Ionic currents in isolated and in situ squid Schwann cells

Abstract

Ionic currents from Schwann cells isolated enzymatically from the giant axons of the squids Loligo forbesi, Loligo vulgaris and Loligo bleekeri were compared with those obtained in situ. Macroscopic and single channel ionic currents were recorded using whole-cell voltage and patch clamp. In the whole-cell configuration, depolarisation from negative holding potentials evoked two voltage-dependent currents, an inward current and a delayed outward current. The outward current resembled an outwardly rectifying K+ current and was activated at -40 mV after a latent period of 5-20 ms following a step depolarisation. The current was reduced by externally applied nifedipine, Co2+ or quinine, was not blocked by addition of apamin or charibdotoxin and was insensitive to externally applied L-glutamate or acetylcholine. The voltage-gated inward current was activated at -40 mV and was identified as an L-type calcium current sensitive to externally applied nifedipine. Schwann cells were impaled in situ in split-open axons and voltage clamped using discontinuous single electrode voltage clamp. Voltage dependent outward currents were recorded that were kinetically identical to those seen in isolated cells and that had similar current-voltage relations. Single channel currents were recorded from excised inside-out patches. A single channel type was observed with a reversal potential close to the equilibrium potential for K+ (E(K)) and was therefore identified as a K+ channel. The channel conductance was 43.6 pS when both internal and external solutions contained 150 mM K+. Activity was weakly dependent on membrane voltage but sensitive to the internal Ca2+ concentration. Activity was insensitive to externally or internally applied L-glutamate or acetylcholine. The results suggest that calcium channels and calcium-activated K+ channels play an important role in the generation of the squid Schwann cell membrane potential, which may be controlled by the resting intracellular Ca2+ level.

Figure 1. Light micrographs under Nomarski optics showing the process of enzyme digestion of the giant axon

A, giant axon preparation (scale bar = 500 μm. B, the same portion of axon 20 min after treatment with trypsin, with indications that the giant axon is shrinking and is separated from the sheath of endoneurial cells by digestion of the basal lamina. C, the giant axon with a layer of Schwann cells, isolated from the sheath using fine scissors and forceps (scale bar = 100 μm). D, single Schwann cell dissociated from the sheath (scale bar = 10 μm).

Figure 7. Single-channel currents recorded from inside-out patches demonstrating that the currents are activated by Ca²⁺ on the cytoplasmic side

The pipette solution was 50 K-100 Na, and the bath solution 150 K-asp and 300 nm Ca²⁺ buffered with EGTA. The upward deflection of the records indicates outward current. A, part of a continuous recording in 300 nm [Ca²⁺] at a membrane potential of +30 mV. B, part of a continuous recording after elevation of [Ca²⁺] to 1 μM. The time constant obtained by a single exponential fit to the histogram is 1.17 ms. C, open time histogram for 60 s recording in 300 nm[Ca²⁺]. D, open-time histogram for 60 s recording after elevation of Ca²⁺ to 1 μM. The time constant obtained by a single exponential fit is 7.76 ms.

Figure 2. Whole-cell currents recorded from a cell internally dialysed with 150 mm K-asp and bathed in one-third strength ASW

A, currents associated with voltage clamp pulses from −90 to +60 mV at 10 mV steps from the holding potential of −50 mV. B, non-linear currents associated with depolarisation after linear current subtraction using the P/4 protocol. The arrow indicates the onset of depolarisation. C, I-V relations at the end of pulses for the records in A (○) and for the records in B (•). Cell capacitance, 131.7 pF.

Figure 3. Pharmacological properties of the Ca²⁺-dependent outward current

Change in outward current after addition of compounds is shown normalized to the initial value. Drugs were applied directly to the recording chamber and allowed to act for at least 5 min. The bars represent the normalised peak outward current (mean ± s.e.m.) generated by a 130 mV depolarising pulse from a holding potential of −60 mV. The internal solution was K-aspartate plus 80 nm free Ca²⁺, external solution was ASW. Cells were monitored for changes in leakage current during application of drugs (n = not less than 5).

Figure 4. Whole-cell membrane currents demonstrating the presence of L-type Ca²⁺ currents recorded from a cell dialysed with 150 mm Cs-asp internally and with one-third strength ASW externally

A, currents associated with voltage pulses from −80 to +100 mV in 10 mV steps from the holding potential of −50 mV. B, currents recorded at 10 min after external application of 10 μM nifedipine. Voltage pulses from −80 to +60 mV were applied from the holding potential of −50 mV. C, nifedipine-sensitive currents obtained by subtracting the records in B from the records in A at corresponding voltages. D, I-V relations at the end of pulses for the records in A (○), B (•) and C (▴). Cell capacitance, 351.2 pF.

Figure 5. Effects of externally applied 3 mm Co²⁺ (A), and 10 μM nifedipine (B) on the outwardly rectifying I_K

Cells were dialysed with 150 K-asp and bathed in one-third strength ASW. Depolarising pulses were applied from the holding potential of −50 mV, and linear currents were subtracted using the P/4 protocol. The lower records in A and B show the currents at 10 min after the Co²⁺, and nifedipine applications, respectively. Cell capacitance: A, 138 pF; B, 242 pF.

Figure 6. Discontinuous single electrode voltage clamp recording (dSEVC) from Schwann cells in situ

A, currents in response to membrane depolarisations from the holding potential of −66 mV (10 mV steps, below). B, leak subtracted currents (P/4) from the same cell as A. C, current-voltage relations of voltage-dependent currents in A (•) and B (○). D, detail of current and voltage from B showing a single exponential fit to the data and the rise-time of the membrane voltage (lower trace). Duty cycle was 2.5 kHz.

Figure 8. I-V relations of single channel currents recorded from excised inside-out patches demonstrate that the Ca²⁺-activated channel is a K⁺-selective channel

Filled circles indicate the current amplitudes obtained with the pipette solution containing 50 K-100 Na and a bathing solution of 150 K-asp, and filled squares with 150 K-asp on both sides. Each point was obtained by averaging the peak values of two Gaussian fits obtained from 5-7 experiments, and the error bars show the magnitude of s.d.