Ligand-gating by Ca2+ is rate limiting for physiological operation of BK(Ca) channels

Abstract

Large conductance Ca(2+)- and voltage-activated potassium channels (BKCa) shape neuronal excitability and signal transduction. This reflects the integrated influences of transmembrane voltage and intracellular calcium concentration ([Ca(2+)]i) that gate the channels. This dual gating has been mainly studied as voltage-triggered gating modulated by defined steady-state [Ca(2+)]i, a paradigm that does not approximate native conditions. Here we use submillisecond changes of [Ca(2+)]i to investigate the time course of the Ca(2+)-triggered gating of BKCa channels expressed in Chinese hamster ovary cells at distinct membrane potentials in the physiological range. The results show that Ca(2+) can effectively gate BKCa channels and that Ca(2+) gating is largely different from voltage-driven gating. Most prominently, Ca(2+) gating displays a pronounced delay in the millisecond range between Ca(2+) application and channel opening (pre-onset delay) and exhibits slower kinetics across the entire [Ca(2+)]i-voltage plane. Both characteristics are selectively altered by co-assembled BKβ4 or an epilepsy-causing mutation that either slows deactivation or speeds activation and reduces the pre-onset delay, respectively. Similarly, co-assembly of the BKCa channels with voltage-activated Ca(2+) (Cav) channels, mirroring the native configuration, decreased the pre-onset delay to submillisecond values. In BKCa-Cav complexes, the time course of the hyperpolarizing K(+)-current response is dictated by the Ca(2+) gating of the BKCa channels. Consistent with Cav-mediated Ca(2+) influx, gating was fastest at hyperpolarized potentials, but decreased with depolarization of the membrane potential. Our results demonstrate that under experimental paradigms meant to approximate the physiological conditions BKCa channels primarily operate as ligand-activated channels gated by intracellular Ca(2+) and that Ca(2+) gating is tuned for fast responses in neuronal BKCa-Cav complexes.

Figure 1.

Gating of BK_Ca channels by voltage and Ca²⁺ in separation. A, Left, Scheme of the piezo-driven fast application system; each of the two barrels could be perfused with several different solutions. Right, Solution exchange (150 mm K⁺ vs 60 mm K⁺, at 10 μm Ca²⁺) with this system determined either at an open pipette or an excised inside-out patch containing BK_Ca channels. Exponential fits (black lines) to rise and decline of the currents through inside-out patches yielded time constants of 0.30 and 0.28 ms, respectively. Current scales are 0.1 nA. B, Voltage and Ca²⁺ gating determined in an inside-out patch excised from a CHO cell expressing BKα. Left, Current response (middle, bottom, and boxed period at expanded timescale) to a voltage step from −120 to 0 mV recorded at the indicated (constant) [Ca²⁺]_i. Note the instantaneous rise in current at the voltage step. Right, Current response to [Ca²⁺]_i steps (indicated by the arrow) from 0 to 1, 3, 10, or 100 μm recorded at a (constant) membrane potential of 0 mV; gray bar denotes three times the value of the exchange time constant (∼0.9 ms). Note the initial delay before the current onset. The reduction in current amplitude at 100 μm Ca²⁺ reflects pore block by the divalent (Ledoux et al., 2008).

Figure 2.

Characterization of Ca²⁺gating: the time domain. A, Currents through BK_Ca channels elicited by [Ca²⁺]_i steps from 0 to 1, 3, 10, or 100 μm at membrane potentials of −50 mV, 0 or 50 mV (upper, middle, and bottom). Increase in amplitude upon withdrawal of the 100 μm Ca²⁺ solution reflects release of the pore block by Ca²⁺. B, Time constants of Ca²⁺ gating obtained from experiments as in A. Rising and declining phases of the currents were fitted with a mono-exponential function; closed symbols (τ_ON) represent fits to the rising phase ([Ca²⁺]_i stepped from 0 μm to 1, 3, 10, or 100 μm Ca²⁺), open symbols (τ_OFF) are results of fits to the decline of the current ([Ca²⁺]_i stepped from 100 μm to 0, 1, 3, or 10 μm Ca²⁺). Data points are mean ± SEM for 3–10 (τ_ON) or 3–16 experiments (τ_OFF). C, Left, Boxed periods of the traces in A at enlarged timescale. Arrows indicate start of effective solution exchange. Right, Pre-onset delay obtained from experiments as on the left and plotted versus the respective [Ca²⁺]_i. Data points are mean ± SEM 7–10 experiments.

Figure 3.

Comparison of voltage- and Ca²⁺-triggered gating: similar steady-states, but distinct time courses. A, Relative steady-state activation of BK_Ca channels determined in inside-out patches and plotted along [Ca²⁺]_i and transmembrane voltage. Intense colors refer to steady-state activation as obtained with Ca²⁺-triggered gating, faint colors are steady-state activation resulting from voltage-triggered gating. Data points are mean ± SEM 6–11 (Ca²⁺ gating) or 5–7 (voltage gating) experiments. Lines are results of fits with a Boltzmann function to the activation curves in both Ca²⁺ and voltage dimension. B, Time constants obtained for voltage (faint colors) and Ca²⁺ gating (intense colors) in the experiments in A and plotted against voltage and [Ca²⁺]_i.

Figure 4.

Effects of co-assembled BKβ4 and the BKα(D369G) mutation on Ca²⁺ gating of BK_Ca channels. A, B, Time constants of Ca²⁺ gating measured at the indicated potentials in inside-out patches from CHO cells expressing wild-type BKα either alone or together with BKβ4 (A) or expressing the BKα(D369G) mutant (B). Symbols are as in Figure 2; data points for τ_ON are mean ± SEM 3–16 (BKα), 5–11 (BKα + BKβ4), and 6–9 (BKα(D369G)) experiments; data points for τ_OFF are mean ± SEM 3–16 (BKα), 6–12 (BKα + BKβ4), and 7–8 (BKα(D369G)). Note the distinct effects of BKβ4 and the D to G mutation on the gating characteristics. C, Pre-onset delay plotted versus [Ca²⁺]_i for BKα and BKα + BKβ4 (left) and BKα and BKα(D369G) (right). Data points are mean ± SEM 7–10 (BKα), 5–9 (BKα + BKβ4), or 3–9 (BKα(D369G)) experiments. Insets, Representative current traces recorded in response to application of Ca²⁺ (horizontal bar) and normalized to maximum. Note the marked reduction in pre-onset delay observed with the BKα(D369G) mutant channels.

Figure 5.

Operation of Cav-associated BK_Ca channels by their Ca²⁺ gating. A, Experimental conditions for analysis of the Ca²⁺ gating in BK_Ca–Cav2.2 complexes. Channel–channel complexes were reconstituted in CHO cells upon coexpression of BKα, BKβ4, Cav2.2α1, α2δ, and Cavβ1b; whole-cell recordings were done with 10 mm EGTA in the pipette to eliminate currents through Cav-free BK_Ca channels (Berkefeld et al., 2006). B, C, Representative currents through BK_Ca–Cav2.2 complexes recorded in whole-cell configuration in response to the indicated “scanning-tail-current protocol” (details in the text). Step to 70 mV activated Cav channels without eliciting Ca²⁺ influx (effective reversal potential for Ca²⁺ slightly below this potential), steps to −40 mV (B; termed conditioning pulse), and 20 mV (C) triggered Ca²⁺ influx (increase in driving force) for defined periods (0.06, 0.12, 0.24, 0.48, 0.96, 1.92, 3.84, 7.68, and 15.36 ms). The final step back to 70 mV switched off the Ca²⁺-inward currents and elicited K⁺-outward currents. Traces in red are responses recorded with a conditioning pulse duration of 0.96 ms (B) or 7.68 ms (C); 0 time point is start of the conditioning pulse. Note the similarity in amplitudes of the two BK_Ca outward currents despite the largely different periods of Ca²⁺ influx. Bottom, Amplitudes of BK_Ca currents recorded 0.2 ms after the final pulse step (I _{BK,instantaneous}, filled symbols; “time” is duration of conditioning pulse plus 0.2 ms) or of BK_Ca peak currents (I _BK,ipeak, open symbols) plotted over the period of Ca²⁺ influx (start of the conditioning pulse). D, BK_Ca currents from an experiment as in B and C with a conditioning pulse of 0.96 ms to −40 mV (blue) and 20 mV (black). E, I_{BK,instantaneous}-time relation from the experiment in B and C at expanded timescale (time points are conditioning interval plus 0.2 ms). Note that the BK_Ca current is larger in amplitude at −40 mV than at 20 mV, and that the time course of channel activation is markedly faster at −40 mV than at 20 mV.

Figure 6.

Determination of the K⁺-current output of BK_Ca–Cav complexes by the properties of BK_Ca Ca²⁺ gating. A–C, I_{BK,instantaneous}-time relations determined for BK_Ca–Cav complexes of the indicated composition in experiments as in Figure 5 but for the conditioning pulse potentials between −40 and 40 mV given by the inset in A. Currents were normalized to the maximum recorded at a conditioning pulse potential of −40 mV; data points are mean ± SEM 13 (BKα), 10 (BKα+ BKβ4), and 6 (BKα(D369G)) experiments. Note the slowing of BK_Ca-channel activation with increasing depolarization and the lack of delay obtained with the BK_Ca(D369G) mutant. D, Overlay of data from A to C recorded at a conditioning pulse potential of 20 mV.

Figure 7.

Acceleration of the BK_Ca channel Ca²⁺ gating upon complex formation with Cav channels. Time course of BK_Ca activation recorded either with Cav-free BK_Ca wild-type and D369G mutant channels upon fast application of 10 and 100 μm Ca²⁺ (at 50 mV) or in Cav2.2-associated BK_Ca channels at 40 mV. Data for complexes are from Figure 6, data for free BK_Ca channels (black traces) are mean ± SEM 8 (10 μm Ca²⁺) and 4 (100 μm Ca²⁺) fast application experiments with excised patches. Note the largely decreased pre-onset delay observed for the BKα(D369G) channels and the Cav2.2-associated BK_Ca channels.