Protein kinases modulate two glycine currents in salamander retinal ganglion cells

Protein kinase modulation of glycine-activated currents was examined in acutely dissociated ganglion cells from tiger salamander retina using whole-cell voltage-clamp techniques. Glycine (100 μM) induced an outward chloride current in cells clamped at 0 mV. Co-application of 50 μM forskolin made the glycine-induced current more transient. The combination of forskolin and glycine reduced the later portion of current response without changing the initial peak amplitude. 3-Isobutyl-1-methylxanthine (IBMX) or 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) produced effects similar to those of forskolin. H-89, a protein kinase A (PKA) inhibitor, blocked the effect of forskolin. A protein kinase C (PKC) activator, OAG (1-oleoyl-2-acetyl-sn-glycerol), also made the glycine response more transient. Unlike PKA analogues, OAG enhanced the glycine peak response without changing the glycine late response. OAG effects were blocked by 1 μM GF-109203X, a PKC inhibitor. Nanomolar concentrations of strychnine selectively blocked the fast phase of the glycine current and reversed the effect of OAG, but not that of forskolin. Conversely, forskolin occluded the effect of 5,7-dichlorokynurenic acid, which selectively suppresses the late phase of the glycine current. The action of OAG was not blocked by 5,7-dichlorokynurenic acid. Thus, through a differential modulation, both protein kinase A and C shorten the decay time of the glycine current. PKA suppresses the slow component, while PKC potentiates the fast component. Phosphorylation of ligand-gated ion channels has been proposed as a possible mode of synaptic plasticity. The glycine receptor, a member of a family of ligand-gated ion channels, has been shown to be phosphorylated by intracellular second messenger-triggered protein kinases, including protein kinase A and C (Ruiz-Gomez, Vaello, Valdivieso & Mayor, 1991; Vaello, Ruiz-Gomez, Lerma & Mayor, 1994). Both kinases alter the magnitudes of glycine-evoked currents; however, there are discrepancies regarding their precise effects. Protein kinase A (PKA) has been reported to enhance the glycine response in Xenopus oocytes injected with rat brain mRNA (Vaello et al. 1994). On the other hand, PKA reduced glycine responses in rat substantia nigra neurones (Inomata, Nabekura & Akaike, 1993). Agopyan, Tokutomi & Akaike (1993) reported that PKA activation reduced the open probability of the glycine receptor in the acutely isolated rat ventromedial hypothalamic neurones, while Song & Huang (1990) reported that PKA increased the channel open probability in the trigeminal neurones of the rat spinal cord. Protein kinase C (PKC) has been shown to increase the glycine current in rat sacral dorsal commissural neurones (Xu, Nabekura & Akaike, 1996), rat substantia nigra (Nabekura, Omura, Horimoto Ogawa & Akaike, 1996), and rat hippocampal neurones (Schonrock & Bormann, 1995), but to decrease current of glycine receptor subunits expressed in Xenopus oocytes (Uchiyama, Hirai, Hishinuma & Akagi, 1994; Vaello et al. 1994). These conflicting results could be due to regional differences in the expression of subunits making up the glycine receptor complex. Molecular biological studies have shown the existence of heterogeneity of glycine receptor subunits in rats, mice and humans, with four different α subunit isoforms and a single β subunit (Grenningloh et al. 1987; Grenningloh, Pribilla, Prior, Multhaup, Beyreuther & Taleb, 1990; Kuhse, Schmieden & Betz, 1990; Akagi, Hirai & Hishinuma, 1991; Handford, Lynch, Baker, Webb, Ford & Sutherland, 1996). The diverse regional and developmental distribution of these subunits has been confirmed by immunohistochemical staining and in situ hybridization in the spinal cord and higher regions of the central nervous system (Triller, Cluzeaud, Pfeiffer, Betz & Korn, 1985; Altschuler, Betz, Parakkal, Reeks & Wenthold, 1986; Malosio, Marqueze-Pouey, Kuhse & Betz, 1991; Kirsch & Betz, 1993). This is relevant to retinal ganglion cells. Greferath, Brandstatter, Wassle, Kirsch, Kuhse & Grunert (1994) have described differential expression of glycine α subunits (α1, α2 and α3) in rat retinal ganglion cells. Recently, we found that retinal ganglion cells possess two types of glycine responses, which can be distinguished by their kinetics and their pharmacology (Han, Zhang & Slaughter, 1997). One has a fast desensitization rate and is blocked by nanomolar concentrations of strychnine. The other, a more sustained current, is suppressed by 5,7-dichlorokynurenic acid (DCKA). This offers an opportunity to simultaneously compare the effects of protein kinases on two distinct glycine currents in the same neurone. Our experiments indicate that PKA reduces the slow component of the glycine current while PKC enhances the fast component of the current. Since the time course of the overall glycine current is the sum of both of these components, the net effect of either kinase is an acceleration of the decay of the glycine response.