BK Channel Regulation of Afterpotentials and Burst Firing in Cerebellar Purkinje Neurons

Abstract

BK calcium-activated potassium channels have complex kinetics because they are activated by both voltage and cytoplasmic calcium. The timing of BK activation and deactivation during action potentials determines their functional role in regulating firing patterns but is difficult to predict a priori. We used action potential clamp to characterize the kinetics of voltage-dependent calcium current and BK current during action potentials in Purkinje neurons from mice of both sexes, using acutely dissociated neurons that enabled rapid voltage clamp at 37°C. With both depolarizing voltage steps and action potential waveforms, BK current was entirely dependent on calcium entry through voltage-dependent calcium channels. With voltage steps, BK current greatly outweighed the triggering calcium current, with only a brief, small net inward calcium current before Ca-activated BK current dominated the total Ca-dependent current. During action potential waveforms, although BK current activated with only a short (∼100 μs) delay after calcium current, the two currents were largely separated, with calcium current flowing during the falling phase of the action potential and most BK current flowing over several milliseconds after repolarization. Step depolarizations activated both an iberiotoxin-sensitive BK component with rapid activation and deactivation kinetics and a slower-gating iberiotoxin-resistant component. During action potential firing, however, almost all BK current came from the faster-gating iberiotoxin-sensitive channels, even during bursts of action potentials. Inhibiting BK current had little effect on action potential width or a fast afterhyperpolarization but converted a medium afterhyperpolarization to an afterdepolarization and could convert tonic firing of single action potentials to burst firing.SIGNIFICANCE STATEMENT BK calcium-activated potassium channels are widely expressed in central neurons. Altered function of BK channels is associated with epilepsy and other neuronal disorders, including cerebellar ataxia. The functional role of BK in regulating neuronal firing patterns is highly dependent on the context of other channels and varies widely among different types of neurons. Most commonly, BK channels are activated during action potentials and help produce a fast afterhyperpolarization. We find that in Purkinje neurons BK current flows primarily after the fast afterhyperpolarization and helps to prevent a later afterdepolarization from producing rapid burst firing, enabling typical regular tonic firing.

Keywords: KCa1.1; action potential clamp; calcium channel; cerebellum; iberiotoxin; paxilline.

Figure 1.

BK and calcium currents evoked by step depolarizations and action potential waveforms. A, Current evoked by a step depolarization from −80 to −20 mV before (black) and after (green) application of 3 μm paxilline, with sodium current blocked by 1 μm TTX and Kv3 potassium current inhibited by 100 μm 4-AP. Replacing extracellular CaCl₂ by MgCl₂ (red trace) eliminated the inward current carried by calcium, as well as a small outward current (seen during the step to −40 mV following that to −20 mV) likely carried by calcium-activated SK channels. B, BK current defined by paxilline inhibition (green), calcium current defined by calcium removal in the presence of paxilline, 4-AP, and TTX (red), and the sum of BK and calcium current (black). C, D, Same as A and B with currents evoked by an action potential waveform.

Figure 2.

BK current requires calcium entry. A, Current evoked by a step depolarization from −80 to +30 mV before (black) and after (red) removal of extracellular calcium (replacement of 1.5 mm CaCl₂ by 4 mm MgCl₂) on a background of 1 μm TTX and 100 μm 4-AP. Application of 3 μm paxilline (green) had no effect with a calcium-free external solution. B, Total calcium-sensitive current (calcium current plus calcium-activated potassium current) defined by removal of calcium (black) and BK current in calcium-free solution (green) defined by the application of paxilline in calcium-free solution. C, D, Same as A and B for currents evoked by an action potential waveform.

Figure 3.

Two components of BK current evoked by depolarizing steps. A, Currents evoked by a 10 ms depolarizing step from −80 to −10 mV with 1 μm TTX and 100 μm 4-AP (black), after the application of 200 nm iberiotoxin (blue), after the addition of 3 μm paxilline (green) in the continuing presence of iberiotoxin, after the addition of 10 mm TEA (gray; in the continuing presence of TTX and 4-AP but omitting iberiotoxin and paxilline), and after the removal of calcium (red; replacement of 1.5 mm CaCl₂ by 4 mm MgCl₂, in presence of TTX, 4-AP, and TEA). B, Iberiotoxin-sensitive BK, iberiotoxin-resistant BK, and calcium current defined by subtraction of records in A. C, Peak iberiotoxin-sensitive BK current, iberiotoxin-resistant BK current, and calcium current for 10 ms steps to voltages −70 to +30 mV. D, Integrated current (nA * ms or pC) during the 10 ms steps. Mean ± SEM: n = 15, IbTx-sensitive BK current; n = 14, IbTx-resistant BK current; n = 13, calcium current.

Figure 4.

BK channel activation in Purkinje neurons is not dependent on calcium-induced calcium release. A, IbTx-sensitive (blue) and IbTx-resistant (green) BK current evoked by a 10 ms step from −80 to −10 mV, measured in the presence of 5 μm ryanodine. B, Peak current evoked by 10 ms steps from −70 to +40 mV in the presence of 5 μm ryanodine (triangles; n = 7) compared with control measurements [circles (replotted from Fig. 3C)]. C, D, Peak currents evoked in the presence of 1 μm thapsigargin (triangles; n = 6) compared with control measurements [circles (replotted from Fig. 3C)]. Mean ± SEM.

Figure 5.

Activation kinetics of iberiotoxin-sensitive BK current, iberiotoxin-resistant BK current, and calcium currents. A, Initial time course of iberiotoxin-sensitive BK current, iberiotoxin-resistant BK current, and calcium current for steps to −30, 0, and +30 mV. Currents are defined as in Figure 3. B, Collected results for activation kinetics measured by time to half-maximal activation. Mean ± SEM: n = 14, IbTx-sensitive BK current and IbTx-resistant BK current; n = 12, calcium current.

Figure 6.

Inactivation kinetics of iberiotoxin-sensitive BK current. A, BK current during 10 ms depolarizations from −40 to +40 mV. Red traces, Fits to single exponential functions over the period of the fitted trace. B, Collected results for percent of peak current remaining at 10 ms. Mean ± SEM, n = 14. C, Time constant of inactivation as a function of voltage. Mean ± SEM, n = 14.

Figure 7.

Deactivation kinetics of iberiotoxin-sensitive BK current, iberiotoxin-resistant BK current, and calcium current. A, Deactivation of iberiotoxin-sensitive BK current, iberiotoxin-resistant BK current, and calcium current at −30, −50, and −70 mV following activation by a 2.5 ms step to −20 mV. Currents defined as in Figure 3. B, Collected results for deactivation kinetics defined by the time to half-maximal decay. Mean ± SEM: n = 14, IbTx-sensitive BK current; n = 15, IbTx-resistant BK current; n = 12, calcium current.

Figure 8.

BK and calcium currents during action potential waveforms from spontaneous firing. A, Action potentials recorded during spontaneous firing of a Purkinje neuron were used as the command waveform in a voltage-clamp recording. Iberiotoxin-sensitive BK current (blue), iberiotoxin-resistant BK current (green), and calcium current (red) were defined by the application of 200 nm iberiotoxin, 3 μm paxilline, and removal of extracellular calcium as in Figure 3. B, Expanded view of currents during the action potential. C, Collected results for integrated current during the action potential (from the threshold to the fast afterhyperpolarization), in the initial portion of the interspike interval (from the fast afterhyperpolarization to 5 ms later), and the remainder of the interspike interval (from 5 ms after the spike to the threshold of the next spike). Mean ± SEM: n = 15, IbTx-sensitive and IbTx-resistant BK current; n = 12, calcium current.

Figure 9.

BK and calcium currents during 134 Hz firing evoked by injection of current. A, Action potentials evoked by a 150 pA current injection were used as the command waveform in a voltage-clamp recording. Iberiotoxin-sensitive BK current (blue), iberiotoxin-resistant BK current (green), and calcium current (red) were defined by the application of 200 nm iberiotoxin and 3 μm paxilline, and by the removal of extracellular calcium as in Figure 3. B, Expanded view of currents during the third action potential. C, Collected results for integrated current during the action potential (from the threshold to the fast afterhyperpolarization) and in the interspike interval (from the fast afterhyperpolarization to threshold of the next spike) for each action potential. Mean ± SEM: n = 15, IbTx-sensitive and IbTx-resistant BK current; n = 12, calcium current.

Figure 10.

BK and calcium currents produced by burst firing. A, Top, A burst of four action potentials elicited by an EPSC-like current injection (see Materials and Methods) was recorded in current clamp and used as the command waveform in voltage-clamp experiments. Bottom, IbTx-sensitive BK, IbTx-resistant BK, and calcium current evoked by burst command waveform. IbTx-sensitive BK current reached its peak value during the third burst spike, whereas calcium current was prominently driven during each action potential. B, Charge measured as current integrated during each spike and interspike interval, including the afterdepolarization following the fourth and last burst spike. Mean ± SEM: n = 15, IbTx-sensitive and IbTx-resistant BK current; n = 12, calcium current.

Figure 11.

Delay between calcium current and IbTx-sensitive BK current activation during action potential waveforms. IbTx-sensitive BK and calcium currents were normalized to their individual peak amplitudes. A–C, The time for each current to reach half its peak amplitude was determined for both spontaneous (A) and evoked (B, C) action potential waveforms. A, IbTx-sensitive BK current activated slightly later than calcium current during spontaneous action potentials (108 ± 22 μs, n = 12). B, A similar delay was found for the first action potential of the evoked train (105 ± 20 μs, n = 12). C, IbTx-sensitive BK current began to activate earlier in action potentials occurring later in the evoked train (56 ± 34 μs, n = 12).

Figure 12.

Iberiotoxin reduces the medium afterhyperpolarization of spontaneous action potentials and can induce bursting. A, Spontaneous firing in control, with 200 nm iberiotoxin, and with 200 nm iberiotoxin plus 3 μm paxilline. Right, Superimposed action potentials in the three conditions. B, In a subset of neurons (6 of 19), iberiotoxin induced spontaneous burst firing. C, Effects of BK inhibition on spontaneous firing rate (n = 13, excluding bursting neurons), spike width (n = 13), the fast afterhyperpolarization (n = 13), and the medium afterhyperpolarization (n = 12). D, Iberiotoxin induction of bursting during injection of −25 pA to hyperpolarize a neuron in which iberiotoxin did not induce bursting during spontaneous firing.

Figure 13.

Iberiotoxin speeds firing evoked by current injection. A, Firing driven by a 200 pA current injection in control, after 200 nm iberiotoxin, and in 200 nm iberiotoxin plus 3 μm paxilline. B, Instantaneous firing frequency for first 11 action potentials during 200 pA current injections in the three conditions. Mean ± SEM: n = 13, control; n = 13, iberiotoxin; n = 10, paxilline. C, Instantaneous firing frequency for first two action potentials during injection of current from 0 to 200 pA. Mean ± SEM: n = 13, control; n = 13, iberiotoxin; n = 10, paxilline.