Figure 4. External divalent cations... | Rockefeller University Press

Figure 4.

$Figure 4. External divalent cations stabilize loop-gating closures induced by hyperpolarization. (A–C) Recordings of a single Cx46 hemichannel in an excised outside-out patch. The patch pipette solution contained standard IPS solution (Materials and methods). (A) Recordings obtained with no added Mg2+ on the extracellular side (bath) of the hemichannel. Shown are 30-s segments of current recorded at membrane potentials of −30, −50, −70, −90, −100, and −130 mV. All points histograms obtained from longer (60–120 s) recordings are shown to the right of each trace. Dashed lines designated C and O in the current traces and the corresponding histograms represent fully closed and fully open states, respectively (currents were not leak subtracted). In the absence of divalent cations, the hemichannel clearly exhibits voltage dependence with hyperpolarization tending toward closure. At large hyperpolarizing voltages, the hemichannel rarely resides in the fully open state and exhibits very noisy flickering (see spread in the histograms away from the fully closed state). (B) Addition of 1 mM Mg2+ to the bath dramatically changed the behavior of the hemichannel. Stable, long-duration closures became evident and considerably more modest voltages produced closure (compare histograms at −90 mV with and without added Mg2+). (C) View of the current trace recorded at −130 mV with no added Mg2+ at an expanded time scale. The solid bar indicates the segment of the trace that was expanded below. The noisy flickering associated with closure can be seen to be occasionally interrupted by full closures that are quiet (indicated by arrows). Asterisk denotes a brief opening event. (D) Plots of Po vs. Vm obtained from the recordings in A and B. Po represents the fraction of time spent in the fully open state (see Materials and methods). These data illustrate that in solutions essentially devoid of divalent cations, voltage dependence remains robust although significantly shifted in the hyperpolarizing direction. Currents were filtered at 2 kHz and data were acquired at 10 kHz.$

External divalent cations stabilize loop-gating closures induced by hyperpolarization. (A–C) Recordings of a single Cx46 hemichannel in an excised outside-out patch. The patch pipette solution contained standard IPS solution (Materials and methods). (A) Recordings obtained with no added Mg²⁺ on the extracellular side (bath) of the hemichannel. Shown are 30-s segments of current recorded at membrane potentials of −30, −50, −70, −90, −100, and −130 mV. All points histograms obtained from longer (60–120 s) recordings are shown to the right of each trace. Dashed lines designated C and O in the current traces and the corresponding histograms represent fully closed and fully open states, respectively (currents were not leak subtracted). In the absence of divalent cations, the hemichannel clearly exhibits voltage dependence with hyperpolarization tending toward closure. At large hyperpolarizing voltages, the hemichannel rarely resides in the fully open state and exhibits very noisy flickering (see spread in the histograms away from the fully closed state). (B) Addition of 1 mM Mg²⁺ to the bath dramatically changed the behavior of the hemichannel. Stable, long-duration closures became evident and considerably more modest voltages produced closure (compare histograms at −90 mV with and without added Mg²⁺). (C) View of the current trace recorded at −130 mV with no added Mg²⁺ at an expanded time scale. The solid bar indicates the segment of the trace that was expanded below. The noisy flickering associated with closure can be seen to be occasionally interrupted by full closures that are quiet (indicated by arrows). Asterisk denotes a brief opening event. (D) Plots of P_o vs. V_m obtained from the recordings in A and B. P_o represents the fraction of time spent in the fully open state (see Materials and methods). These data illustrate that in solutions essentially devoid of divalent cations, voltage dependence remains robust although significantly shifted in the hyperpolarizing direction. Currents were filtered at 2 kHz and data were acquired at 10 kHz.

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