 pA/mV, p=0.002). Line and error bars represent mean ± SEM, * denotes statistical significance.