Voltage-dependent calcium and potassium channels in retinal glial cells

Abstract

Glial cells, which outnumber neurones in the central nervous system, have traditionally been considered to be electrically inexcitable and to play only a passive role in the electrical activity of the brain. Recent reports have demonstrated, however, that certain glial cells, when maintained in primary culture, possess voltage-dependent ion channels. It remains to be demonstrated whether these channels are also present in glial cells in vivo. I show here that Müller cells, the principal glial cells of the vertebrate retina, can generate 'Ca2+ spikes' in freshly excised slices of retinal tissue. In addition, voltage-clamp studies of enzymatically dissociated Müller cells demonstrate the presence of four types of voltage-dependent ion channels: a Ca2+ channel, a Ca2+-activated K+ channel, a fast-inactivating (type A) K+ channel and an inward-rectifying K+ channel. Currents generated by these voltage-dependent channels may enhance the ability of Müller cells to regulate extracellular K+ levels in the retina and may be involved in the generation of the electroretinogram.

Fig. 1

Regenerative ‘Ca²⁺ spikes’ evoked by depolarizing current pulses in Müller cells of the salamander. a, b, Voltage recordings from a cell in a retinal slice. The regenerative response in control perfusate (a) increased significantly when [Ca²⁺] was raised from 2 to 20 mM (b). The plateau of the action potential lasted for several seconds following stimulation. The cell was pressure-injected with Cs⁺ and TEA to reduce the resting K⁺ conductance and bathed in pH 6.8 perfusate to de-couple the cell from other Müller cells. c-e, Recordings from a dissociated Müller cell in 6 mM Ba²⁺. Current pulses evoked a large sustained Ba²⁺ spike (c) which was substantially reduced by addition of 1 mM Cd²⁺ (d). The regenerative response returned when the Cd²⁺ was washed out (e). In both cells the resting potential was maintained at -70 mV by injection of small continuous currents. Current pulse amplitudes are indicated below each series of traces.

Fig. 2

Voltage-clamp records of voltage-dependent currents in dissociated salamander Müller cells. a, Inward Ca²⁺ current. The cell potential was stepped to the value which evoked maximal inward current (+15 mV in 20 mM Ba²⁺ and +5 mV in mM Ba²⁺). The inward current increased when [Ba²⁺] was raied from 2 to 20 mM and was abolished by addition of 2 mM Cd²⁺. The perfusate contained 5 mM TEA to reduce Ca²⁺-activated K⁺ currents. Recordings made from a cell with endfoot intact. b, Outward Ca²⁺-activated K⁺ current. In 10 mM Ca²⁺ perfusate (10 Ca), a slowly developing outward current was superimposed on the inward Ca²⁺ current. 1 mM TEA reduced the outward current while 2 mM Cd²⁺ eliminated both the inward and outward currents. Recordings from a cell lacking its endfoot process. c, Transient outward (type A) K⁺ current. The transient current was eliminated when the voltage step was preceded by a 2-s prepulse to -40 mV. d, Transient current shown in isolation by subtracting records preceded by a prepulse from records without a prepulse. The ‘control’ is the difference of the two records in c. The transient current in control perfusate was unaltered by 5 mM TEA but abolished by 5 mM 4-AP. Records from c and d were from the same endfoot-shorn Müller cell. e, Inward-rectifying K⁺ current evoked by depolarizing and hyperpolarizing voltage steps in 80 mM K⁺ perfusate. Holding potential (E_h), -4 mV, the membrane potential of the cell in 80 mM K⁺. Recordings from an endfoot-shorn cell. In a-e, inward currents are shown as downward deflections. Capacitive transients were reduced using transient cancellation circuitry. In a and b, linear contributions of the ‘leakage current’ were removed by subtracting a scaled version of records evoked by depolarizations to ∼ -60 mV.

Fig. 3

Current-voltage plots of the voltage-dependent ionic currents illustrated in Fig. 2. a, The Ca²⁺ current relation (Ca) was obtained from an intact Müller cell bathed in perfusate containing 2 mM Ba²⁺ and 5 mM TEA. It represents peak inward current, corrected for an extrapolated linear leakage current. E_h, -80 mV. The Ca²⁺-activated K⁺ current relation (K_Ca) was obtained from an endfoot-shorn cell in control perfusate and represents the steady-state current attained during a 3.5-s voltage pulse. The small outward current on the K_Ca curve between -80 and -30 mV is inward rectifier current. E_h, -90 mV. The transient (type A) K⁺ current relation (K_A) was obtained from an endfoot-shorn Müller cell in control perfusate. Current amplitude was measured between the peak of the response and the value at the end of an 800 ms pulse. E_h, -90 mV. b, The inward rectifier K⁺ current relation was recorded from an endfoot-shorn cell bathed sequentially in 2.5, 16 and 80 mM K⁺ perfusate. Current amplitudes represent the maximal values attained during an 800-ms voltage step. E_h, -80, -40 and -4 mV, the cell membrane potential in the three perfusates. The dashed line and points a-c in b are used to illustrate changes in membrane conductance caused by a localized [K⁺]_o increase. See text for details. Note differences in the vertical scales for the four currents.