Properties of Slo1 K+ channels with and without the gating ring

Abstract

High-conductance Ca(2+)- and voltage-activated K(+) (Slo1 or BK) channels (KCNMA1) play key roles in many physiological processes. The structure of the Slo1 channel has two functional domains, a core consisting of four voltage sensors controlling an ion-conducting pore, and a larger tail that forms an intracellular gating ring thought to confer Ca(2+) and Mg(2+) sensitivity as well as sensitivity to a host of other intracellular factors. Although the modular structure of the Slo1 channel is known, the functional properties of the core and the allosteric interactions between core and tail are poorly understood because it has not been possible to study the core in the absence of the gating ring. To address these questions, we developed constructs that allow functional cores of Slo1 channels to be expressed by replacing the 827-amino acid gating ring with short tails of either 74 or 11 amino acids. Recorded currents from these constructs reveals that the gating ring is not required for either expression or gating of the core. Voltage activation is retained after the gating ring is replaced, but all Ca(2+)- and Mg(2+)-dependent gating is lost. Replacing the gating ring also right-shifts the conductance-voltage relation, decreases mean open-channel and burst duration by about sixfold, and reduces apparent mean single-channel conductance by about 30%. These results show that the gating ring is not required for voltage activation but is required for Ca(2+) and Mg(2+) activation. They also suggest possible actions of the unliganded (passive) gating ring or added short tails on the core.

Keywords: BK channel; Kv1.4; iberiotoxin; tetraethylammonium; β1 subunit.

Fig. 1.

Slo1 channel constructs used in this study. The Slo1 channel constructs used in this study are based on the mouse mbr5 cDNA (12) and the mouse Shaker family Kv1.4 channel (25). The “Slo1 core and tail” refers to the first 342 and the last 827 amino acid residues. The “Kv1.4 tail” refers to the last 74 amino acid residues of Kv1.4. The different channel constructs are designated as follows: Slo1-WT is Slo1 full-length WT; Slo1C-KvT is a Slo1 core with a 74-residue Kv1.4 tail; Slo1C-Kv-minT is a Slo1 core with a Kv1.4 11-residue mini tail; Slo1C-KvT_NAFQ is a Slo1 core with a 74-residue Kv1.4 tail with NAFQ substituted for KKFR in the tail; Slo1C-KvT R207E is Slo1C-KvT with R207E in S4 in the core.

Fig. 2.

Slo1 constructs without gating rings express large currents in Xenopus oocytes. (A) Whole-cell current recordings from oocytes injected with cRNAs of the indicated constructs (see Fig. 1 and legend). Currents were not observed when a stop codon was placed immediately after the tetramerization domain at position 342 (Slo1C-Stop). With a two-electrode voltage clamp, oocytes were held at −70 mV, and 20-ms step pulses were applied from −70 mV to 250 mV in 10-mV increments followed by a step to 0 mV to see outward tail currents in an ND96 bath solution. Not all traces are shown. (B) Currents recorded from inside-out macropatches from oocytes injected with the constructs in A. Asymmetric K⁺ with 10 mM K⁺ in pipette and 140 mM K⁺ on the inside of membrane was used. A 50-ms prepulse to −100 mV was followed by 20-ms step pulses from −100 to 240 mV in 20-mV increments followed by a step to 0 mV for 10 ms. Arrows indicate prominent tail currents observed for Slo1C-KvT. (C) The G–V curve for Slo1-WT is left-shifted compared with Slo1C-KvT and Slo1C-Kv-minT. Data were obtained from Inside-out macropatch recordings in symmetrical 140 mM K⁺.

Fig. 3.

Verification that the Slo1C-KvT construct without a gating ring is expressed and functional. The R207E mutation in S4 in the voltage sensor of Slo1-KvT left-shifted the voltage-dependent activation of Slo1C-KvT as expected from previous Slo1-WT experiments (22, 23), indicating that the isolated core of Slo1 channels without gating rings is being expressed. (A) Sequence of S4 (Upper) in the voltage sensor of Slo1 and with the R207E mutation (Lower). (B) Currents from inside-out macropatches from oocytes injected with Slo1C-KvT and Slo1C-KvT-R207E. G–V plots (n = 5) are shown on the right. The voltage protocol was −80 mV for 20 ms followed by a 40-ms voltage step of −80 to +295 mV (in 25-mV increments), followed by steps to 0 mV for 20 ms to measure tail currents. Asymmetric K⁺ with 10 K⁺ in pipette and 150 K⁺ at intracellular side was used. (C) Whole-cell currents recorded from Slo1C-KvT and Slo1C-KvT-R207E channels. Oocytes were held at −70 mV and 20-ms step pulses applied from −90 mV to 240 mV with a step back to 0 mV. G–V plots are shown on the right. Solutions are as in Fig. 2 for whole-cell recording.

Fig. 4.

The gating ring is required for activation by Ca²⁺ and Mg²⁺. (A) Representative single-channel current activity from Slo1-WT and Slo1C-Kv-minT. Inside-out patches were held at +80 mV, and the intracellular side of membrane was exposed to Ca²⁺ or Mg²⁺ in the sequence indicated. Symmetrical 150 mM K⁺ was used. Open (O) and closed (C) current levels are indicated. Ca²⁺ (100 µM) or (10 mM Mg²⁺) activates Slo1-WT channels but has little effect on Slo1C-Kv-minT channels. Mg²⁺ (10 mM) decreased single-channel conductance (47) for both Slo1-WT and Slo1C-Kv-minT. (B) Ca²⁺ and Mg²⁺ significantly increase Po in Slo1-WT channels (P < 0.0001, n = 5 for Ca²⁺ and P < 0.05, n = 6 for Mg²⁺, paired t tests before normalization) but have insignificant effects on Slo1C-Kv-minT channels (P > 0.1, n = 4 in each case). Note log scale on ordinate. (C) Current traces from inside-out patches ramped from −90mV to 90 mV in the absence and presence of Ca²⁺ or Mg²⁺ as indicated. Slo1C-KvT and Slo1C-Kv-MinT currents are not detectably activated by 200 µM Ca²⁺ or 10 mM Mg²⁺. Note differences in scale bars for Slo1-WT. Increasing single-channel activity in the ramps at positive voltages indicates voltage sensitivity in all channel constructs. Symmetrical 140 K⁺ was used.

Fig. 5.

The gating ring is required for Ca²⁺ and Mg²⁺ to left-shift the G–V curves. (A and B) Currents recorded from inside-out macropatches from Slo1-WT channels (A) or from Slo1C-Kv-minT channels (B) with the G–V curves plotted to the right. The potential was held at 0 mV, stepped to −100 mV for 50 ms, and then stepped from −100 mV to 240 mV in 20-mV increments followed by a step to −80 mV to measure tail currents (Left). Symmetrical 140 K⁺ was used.

Fig. 6.

Open-interval duration, burst duration, and single-channel conductance are reduced in SloC-Kv-minT channels. (A and B) Single-channel recordings at +80 mV from Slo1-WT channels (A) and from Slo1C-Kv-minT channels without the gating ring (B). Note reduction of current amplitudes in B. (C) Bar graphs showing the decrease in mean open-interval and burst duration for Slo1C-Kv-minT channels. Symmetrical 150 mM K⁺ was used.