K ⁺ ‐independent Kir blockade by external Cs ⁺ and Ba ²⁺

Figures

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  FIGURE 1. External Cs+ blocks both the inward and outward components of the Kir current, but only the inward component of the Kv currents. (a) TEA‐sensitive outward K+ currents activated by a classical 10 mV step voltage‐clamp protocol. Traces are shown after the subtraction from the control recordings of the currents in the presence of TEA 20 mM. Inset: Mean TEA‐sensitive outward peak currents plotted against the membrane potential, in the absence (black circles) and in the presence (red circles) of external Cs+20 mM (mean ± SEM, n = 4). (b) 4AP‐sensitive K+ currents, obtained as in (a) by subtraction from the control recordings of the currents in the presence of 4AP 3 mM. Inset: Mean 4AP‐sensitive K+ outward peak currents plotted against the membrane potential, in the absence (black symbols) and in the presence (red symbols) of external Cs+20 mM (mean ± SEM, n = 3). Triangles and circles refer to the two peaks visible on each trace (see text). (C) TEA‐sensitive “tail‐currents” were recorded after a 80 mV, 20 ms activating step in the absence (black traces) and in the presence (red traces) of external Cs+20 mM. (d) 4AP‐sensitive “tail‐currents” were recorded after a 80 mV, 20 ms…

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  FIGURE 2. Effect of external Cs+ and Ba2+ on the outward component of the Kir current. (a) Averaged Kir currents (n = 5 for each K+ gradient) generated by a voltage‐clamp ramp protocol (150 mV/s) under control conditions and with 1, 3, or 6 mM of external Cs+, in different K+ concentration gradients: [K+]i = 110 /[K+]o =3 mM (grey traces), and [K+]i = 110/[K+]o = 10 mM (light grey traces). (b) Averaged Kir currents (n = 5 for each K+ gradient) in control conditions and with 5, 20 or 40 µM of external Ba2+, in different K+ gradients: [K+]i = 110/[K+]o = 3 mM (black traces), and [K+]i = 110/[K+]o = 10 mM (light grey traces). (c) Mean fractional outward currents (n = 5 for each K+ gradient) remaining after Cs+ addition, plotted against the membrane potential for three K+ concentration gradients: [K+]i = 150/[K+]o = 3 mM (black traces), [K+]i = 110/[K+]o = 3 mM (grey traces), and [K+]i = 110/[K+]o = 10 mM (light grey traces). (d) Mean fractional outward currents (n = 5 for each K+ gradient) remaining after Ba2+ addition, plotted against the membrane potential for three K+ concentration gradients: [K+]i = 110/[K+]o = 3 mM (black traces), [K+]i = 110/[K+]o = 10 mM (light grey traces), and [K+]i…

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  FIGURE 3. Cs+ competes with Ba2+ in a blockade of the outward Kir current. (a) Averaged Kir currents (n = 6) recorded during a ramp potentials (150 mV/s) in external medium containing 15 µM of Ba2+ ([K+]o = 3/[K+]i = 110 mM), in the absence and in the presence of 1, 3 and 6 mM of Cs+. (b) Mean fractional outward currents (n = 6) remaining after addition of Cs+ in the absence (grey traces, same data as in Figure 2c) or in the presence of Ba2+ 15 µM. (black traces, relative to the Ba2+ alone condition). (c) Fractional currents, extracted from (b) at various membrane potentials (circles), plotted against the Cs+ concentration. The solid lines are fits to Equation (1), with n = 1.4. (d) Cs+ apparent dissociation constants computed from the fitted curves shown in (c), in the presence of Ba2+ (black symbols) and in control conditions (grey symbols, same data as in Figure 2g). The dashed line represents the theoretical apparent affinity given by Equation (2)

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  FIGURE 4. Effect of external Cs+on the inward component of the Kir current. (a) Averaged Kir currents (n = 6 for each K+ gradient) generated by a ramp potentials (−125 mV/s) under control conditions and with 10, 100, and 500 µM of external Cs+, in different K+ concentration gradients: [K+]i = 110/[K+]o = 3 mM (black traces) and [K+]i = 110/[K+]o = 10 mM (light grey traces) (b) Mean fractional inward currents (n = 6 for each K+ gradient) plotted against the membrane potential, in three K+ gradients: [K+]i = 110/[K+]o = 3 mM (black traces), [K+]i = 150/[K+]o = 10 mM (grey traces), and [K+]i = 110/[K+]o = 10 mM (light grey traces). (c) Mean fractional currents in [K+]i = 110/[K+]o = 10 mM, plotted against the Cs+ concentration as derived from the fractional currents presented in (b) at various membrane potentials; solid lines are fits to Equation (1), with n = 1. (d) Fractional currents resulting from computation by Equation (4) of data shown in (c). This operation subtracts from the fractional currents the part due to the K+‐independent blockade described in Figure 2. The solid lines are fits to Equation (1), with n = 1. (e) Cs+ dissociation constants plotted against the membrane potential,…

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  FIGURE 5. Effect of external Ba2+ on the inward component of the Kir current. (a) Averaged Kir currents (n = 5 for each K+ gradient) recorded during a ramp potentials (−125 mV/s) in the absence and with 5, 20 and 40 µM of external Ba2+, in two different K+ gradients: [K+]i = 110/[K+]o = 3 mM (black traces), and [K+]i = 110/[K+]o = 10 mM (light grey traces). (b) Mean fractional inward currents (n = 5 for each K+ gradient) plotted against the membrane potential, in the presence of the same Ba2+ and K+ concentrations as in (a). (c) Mean fractional inward currents plotted against the Ba2+ concentration, in [K+]i = 110/[K+]o = 3 mM. The solid lines are fits to Equation (1), with n = 1.3. (d) Fractional currents computed from data shown in (c) using Equation (4). The solid lines are fits to Equation (1), with n = 1. (e) Ba2+ dissociation constants plotted against the membrane potential. Circle symbols represent the apparent KD values related to the inward Kir blockade, calculated from fits of Equation (1) as shown in (c). Triangle symbols correspond to the KD values computed from fits of fractional currents as presented in (d), after subtraction of the K+‐independent blockade. The solid lines…

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  FIGURE 6. Competition in a blockade of the inward Kir currents. (a) Averaged Kir currents (n = 6) recorded during ramp potentials (−125 mV/s) in external medium containing 100 µM of Cs+ ([K+]o =3/[K+]i = 110 mM), in the absence and in the presence of 5, 20 and 40 µM of Ba2+. (b) Mean fractional inward currents (n = 6) remaining after addition of Ba2+ in presence of Cs+100 µM (black traces, relative to the Cs+ alone condition). Fractional currents in absence of Cs+ are shown in grey (same data as in Figure 5b). (c) Fractional inward currents plotted against the Ba2+ concentration, after subtraction by Equation (4) of the K+‐independent blockade due to Ba2+. The solid lines are fits of Equation (1), with n = 1. (d) Ba2+ dissociation constants plotted against the membrane potential. Black symbols represent the KD(v) relationship in the presence of Cs+, whereas grey symbols correspond to the control conditions (same data as in Figure 5e). Circles represent the apparent KD(v) relationships calculated before the subtraction of the K+‐independent blockade. Triangles correspond to the KD(v) values calculated after subtraction of the K+‐independent blockade, from fits of Equation (1) to data shown…

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  FIGURE 7. Schematic representation of the dual Kir blockade. (a) Obstruction of the selectivity filter. When K+ (green dots) flux is outwardly directed (left panel), external Cs+ or Ba2+ (red dots) cannot enter the ions file and cannot obstruct the selectivity filter. When K+ flux is reversed from outward to inward (subsequent panels), the blocking cation enters the permeant ion queue, but bumps onto an impassable energy barrier and blocks the inward K+ current. (b) Schematic representation of the equilibrium between different channel states in the context of the dual blockade, and when K+ flux is inwardly directed. Obstruction of the selectivity filter is represented by the blocking cation (red dot) inside the selectivity filter. A K+‐independent blockade is represented by a green latch that closes the channel below the selectivity filter when the ligand (red dot) binds an extracellular site

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  FIGURE 8. Kir channel functioning may provide the K+‐independent effects of external Cs+ and Ba2+ with a voltage‐dependency. (a) Equilibrium scheme where the ligand L binds two‐channel states Kir′ and Kir′′ linked by a voltage constant K(v), with different affinities KD′ and KD′′ respectively. (b) Equation (6) Theoretical fractional Kir currents remaining in presence of the ligand L, as a function of the L concentration, of the K(v) constant, and of the dissociation constants KD′ and KD′′. (c) Experimental fractional currents remaining in presence of external Cs+ shown in Figure 2c. The black line is a fit to Equation (6) performed on the lowest Cs+ concentration (1 mM). The K(v), KD′ and KD′′ constants given by the fit were used to calculate the other relationships (grey lines) simply by changing the Cs+ are concentration in Equation (6). (d) Experimental fractional currents remaining in presence of external Ba2+ shown in Figure 2d. The black line is a fit to Equation (6) performed on the lowest Ba2+ concentration (5 µM). The K(v), KD′ and KD′′ constants given by the fit were used to calculate the other relationships (grey lines) simply by changing the Ba2+ concentration in Equation (6)