Iberiotoxin-sensitive and -insensitive BK currents in Purkinje neuron somata - PubMed (original) (raw)

Iberiotoxin-sensitive and -insensitive BK currents in Purkinje neuron somata

Mark D Benton et al. J Neurophysiol. 2013 May.

Abstract

Purkinje cells have specialized intrinsic ionic conductances that generate high-frequency action potentials. Disruptions of their Ca or Ca-activated K (KCa) currents correlate with altered firing patterns in vitro and impaired motor behavior in vivo. To examine the properties of somatic KCa currents, we recorded voltage-clamped KCa currents in Purkinje cell bodies isolated from postnatal day 17-21 mouse cerebellum. Currents were evoked by endogenous Ca influx with approximately physiological Ca buffering. Purkinje somata expressed voltage-activated, Cd-sensitive KCa currents with iberiotoxin (IBTX)-sensitive (>100 nS) and IBTX-insensitive (>75 nS) components. IBTX-sensitive currents activated and partially inactivated within milliseconds. Rapid, incomplete macroscopic inactivation was also evident during 50- or 100-Hz trains of 1-ms depolarizations. In contrast, IBTX-insensitive currents activated more slowly and did not inactivate. These currents were insensitive to the small- and intermediate-conductance KCa channel blockers apamin, scyllatoxin, UCL1684, bicuculline methiodide, and TRAM-34, but were largely blocked by 1 mM tetraethylammonium. The underlying channels had single-channel conductances of ∼150 pS, suggesting that the currents are carried by IBTX-resistant (β4-containing) large-conductance KCa (BK) channels. IBTX-insensitive currents were nevertheless increased by small-conductance KCa channel agonists EBIO, chlorzoxazone, and CyPPA. During trains of brief depolarizations, IBTX-insensitive currents flowed during interstep intervals, and the accumulation of interstep outward current was enhanced by EBIO. In current clamp, EBIO slowed spiking, especially during depolarizing current injections. The two components of BK current in Purkinje somata likely contribute differently to spike repolarization and firing rate. Moreover, augmentation of BK current may partially underlie the action of EBIO and chlorzoxazone to alleviate disrupted Purkinje cell firing associated with genetic ataxias.

Keywords: EBIO; Kca; SK; action potential; calcium-activated potassium; cerebellum; voltage-clamp.

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Figures

Fig. 1.

Fig. 1.

Total Ca and Ca-activated K (KCa) current in Purkinje somata during steps and trains. A: representative raw currents evoked by 20-ms steps from −65 mV in 5-mV increments, in control (ctrl) solution (top), iberiotoxin (IBTX; middle), and in 300 μM Cd (bottom). Δ, Step increment. B: current-voltage relations for the maximal step depolarization-evoked currents for all three conditions (n = 11). C, left: Cd-sensitive currents (summed Ca and KCa), obtained as the difference of records evoked in control and Cd, by 50- (top) or 100-Hz (bottom) 1-s trains of 1-ms pulses to +20 mV [holding potential (_V_hold) = −60 mV]. Right: the first few responses on an expanded time base. D: peak train-evoked Cd-sensitive currents, normalized to the response to the first pulse (n = 8). For 100 Hz, every second point is plotted. Values in all figures are plotted as mean ± standard error.

Fig. 2.

Fig. 2.

Inactivation of IBTX-sensitive large-conductance KCa currents. A: difference currents from Fig. 1, illustrating IBTX-sensitive currents (left) and IBTX-insensitive, Cd-sensitive currents (right) evoked by 20-ms steps from −65 mV in 5-mV increments. Scale applies to both sets of traces. Insets: tail currents for each set of traces on expanded time base; same scale and voltage steps for both insets. B: current-voltage relation for peak IBTX-sensitive and IBTX-insensitive currents (n = 11). Note that, because the peaks occur at different times, the sum of the two curves is greater than the peak raw currents in Fig. 1_B_. C: representative IBTX-sensitive currents elicited by 50 (top) or 100 Hz (bottom) 1-s trains of 1-ms pulses to +20 mV (_V_hold = −60 mV). D: expansion of the first three responses to 50-Hz steps (black). Dotted line is baseline (80 pA). Vertical scale applies to all panels. E: peak currents normalized to the first response in the train (n = 7). For 100 Hz, every second point is plotted.

Fig. 3.

Fig. 3.

IBTX-insensitive currents. A: representative IBTX-insensitive, Cd-sensitive (summed Ca and KCa) currents evoked by 20-ms steps from −65 mV in 5-mV increments. Inset: tail currents on expanded time base. Only records before the turnover of the current-voltage curve are included for clarity. B: current-voltage relation for peak step-evoked currents (n = 11). C: currents evoked with constant Ca (30 μM intracellular Ca; 30 μM extracellular Cd) by voltage steps from −65 mV to +60 mV, in −10-mV increments, in control (IBTX-free) solutions (left) and IBTX (middle). Right: IBTX-sensitive current obtained by subtraction. D: mean current-voltage relations (n = 5), indicating the peak current in control (upward triangles), the instantaneous current in control (downward triangles), and the peak current in IBTX (circles). In IBTX, no data were recorded at −50 and −60 mV. E: Cd-sensitive currents evoked with standard extracellular Ca and intracellular EGTA in IBTX (left), in IBTX with 1 mM tetraethylammonium (TEA; middle). Right: Cd and TEA-sensitive, IBTX-insensitive current obtained by subtraction. F: mean current-voltage relations for the three conditions (n = 9). G: macroscopic current-voltage relation for an inside-out patch exposed to either 0 or 30 μM Ca intracellularly, with IBTX and Cd on the extracellular face. Currents were measured as the average current amplitude between 300 and 400 ms of the voltage step. H: representative IBTX-insensitive single K channels recorded at −90 mV in 0 or 30 μM Ca. Conductance, 164 pS. Same patch as in _G. E_K, K equilibrium potential.

Fig. 4.

Fig. 4.

Lack of effect of small-conductance KCa antagonists on IBTX-insensitive current. A, left: representative raw (unsubtracted) responses of IBTX-insensitive Ca and KCa currents evoked by 10-ms depolarizing steps from −65 mV to −20 mV, before (black) and after (gray) antagonist application (as labeled). Calibration bars apply to all traces. Right: summary of peak outward current amplitudes for each cell before and after antagonist. Individual cells, open symbols; mean values, closed symbols. Only curare produced a statistically significant decrease in the current. B, left, top: raw currents evoked by 50 trains of depolarizing pulses as in Fig. 1, in control (IBTX-containing) solutions (black) and in 200 nM apamin (gray). The black trace is largely obscured by the gray. Left, bottom: difference current, illustrating the apamin-sensitive component. Middle: expanded response to the last depolarizing pulse from the left traces. Arrow, time of maximal outward current after the train, from which measurements of posttrain current were taken. Note that the difference current is tiny and inward during the step, likely from a small rundown of Ca current. Right: peak posttrain current in 4 cells, before and after apamin. BMI, bicuculline methiodide; TRAM, 1-[(2-chlorophenyl)diphenylmethyl]-1_H_-pyrazole.

Fig. 5.

Fig. 5.

Augmentation of IBTX-insensitive, Cd-sensitive current by 1-ethyl-2-benzimidazolinone (EBIO). A, left: representative Cd-sensitive current recorded in IBTX evoked by a 20-ms step from −65 to −25 mV before (gray) or during (black) application of 20 μM EBIO. B: current-voltage relationships for the step (left) and tail (right) currents before (closed symbols) or after (open symbols) application of EBIO (n = 6). C: half-activation (_V_1/2) and slope factor (k) of the conductance-voltage curve, obtained from tail currents, with and without EBIO (n = 6). D, left: responses to a 50-Hz, 1-s train of 1-ms pulses to +20 mV (_V_hold = −60 mV) before (top) or during (bottom) application of EBIO. Right traces are expansions of the current evoked by the final step. E: expansion of boxed region in D. F: interstep currents following the 20th pulse in D. G: peak interstep currents, measured 1 ms before the next step, during 50- or 100-Hz stimulation (n = 6). NS, nonsignificant.

Fig. 6.

Fig. 6.

Modulation of spontaneous firing by EBIO. A: spontaneous action potentials of a Purkinje cell body before and during application of 10 μM EBIO (bar). B: a single action potential showing the small increase in the afterhyperpolarizations (AHP) in EBIO (gray). C: relationship between spontaneous firing rate (left), AHP amplitude (middle), and the maximal rate of rise and decay of a spike (dV/d_t_; right) in the absence or presence of EBIO (n = 18).

Fig. 7.

Fig. 7.

Modulation of the input-output curve by EBIO. A: spontaneous and driven action potentials evoked by 200-ms current injections (amplitudes as labeled) before (left) and after (right) application of 10 μM EBIO. Calibration bars apply to all traces. B: input-output curves in the absence (closed symbols) or presence (open symbols) of EBIO (n = 7).

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