A role for a TASK-1-like conductance in GABAergic modulation of fast synaptic transmission
Background (leak) K+ currents control neuronal excitability and shape action potentials by controlling the resting membrane potential (Goldstein et al. 2001). Enhancement of leak conductances stabilises cells at hyperpolarised potentials, while inhibition leads to membrane depolarisation and excitation. Background currents are carried by members of the K2P family of tandem-pore-domain K+ channels, of which at least 14 members have been cloned (Goldstein et al. 2001, 2003). K2P channel activity is tightly controlled by intracellular factors such as cyclic nucleotide levels and metabolic status, and by extracellular factors such as neurotransmitters, including 5-HT, noradrenaline, substance P, glutamate, TRH and ACh (Millar et al. 2000; Talley et al. 2000; Goldstein et al. 2001). In hypoglossal motoneurones, where TASK-1 is abundantly expressed, native TASK-1-like currents were inhibited by agonists at several G-protein-coupled neurotransmitter receptors (Talley et al. 2000). This provides a mechanism whereby neurotransmitters can regulate neuronal excitability and provide slow regulation of fast neurotransmission via intracellular effectors. Here, we demonstrate that a selective agonist at metabotropic receptors for the inhibitory neurotransmitter GABA activates a K+-selective background conductance in presynaptic chemoreceptor cells of the rat carotid body. Block of this conductance by either anandamide (Maingret et al. 2001) or Ba2+ ions, alongside data demonstrating that baclofen activates a linear K+ conductance under conditions where voltage- and Ca2+-dependent K+ channels are inhibited, suggests that this effect is attributable to activation of a tandem-pore-domain K+ channel with characteristics of TASK-1. This link between GABAB receptors and this background K+ channel provides a novel mechanism for regulating neuronal excitability and synaptic signalling.
In the present study, we examined the effects of several GABAB receptor antagonists on the depolarising responses to hypoxia in both isolated type I clusters and in petrosal neurons juxtaposed to type I clusters in co-culture. The responses to hypoxia obtained in these studies were modest, particularly when compared to those obtained previously by others when recording hypoxic responses in single type I cells (e.g. Buckler & Vaughan Jones, 1994). The reasons for this are not fully understood, but one possibility is the operation of autoreceptor feedback mechanisms within clusters, such as that reported here, that serve to limit the degree of depolarisation during hypoxia. Such mechanisms are expected to be most effective when recording from clusters as opposed to single cells. In general, the receptor potential in a chemoreceptor cluster reflects a balance between inhibitory (e.g. GABA) and excitatory (e.g. 5-HT; Zhang et al. 2003) feedback influences, and this may vary in culture from one cluster to another.
A recent study demonstrated the activation by baclofen of a background K+ conductance in mouse cerebellar purkinje neurones with pharmacological characteristics of the background channel THIK-1 (Rajan et al. 2001; Bushell et al. 2002). Furthermore, we recently demonstrated the O2 sensitivity of a similar background K+ current in glossopharyngeal (GPN) neurones (Campanucci et al. 2003). However, the THIK-1-like, O2-sensitive K+ current in GPN neurones is anandamide-insensitive (Campanucci et al. 2003), making it unlikely that these channels mediate O2 and/or baclofen sensitivity in type I cells.
GABAB receptors linked to TASK-1 provide presynaptic autoregulatory feedback during hypoxia
GABA is a well characterised inhibitory CNS neurotransmitter and its effects at presynaptic metabotropic GABAB receptors are thought to underlie, amongst other processes, autoregulation of neurotransmitter release. In many cases regulation involves receptors coupled by G-proteins to plasmalemmal Ca2+ and K+ channels (Misgeld et al. 1995; Bowery & Enna, 2000) or to the exocytotic machinery itself (Wu & Saggau, 1997). Here, we present immunohistochemical evidence for the presence of GABA in presynaptic type I cells of the rat carotid body. Examination of type I cell clusters in co-culture with their postsynaptic (petrosal neurone) partners revealed that selective inhibitors of GABAB receptor function markedly enhanced synaptic transmission in response to hypoxia. This is attributable, at least in part, to a presynaptic mechanism since GABAB receptor blockade also enhanced the hypoxia-induced depolarisation or receptor potential in type I cells cultured alone.
The mechanism by which GABAB receptor inhibition enhances the efficacy of hypoxic chemotransmission involves modulation of the activity of PKA, since blockers of this kinase (and not of PKC) inhibited the enhancing effect of the GABAB receptor blocker hydroxysaclofen on presynaptic depolarisation. Evidence from this study further points to a mechanism involving GABA-mediated activation of a background K+ channel, with properties similar to those of TASK-1, during hypoxia. Inhibition of this TASK-1-like K+ channel (Buckler et al. 2000), is thought to be at least partly responsible for the hypoxic depolarisation or receptor potential in type I cells. The rat isoform of TASK-1 possesses two C-terminal consensus sites for phosphorylation by PKA and furthermore, current through TASK-1 is inhibited by stimulation of this kinase (Leonoudakis et al. 1998; Lopes et al. 2000). Neuronal GABAB receptors couple to the inhibitory G protein, Gi (Isaacson, 1998; Leaney & Tinker, 2000), leading to K+ channel activation (Misgeld et al. 1995). Moreover, Gi has also been shown to couple functionally to a GABAB receptor-adenylyl cyclase system (Nishikawa et al. 1997). In our system, inhibition of Gi by pretreatment with PTX abolished presynaptic sensitivity to GABAB receptor blockade. We suggest therefore that in the rat carotid body, presynaptic GABAB receptor activation by GABA released from type I cells couples to Gi, resulting in inhibition of PKA activity and activation of background K+ channels. This pathway is enhanced during hypoxic stimulation, due to depolarisation-evoked GABA release which leads to activation of presynaptic GABAB autoreceptors and subsequently activation of the TASK-1-like conductance via Gi-mediated inhibition of PKA. This cascade gives rise to a hyperpolarisation which effectively blunts the depolarising receptor potential due to hypoxia. The end result is an autoregulatory feedback mechanism (see Fig. 7 for schematic representation of this cascade) that modulates the release of neurotransmitters from the receptor cells during hypoxia via convergence of two separate signalling pathways onto the same K+ channels. We further suggest that the basal activation of GABAB receptors exerts control over the excitability of presynaptic type I cells, since blockade of this constitutive activity with hydroxysaclofen enhanced the excitability of presynaptic type I cells in which spontaneous activity was observed. Taken with our data, this suggests that basal GABAB receptor activation stimulates Gi, causing inhibition of adenylate cyclase and a reduction in cAMP levels, which would reduce type I cell excitability via the activation of the TASK-1-like conductance.
Figure 7. Schematic representation of the autoregulatory pathways involved in the GABA-mediated regulation of neurotransmitter release from type I cells during hypoxia
Via an as yet uncharacterised intracellular pathway, hypoxia inhibits TASK-1-like background channels in type I cells, leading to membrane depolarisation and ultimately (broken arrow) neurotransmitter release. In this process GABA (black circles) is released from type I cells, and acts at presynaptic GABAB receptors on either the same type I cell (autocrine) or on an adjacent type I cell (paracrine) in the cluster. This causes stimulation of the pertussis toxin-sensitive inhibitory G protein Gi, causing inhibition of protein kinase A (PKA) and subsequently activation of TASK-1. This would serve to hyperpolarise the type I cell and limit the degree of depolarisation during exposure to hypoxia, regulating the further release of transmitters. GABA may also act at postsynaptic ionotropic or metabotropic GABA receptors to modulate chemoreceptor output. For clarity, the involvement of other K+ channels and neurotransmitters in chemotransmission, and the intracellular events leading to transmitter release, have been omitted.
Download figure to PowerPoint
In the rabbit carotid body dopamine, acting at presynaptic D2 receptors, exerts autoregulatory feedback to inhibit the further release of this transmitter (Bairam et al. 2000), presumably via the inhibition of voltage-dependent Ca2+ currents (Benot & Lopez-Barneo, 1990). The presence of dopamine and presynaptic D2 receptors (Gauda et al. 1996; Donnelly, 2000) suggests that similar autoreceptor feedback loops may control neurotransmitter output in the rat carotid body, although this has not been directly tested. Since dopamine D2 receptors are linked to inhibition of adenylate cyclase and reduce cellular cAMP levels upon activation, it is possible that dopamine acting via presynaptic D2 receptors elicits a similar autoinhibitory feedback loop as that evoked by GABAB receptor activation. To support this possibility, D2 receptor activation has been shown in many cases to activate neuronal K+ conductances (Lacey et al. 1987; Freedman & Weight, 1988; Casteletti et al. 1989). On the other hand, recent studies from this laboratory indicate that paracrine release of 5-HT from clustered type I cells produces the opposite effect and augments the receptor potential via PKC-mediated inhibition of K+ channels (Zhang et al. 2003).
Autoreceptor regulation of neurotransmitter release in the CNS is mediated in part by activation of presynaptic GABAB receptors which modulate voltage-gated Ca2+ channels (Misgeld et al. 1995; Bowery & Enna, 2000). However, in the present studies GABAB receptor activation was without effect on Ca2+ channel activity in presynaptic type I cells. In the neonatal rat carotid body, Ca2+ current is carried almost exclusively by L-type channels (Stea et al. 1995; Peers et al. 1996). Although the inhibitory effects of GABAB receptor activation on neuronal N- (e.g. Lambert & Wilson, 1996) and P/Q-type (e.g. Chen & van den Pol, 1998) Ca2+ channels are well documented, there is little evidence to suggest that L-type Ca2+ channels are modulated by GABAB receptors. Moreover in some neurones which express multiple Ca2+ channel subtypes, effects of GABAB receptor activation on L-type Ca2+ currents are negligible (Harayama et al. 1998) or non-existent (Doze et al. 1995) compared to effects on N- and P/Q-type channels. Also consistent with our data, there is no evidence of regulation by PKA of L-type Ca2+ channels in type I cells from other species (Summers et al. 2000). Thus, autoregulation of neurotransmitter release from type I cells is mediated by presynaptic activation of a K+ current rather than Ca2+ channel inhibition. Activation of K+ conductances by GABAB receptor agonists has mostly been described in postsynaptic CNS neurones (Misgeld et al. 1995), while other studies have demonstrated no effect of GABAB receptor activation on presynaptic K+ conductances (Isaacson, 1998). However, Wagner & Dekin (1993, 1997) demonstrated the regulation by cAMP and activation by baclofen of K+ channels in presynaptic respiratory neurones with pharmacological and biophysical characteristics similar (although not identical) to those described for background K+ channels. Since TASK-1 is expressed in respiratory neurones (Bayliss et al. 2001), it is possible that GABAB receptors mediate presynaptic regulation of TASK-1 in these cells. Furthermore, background K+ channels and GABAB receptors are co-expressed in a variety of neuronal cell types and these findings open the possibility that GABAB autoreceptors linked to background channels may be a general mechanism for controlling CNS excitability.