Selectivity filter gating in large-conductance Ca(2+)-activated K+ channels

Abstract

Membrane voltage controls the passage of ions through voltage-gated K (K(v)) channels, and many studies have demonstrated that this is accomplished by a physical gate located at the cytoplasmic end of the pore. Critical to this determination were the findings that quaternary ammonium ions and certain peptides have access to their internal pore-blocking sites only when the channel gates are open, and that large blocking ions interfere with channel closing. Although an intracellular location for the physical gate of K(v) channels is well established, it is not clear if such a cytoplasmic gate exists in all K(+) channels. Some studies on large-conductance, voltage- and Ca(2+)-activated K(+) (BK) channels suggest a cytoplasmic location for the gate, but other findings question this conclusion and, instead, support the concept that BK channels are gated by the pore selectivity filter. If the BK channel is gated by the selectivity filter, the interactions between the blocking ions and channel gating should be influenced by the permeant ion. Thus, we tested tetrabutyl ammonium (TBA) and the Shaker "ball" peptide (BP) on BK channels with either K(+) or Rb(+) as the permeant ion. When tested in K(+) solutions, both TBA and the BP acted as open-channel blockers of BK channels, and the BP interfered with channel closing. In contrast, when Rb(+) replaced K(+) as the permeant ion, TBA and the BP blocked both closed and open BK channels, and the BP no longer interfered with channel closing. We also tested the cytoplasmically gated Shaker K channels and found the opposite behavior: the interactions of TBA and the BP with these K(v) channels were independent of the permeant ion. Our results add significantly to the evidence against a cytoplasmic gate in BK channels and represent a positive test for selectivity filter gating.

Figure 1.

Open-state block of BK channels by the BP in K⁺ solutions. (Top inset) Time course of raw BK channel currents recorded in 100 µM Ca²⁺ in the absence (black) and presence (red) of 2 µM BP at the potentials indicated. Calibration: 0.4 nA, 50 msec. Zero-current level indicated by dashed line. (Main) Voltage dependence of BK channel activation (■) and fraction of channels not blocked by the BP (○) in 100 µM Ca²⁺. (Inset) Raw current recorded at + 60 mV from a holding potential of −80 mV in the absence (black) and presence (red) of 2 µM BP. Calibration: 2 nA, 50 msec. Zero-current level indicated by dashed line. (Boxed inset) Magnified view of the currents after repolarization to −80 mV, with the peak current in the BP scaled to match that of the control record. Calibration: 1 nA, 25 msec.

Figure 2.

Open-state block of BK channels by TBA in K⁺ solutions. (Top insets) Time course of raw BK channel currents recorded in 100 µM Ca²⁺ in the absence (black) and presence (red) of 10 mM TBA at the potentials indicated. Calibration: 0.4 nA, 50 msec. The zero-current level is indicated by the dashed line. (Main) Voltage dependence of BK channel activation in 100 µM Ca²⁺ (■) and in 10 µM Ca²⁺ (□). Also shown is the voltage dependence of the fraction of channels not blocked by 10 mM TBA in 100 µM Ca²⁺ (•) and in 10 µM Ca²⁺ (○). Solid lines, fits of the Boltzmann equation to the data; dashed lines, spline fits to the data.

Figure 3.

State-independent block of BK channels by TBA in Rb⁺ solutions. (A; insets) Time course of raw BK channel currents recorded in 100 µM Ca²⁺ in the absence (black) and presence (red) of 10 mM TBA at the potentials indicated. The voltage level before depolarization was −100 mV, and after the depolarization it was −80 mV (see Materials and methods). Calibration: 0.12 nA, 50 msec. Zero-current level indicated by dashed line. (Main) Steady-state current–voltage relation before (■), during (•), and after (□) the application of 10 mM TBA in Rb⁺ solutions. (B) Voltage dependence of BK channel activation (■) and fraction of channel not blocked by TBA (○) in 100 µM Ca²⁺.

Figure 4.

State-independent block of BK channels by TBA in Rb⁺ solutions. Pooled data. Voltage dependence of BK channel activation (■) and fraction of channels not blocked by TBA (○) in 100 µM Ca²⁺. Mean data shown with standard error limits if larger than symbol. Data from six to nine experiments, except n = 3 at −90 mV.

Figure 5.

Time course of BP block of BK channels in K⁺ and Rb⁺ solutions. Raw currents at the indicated potentials in the absence (black) and presence (red) of 2 µM BP recorded in 100 µM Ca²⁺ in K⁺ (left; calibration: 1.2 nA, 50 msec) and Rb⁺ (right; calibration: 0.25 nA, 50 msec) solutions. The holding potential for the K⁺ solutions was −80 mV. For the Rb⁺ solutions, the voltage level before depolarization was −100 mV, and after the depolarization it was −80 mV. The tail currents upon repolarization have been truncated for clarity.

Figure 6.

State-independent block of BK channels by BP in Rb⁺ solutions. (A; insets) Time course of raw BK channel currents recorded in 100 µM Ca²⁺ in the absence (black) and presence (red) of 2 µM BP at the potentials indicated. Calibration: 0.1 nA, 50 msec. (Main) Steady-state current–voltage relation before (■), during (•), and after (□) application of 2 µM BP in Rb⁺ solutions. (B) Voltage dependence of BK channel activation (■) and fraction of channels not blocked by BP (○) in 100 µM Ca²⁺. (Inset) BK currents in response to repolarization to −80 mV from +40 mV in the absence (black) and presence (red; average of four records) of 2 µM BP, with the peak current in the BP scaled to match that of the control record. Calibration: 0.2 nA, 10 msec.

Figure 7.

State-independent block of BK channels by BP in Rb⁺ solutions. Pooled data. Voltage dependence of BK channel activation (■) and fraction of channels not blocked by 2 µM BP (○) in 100 µM Ca²⁺. Mean values from four experiments shown with standard error limits if larger than symbol. (Inset) Voltage dependence of the time constant of deactivation in the absence (■) and presence (○) of the BP. Mean values from three experiments shown with standard error limits if larger than symbol.

Figure 8.

Open-state block of Shaker K channels by the BP in K⁺ and Rb⁺ solutions. (A) Block of Shaker channels by 2 µM BP in K⁺ solutions. (Top inset) Currents recorded at −40 mV from a holding potential of −100 mV in the absence (black) and presence (red) of 2 µM BP. Calibration: 0.2 nA, 100 msec. (Main) Voltage dependence of Shaker channel activation (■) and fraction of channels not blocked by 2 µM BP (○). (Inset) Shaker channel currents in response to repolarization to −100 mV (from +20 mV) in the absence (black) and presence (red) of 2 µM BP, with the peak current in the BP scaled to match that of the control record. Calibration: 0.75 nA, 20 msec. (B) Block of Shaker channels by 2 µM BP in Rb⁺ solutions. (Top inset) Currents recorded at −40 mV from a holding potential of −100 mV in the absence (black) and presence (red) of 2 µM BP. Calibration: 0.4 nA, 100 msec. (Main) Voltage dependence of Shaker channel activation (■) and fraction of channels not blocked by 0.5 µM BP (○). Mean values from three experiments shown with standard error limits if larger than symbol. (Inset) Shaker channel currents in response to repolarization to −120 mV (from +20 mV) in the absence (black) and presence (red) of 2 µM BP, with the peak current in the BP scaled to match that of the control record. Calibration: 1.2 nA, 20 msec. Dashed line in all insets represents the zero-current level.