Activity- and target-dependent regulation of large-conductance Ca2+-activated K+ channels in developing chick lumbar motoneurons

Abstract

The functional expression of large-conductance (BK-type) Ca2+-activated K+ (K(Ca)) channels was examined in developing chick lumbar motoneurons (LMNs) between embryonic day 6 (E6) and E13 using patch-clamp recording techniques. The macroscopic K(Ca) current of E13 LMNs is inhibited by iberiotoxin and resistant to apamin. The average macroscopic K(Ca) density was low before E8 and increased 3.3-fold by E11, with an additional 1.8-fold increase occurring by E13. BK-type K(Ca) channels could not be detected in inside-out patches from E8 LMNs but were readily detected at E11. The density of voltage-activated Ca2+ currents did not change between E8 and E11. Surgical ablation of target tissues at E5 caused a significant reduction in average K(Ca) density in LMNs measured at E11. Conversely, chronic in ovo administration of d-tubocurarine, which causes an increase in motoneuron branching on the surface of the muscle target tissue, evoked a 1.8-fold increase in average LMN K(Ca) density measured at E11. Electrical activity also contributed to developmental regulation of LMN K(Ca) density. A significant reduction in E11 K(Ca) density was found after chronic in ovo treatment with the neuronal nicotinic antagonist mecamylamine or the GABA receptor agonist muscimol, agents that reduce activation of LMNs in ovo. Moreover, 3 d exposure to depolarizing concentrations of external K+ to LMNs cultured at E8 caused an increase in K(Ca) expression. Conversely, tetrodotoxin caused a decrease in K(Ca) expression in cultured E8 LMNs developing for 3 d in the presence of neurotrophic factors that promote neuronal survival in the absence of target tissues.

Fig. 1.

Properties of K_Ca currents in E11 LMNs. A, Outward currents were evoked in control and Ca²⁺-free saline (left traces) by 25 msec depolarizing pulses to +30 mV from a holding potential of −40 mV (bottom left). Net macroscopic K_Ca was obtained after digital subtraction of raw traces (right trace). B, Mean macroscopic K_Caconductance as a function of voltage in 13 LMNs. A decline in conductance at command potentials positive to +50 is predicted for a Ca²⁺-dependent current. C, Effect of iberiotoxin (200 nm) and apamin (1 μm) on macroscopic K_Ca currents. Dissociated E11 LMNs were treated with these toxins for at least 30 min before whole-cell recordings. Control LMNs were not exposed to toxins before recording.

Fig. 2.

Tail currents and analysis of K_Cadeactivation kinetics in LMNs. A, Tail currents from the same neuron represented in Figure 1 evoked by the voltage-clamp protocol are shown below the current traces. The decay phases of the tail currents were fitted with single-exponential curves.B, Plot of Ca²⁺-dependent tail current amplitude as a function of voltage. The tail currents become undetectable at −70 mV, close to the calculatedE_K of −78 mV. C, Plot of mean tail current decay time constant as a function of voltage showing that deactivation kinetics are only weakly voltage-dependent over this range of test potentials (n = 7 cells).

Fig. 3.

Biophysical properties of large-conductance Ca²⁺-dependent K⁺ channels recorded in E11 LMNs. A, In a typical patch held at 0 mV, ion channels are quiescent in Ca²⁺-free medium but become active after bath application of saline containing 5 μm free Ca²⁺. B, Current–voltage relationship for K_Ca channels in LMNs. The reversal potential of unitary currents was determined by a voltage ramp (from −60 to 60 mV at 0.6 V/sec). Unitary currents reversed close to the E_K (−35 mV) calculated for these ionic conditions. C, All-point histograms from the patch shown in A fitted as the sum of two Gaussian functions (dotted line). Unitary current was determined as the difference in the peaks of the all-point histogram. Data from all of the patches analyzed in this way (n = 7) yielded a mean unitary conductance of 115 pS under these ionic conditions.D, Open-time histogram (bin width, 0.1 msec) with a superimposed fitted single-exponential curve (dotted line) with a time constant of 0.5 msec. E, Probability of K_Ca channel opening (p_o) over time in the presence of 5 μm-free Ca²⁺and 0 mV. The averagep_o for this patch (dotted line) was 0.20 (p_o epoch interval, 50 msec). Recordings were filtered at 2 kHz, and data were digitized at 10 kHz before analysis.

Fig. 4.

Developmental changes in the expression of macroscopic K_Ca in acutely isolated LMNs. A, Representative currents in E8 and E11 LMNs recorded in control and Ca²⁺-free saline. B, Mean K_Ca density between E6 and E13. In this and subsequent figures, error bars represent SEM, and the number of cells recorded is given above each bar. Note the significant increase in mean current density between E8 and E11, with an additional increase at E13.

Fig. 5.

Histograms of K_Ca current densities in E8, E11, and E13 LMNs. Note the rightward shift in the number of LMNs expressing higher current densities with increasing developmental stage.

Fig. 6.

Voltage-activated Ca²⁺ currents in E8 and E11 LMNs. A, Representative current traces in control and Ca²⁺-free saline. Total Ca²⁺ currents were obtained by digital subtraction (control, Ca²⁺-free), with representative examples shown on the right. Currents were evoked after a 250 msec step to +30 mV from a holding potential of −40 mV (left, bottom trace). B, Data compiled from many cells indicate no change in mean Ca²⁺ current density between E8 and E11.

Fig. 7.

Effect of growth conditions on the expression of K_Cain vitro. LMNs were dissociated on E8 and maintained in culture for 72 hr in the presence of hindlimb myotubes (A), the survival factors CNTF (40 ng/ml), NT4 (10 ng/ml), and CPT-cAMP (1 μm) (B), or 50 mm extracellular K⁺ ions (C). To examine the role of electrical activity on K_Caexpression, we added 1 μm TTX to culture media. Control neurons were cultured for 3 hr or overnight before whole-cell recordings. ^#p < 0.05 versus 3 hr with muscle;^##p < 0.05 versus 12 hr in 50 mm K⁺;^###p < 0.05 versus 3 hr in CNTF; ^####p < 0.05 versus 72 hr in CNTF.

Fig. 8.

Effects of electrical activity and target tissue interactions on the functional expression of K_Ca in LMNs developing in vivo. A, Inhibition of LMNs by in ovo application of muscimol or mecamylamine decreases K_Ca density, suggesting a role for activity in regulation of K_Ca. In contrast, in ovotreatment with the neuromuscular blocker d-tubocurarine significantly increased K_Ca density, consistent with a role for target tissue interactions in K_Ca regulation.B, Removal of target tissues reduced K_Cadensity in LMNs compared with sham-operated controls.^#p < 0.05 versus vehicle;^##p < 0.05 versus control.