Other Voltagedependent Ion Channels

There has been a great deal of speculation as to the significance of voltagedependent ion channels on glial cells. Much of this has been engendered by results obtained in studies on the current-voltage (I-V) relation of the individual channels, which suggest that many of them are inactive under normal physiological conditions. Because the glial cell membrane potential is highly dependent upon [K+]o, there is the assumption that the potassium efflux resulting from neuronal activity is too small to depolarize the glial cell membrane sufficiently to cause channel activation.

There is, however, an important caveat. Estimates of [K+]o are often obtained from measurements in the extracellular space with relatively large K+-sensitive electrodes. These and other ion-specific microelectrodes (ISM) typically taper to tip diameters of ~1.5 um, but create a large "dead" space

K* decrease

(Receptor layer)

K+ increase

Figure 5.6. Schematic drawing of potassium buffering currents resulting from the light-evoked extrusion of K+ at synaptic sites within the IPL and OPL. The current path will depend upon the membrane distribution of IK(IR) and can lead to the deposition of K+ in the vitreous, as well as in the distal retina. In this diagram, K+ enters the Müller cell in the plexiform layers, and exits in regions where K+ is unchanged (the vitreous) or reduced by neural activity (the sub-retinal space).

K+ increase

K* decrease

(Receptor layer)

K+ increase

K+ increase

K+ exit

K+ entry

K+ exit

K+ entry

K+ exit around the electrode and cause damage as they penetrate the tissue to reach the recording site (often poorly defined). Moreover, the ISM records "net" changes in ion activity, and the measured values may be reduced by active uptake mechanisms. Collectively, these factors probably lead to gross underestimates of the changes in occurring within the confines of the synaptic clefts (cf. Orkand, 1980; Ripps and Wikovsky, 1985). Consequently, there may be large potential changes across local regions of the glial cell membrane that go undetected by the recording electrode.

5.3.1. The Delayed Rectifier Channel (KDR)

The outward current carried by the delayed rectifier potassium channel provides a good example of why there is confusion regarding the role of most voltage-activated channels in glial cells. First described in the squid giant axon, where it serves to repolarize the cell following discharge of the action potential, its presence on astrocytes both in culture and in situ has

Figure 5.7. Whole cell recordings of a sustained (noninactivating), outwardly rectifying K+ current in rabbit Müller cells. A. Slowly activating outward currents elicited by depolarizing voltage steps from -80 to +60 mV in 10 mV increments. B. The I-V curve for this delayed rectifier (IK(DR)) shows that the threshold of activation is about -30 mV, well beyond the normal operating range of Müller cells (Chao et al., 1994a). (Copyright 1994 Springer-Verlag, reprinted with permission.)

Figure 5.7. Whole cell recordings of a sustained (noninactivating), outwardly rectifying K+ current in rabbit Müller cells. A. Slowly activating outward currents elicited by depolarizing voltage steps from -80 to +60 mV in 10 mV increments. B. The I-V curve for this delayed rectifier (IK(DR)) shows that the threshold of activation is about -30 mV, well beyond the normal operating range of Müller cells (Chao et al., 1994a). (Copyright 1994 Springer-Verlag, reprinted with permission.)

been amply documented (Bevan and Raff, 1985; Bevan et al., 1985; Nowak et al., 1987; Tse et al., 1992; Steinhauser, 1993; Steinhauser et al., 1994). Delayed rectifier channels mediating the outward current in different cell types appear to be pharmacologically and structurally distinct (cf. Hille, 1992). However, the channels are typically inactive at the resting membrane potential of glial cells and activate when the membrane is depolarized to about -30 mV; with prolonged depolarization, the outwardly-rectifying K+ current is well maintained.

Recently, the presence of a similar type of voltage-dependent K+ channel was revealed with whole-cell voltage-clamp recordings from isolated mammalian Müller cells (Chao et al., 1994a, 1997; Reichelt et al., 1993). In rabbit, the KDR channels required depolarizing voltages (--30 mV) for activation (Fig. 5.7), whereas in guinea pig and mouse Müller cells, the channels were activated when membrane depolarization was positive to -20 mV and 0 mV, respectively.

It is hard to speculate about a possible role for these channels in Müller cells. Although it has been suggested that they may be involved in promoting cell proliferation following neural injury (Chiu and Wilson, 1989; Chao et al., 1997), no plausible explanation of how this is accomplished has been put forth.

A-type K+ currents activate and inactivate quickly, properties that enable them to regulate the frequency of repetitive firing in spontaneously active neurons (Connor and Stevens, 1971). Once again, there appears to be no rational explanation for their presence on glial cells. Nevertheless, transient outward currents with characteristics similar to those of neuronal Ka have been described in salamander (Fig. 5.5), guinea pig, and rabbit Müller cells (Newman, 1985b; Chao et al., 1994a; Reichelt et al., 1993) as well as in other types of glia in the CNS (cf. Bevan et al., 1987; Barres et al., 1990; Ritchie, 1992).

It is not too difficult to distinguish between currents carried by KA and those due to KDR channels. Like the delayed rectifier, the "A" current is activated only at depolarized membrane potentials (--30 mV), but the KA-

mediated current is far more transient and rapidly inactivates. In Müller cells, the A current appears to be significantly less sensitive to the convulsant 4-aminopyridine (4-AP) and its derivatives, as well as to external tetra-ethylammonium (Chao et al., 1994a). Moreover, activation of KA channels typically requires a brief hyperpolarization preceding the depolarization. Retinal glia are rarely exposed to patterns of activity that could induce activation of KA, e.g., large, rapid fluctuations in K+o resulting from recurrent action potentials, and thus the functional significance of KA channels on Müller cells is far from clear.

5.3.3. Ca2+ Channels and Ca2+-activated K+ Channels

Voltage-dependent Ca2+ channels resembling the L-type neuronal channel were first described in cultures of rat brain astrocytes (MacVicar, 1984), and subsequently shown to be present in Müller cells of salamander retina (Fig. 5.5; Newman, 1985b). More recently, two classes of Ca2+ channel, the L-type and T-type (Fig. 5.8), were identified in cultures of Müller cells from donor eyes (Puro and Mano, 1991a). These channels are readily distinguished by their kinetics and pharmacology. L-type currents are prolonged, and blocked by 1,4-dihydropyridines; T-type currents are transient, inactivate within tens of msec, and are insensitive to dihydropyridines. Interestingly, the expression of calcium channels in glia appears to be dependent upon extrinsic factors derived from serum (Barres et al., 1989b), cocultured neurons (Corvalan et al., 1990; Puro and Mano, 1991a; Puro, 1994a), or exposure to agents that increase intracellular cAMP (MacVicar, 1984).

Activation of both types of voltagedependent Ca2+ channel requires membrane depolarization to about -50 mV, again seemingly beyond the normal operating range of Müller cells. However, there is evidence that the depolarization-induced rise in [Ca2+] seen in astrocytes in situ is an indirect effect, resulting from the activation of metabotropic receptors by the neuronal release of neurotransmitters, e.g., glutamate (Carmignoto et al., 1998). Regardless of how the rise in [Ca2+] is brought about, the

depolarization-induced influx of calcium may lead to activation of Ca2+-sensitive potassium channels (Fig. 5.5) and thus contribute to the buffering capacity of glial cells (Quandt and MacVicar, 1986). Indeed, the process may be further enhanced in human Müller cells by a voltage-insensitive, calcium-activated, nonspecific cation channel that responds to the rise in [Ca2+] (Puro, 1991a).

5.3.4. Sodium Channels

Perhaps even less well understood is the finding that glial cells express voltage-activated, tetrodotoxin-sensitive Na+ channels indistinguishable from those used by nerve cells to generate action potentials (cf. Bevan et al., 1985; Barres et al., 1989a; Sontheimer and Waxman, 1992). Their functional significance in glial cells remains a mystery, although various suggestions have been put forth. One popular idea is that the influx of Na+ through these channels may fuel the (Na+, K+)-ATPase that subserves K+ uptake. Another is that a rise in [Na+]i could produce graded potentials that serve to activate various intracellular signaling cascades (Black and Waxman, 1996). There is little reason for thinking these events occur under normal conditions; the I-V relationship for astrocytes shows that the threshold for activation of the sodium current is about -40 mV, and the maximal current is elicited at approximately -20 mV (Bevan et al., 1985).

Similar voltage-dependent sodium channels have been found in Müller cells of human and other (but not all) mammalian species (Chao et al., 1993, 1994b). As with astrocytes, threshold activation occurs at about -40 mV, and the peak current is reached at -12 mV (Fig. 5.9). Because these levels of depolarization would not be expected under normal physiological conditions, the sodium channels on Müller cells are almost certainly not involved in impulse propagation, nor is there evidence that other types of glia are capable of establishing regenerative Na+ spikes (cf. Ritchie, 1987,

Figure 5.8. Calcium currents obtained from whole-cell patch clamp recordings on isolated human Müller cells are predominantly a slowly-inactivating L-type current, or a transient T-type current. A. Currents evoked by voltage steps from a holding potential of -100 mV to the potentials indicated to the right of the current traces reveal the presence of a L-type inward current. B. Peak currents plotted against voltage for the cell depicted in A show that the threshold for activation is about -35 mV, and the maximum current is reached when the membrane potential is depolarized to approximately 0 mV. C. Another Müller cell responded to depolarizing pulses (from a holding potential of -100 mV) with transient T-type calcium currents. D. The peak current-voltage relation shows that the activation threshold is at about -50 mV, and Imax again occurs in the region of 0 mV (Puro and Mano, 1991a). (Copyright 1991 The Journal of Neuroscience, reprinted with permission.)

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