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Regulation of the slow afterhyperpolarization in enteric neurons by protein kinase A
Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 258 – 263 www.elsevier.com/locate/autneu
Review
Regulation of the slow afterhyperpolarization in enteric neurons by protein kinase A Craig B. Neylon *, Christopher J. Fowler, John B. Furness Department of Anatomy and Cell Biology, University of Melbourne, Parkville 3010, Australia Received 18 January 2006; accepted 27 February 2006
Abstract The slow after-hyperpolarization (sAHP) following the action potential is an important determinant of the firing patterns of enteric neurons. The channel responsible for the sAHP thus serves as a critical control point at which neurotransmitters and inflammatory mediators modulate gut motility. Many of these receptor-evoked pathways are known to inhibit the sAHP and, thus, excite enteric neurons. They act through protein kinase A (PKA) which is a strong inhibitor of the sAHP current while protein phosphatases enhance the current. Increasing evidence suggests that the sAHP is mediated by the opening of intermediate-conductance Ca-activated potassium (IK) channels. This neuronal IK channel, previously known to be expressed in a variety of non-excitable cells, is strongly influenced by protein kinases. Investigation of the molecular basis for the modulation of IK channels by protein phosphorylation indicates that there are multiple mechanisms of channel control. Inhibition of channel activity by PKA involves phosphorylation sites located within the calmodulin-binding domain of the channel. The localization of these sites within the region involved in Ca2 + activation suggests that PKA-mediated phosphorylation of the channel opposes the conformational changes caused by binding of Ca/calmodulin, which would otherwise lead to opening of the channel. We suggest that the channel exists as a macromolecular complex involving calmodulin, protein kinases, protein phosphatase and possibly other proteins. The regulation of the channel through kinases and phophatases results in exquisite control of neuronal firing and subsequent modulation of enteric reflexes. D 2006 Published by Elsevier B.V. Keywords: Enteric nervous system; Afterhyperpolarization; Calcium-activated potassium channels; Protein kinase A; Phosphorylation
Contents 1. Introduction. . 2. Conclusion . . Acknowledgements References . . . . .
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1. Introduction Neurotransmitters modulate the firing properties of many neurons by stimulating second messenger cascades that phosphorylate ion channels. Phosphorylation exerts imme-
* Corresponding author. E-mail address: [email protected] (C.B. Neylon). 1566-0702/$ - see front matter D 2006 Published by Elsevier B.V. doi:10.1016/j.autneu.2006.02.028
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diate and/or long term changes in channel properties, leading to effects on the electrical activity of the neurons and downstream effects on physiological function. Regulation of neuronal ion channels by phosphorylation can also be mediated by hormones and cytokines and these may contribute to the changes in excitability of enteric neurons in pathological conditions (Linden et al., 2003). It is therefore important to understand the mechanisms by which phosphorylation modulates neuronal ion channels as these
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mechanisms are likely to underlie changes in neuronal function in disease. Ca2+-activated potassium channels (KCa channels) are important targets for phosphorylation because they regulate the firing properties of neurons. KCa channels are responsible for afterhyperpolarizing potentials (AHP) that follow the action potentials. Because they change the amplitude and duration of the AHP; they can have a dramatic effect on the frequency of action potential generation. Phosphorylation is known to modulate the AHP channel in enteric neurons, however it is only recently that we are beginning to understand the molecular mechanisms at the single channel level. This new data is defining the roles of protein kinases and phosphatases in channel regulation and the structural domains within the channels that are targeted by these signaling molecules. In order to understand the roles of KCa channel phosphorylation in regulating neuronal firing, it is important to describe the different classes of channels involved in generating the AHP. Three distinct KCa channel types are involved which vary in their electrophysiological and pharmacological properties. Large-conductance (BK) Ca2+activated K+ channels contribute to fast AHPs, that last only a few msec and are continuous with the repolarizing phase of the action potential. These voltage-dependent channels are activated by the depolarization during the action potential and by Ca2+ entry, which is through N-type Ca2+ channels in enteric neurons. BK channels are involved mainly in action potential repolarization and close rapidly when membrane potential returns to resting levels. A medium AHP (mAHP), that is observed in some sympathetic and CNS neurons, is generated by a voltageinsensitive, Ca2+-dependent current, that is blocked by apamin. Apamin-sensitive currents are generated by the small-conductance (SK) Ca2+-activated K+ channel family that are products of three closely-related genes; SK1, SK2 and SK3. The predominant contributor to the mAHP current in hippocampal CA1 neurons is the SK2 channel (Bond et al., 2004). A third type of AHP current, the slow AHP (sAHP), lasts in the order of seconds and is not sensitive to apamin or low concentrations of TEA. The sAHP is found in CA1 pyramidal cells of the hippocampus (Alger and Nicoll, 1980; Hotson and Prince, 1980; Lancaster and Adams, 1986; Pedarzani et al., 1998; Sah, 1996; Sah and Clements, 1999; Schwartzkroin and Stafstrom, 1980), sympathetic neurons of the celiac ganglion (Cassell and McLachlan, 1987), cortical neurons (Schwindt et al., 1988), amygdala neurons (Faber and Sah, 2002) and AH neurons in the enteric nervous system (Hirst et al., 1974, 1985; North and Tokimasa, 1987). Identification of the channel(s) responsible for the sAHP has been elusive. For example, there is no conclusive evidence for involvement of any of the SK channel proteins in generating the sAHP in the hippocampus (Bond et al., 2004; Sah and Faber, 2002; Villalobos et al., 2004). The sAHP in the enteric nervous system is expressed predominantly in intrinsic primary
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afferent neurons (IPANs, also known as AH neurons in the guinea-pig). Because AH neurons are the first neurons in the enteric circuit, receiving information from the mucosa and connecting to other neurons in the intestinal wall, the sAHP sets the timing of intrinsic intestinal reflexes (Furness et al., 2004a). By regulating action potential generation, sAHP channels control the activity of the enteric network and its inhibition severely impacts on intestinal motility. Thus, regulation of the sAHP channel by phosphorylation is an important mechanism in the overall control of intestinal function. While the identity of the channels underlying the sAHP in central neurons is unknown, the electrophysiological properties of the sAHP channel in enteric neurons are similar to those of the intermediate-conductance potassium (IK) channel, that is expressed in many non-excitable cells. The current in AH neurons is insensitive to apamin and low concentrations of TEA, is independent of membrane potential, is potently activated by Ca2+, and has a single channel conductance in the IK range (Vogalis et al., 2002). Compelling evidence that the IK channel is responsible was obtained by development of IK channel-specific antisera which showed localization of positive immunoreactivity to the same neurons (Dogiel Type II) that exhibit the pronounced sAHP and identification of a protein on Western blots that is similar in molecular weight to the IK channel monomer found in other tissues (Furness et al., 2004b, 2003; Neylon et al., 2004b). The current is blocked by the IK channel blocker, clotrimazole (Neylon et al., 2004b; Nguyen et al., 2005). However there are some notable differences including the kinetics of the Ca2+ dependence of the channel (Sah and Clements, 1999; Sah and Davies, 2000; Vogalis et al., 2002) which is not consistent with cloned IK channels expressed in heterologous systems (Ishii et al., 1997; Neylon et al., 1999). The reasons for these differences are not known, however they may be due to differences in the properties of native and exogenously expressed channels, the presence of different channel isoforms, or influence of partner proteins. In all neurons in which the sAHP is expressed, phosphorylation plays a critical role in its regulation. Modulation of the sAHP by phosphorylation is a primary function for many metabotropic neurotransmitters including acetylcholine, noradrenaline, serotonin, histamine, dopamine (Pedarzani and Storm, 1995) and the PKA pathway appears to be a major protein kinase signaling mechanism involved. Many neurotransmitter receptors are coupled to adenylate cyclase which increases neuronal cAMP levels (Zafirov et al., 1985) and suppresses the sAHP current (Andrade and Nicoll, 1987; Madison and Nicoll, 1986; Nicoll, 1988; Pedarzani and Storm, 1993, 1995). The elevation of intraneuronal cAMP levels (Bertrand and Galligan, 1995; Nemeth et al., 1986; Palmer et al., 1986; Zafirov et al., 1993, 1985) and activation of PKA (Erdemli et al., 1998; Lancaster and Batchelor, 2000; Pedarzani and Storm, 1993, 1995; Torres et al., 1995) is responsible for
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suppression of the AHP current. In myenteric AH neurons, several neurotransmitters including histamine (Xia et al., 1996), 5-hydroxytryptamine (Xia et al., 1994) and substance P suppress Ca2+-activated K+ conductance (Palmer et al., 1987; Xia et al., 1997). The activation of PKA by these receptors presumably leads to phosphorylation of the Ca2+activated K+ channel itself or key proteins involved in its regulation. This activity appears to be unique to the sAHP channel since the channels that underlie the mAHP do not appear to be modulated (Sah and McLachlan, 1993; Schwindt et al., 1988). It should be noted that the mAHP is not readily revealed or is absent in enteric AH neurons. It has been suggested that PKA phosphorylates the channel protein directly (Nicoll, 1988; Shuster et al., 1985), but this remains to be determined experimentally. It is clear that PKA can act directly on proteins that are integral to or anchored to the intracellular face of the membrane because a reduction in channel openings is produced by exposure of the inside – out patch of enteric AH neurons to the catalytic subunit of PKA (Vogalis et al., 2003). The first evidence for a direct modulation of the channel by PKA was obtained using recombinant IK channels heterologously expressed in Xenopus oocytes (Neylon et al., 2004a). PKA induces a rapid inhibition of macroscopic IK channel current when applied to the cytoplasmic side of the patch and this effect is abolished by mutation of a series of PKA phosphorylation sites (Neylon et al., 2004a). Experiments using recombinant partial sequences of the rat IK channel suggest that serine332 (equivalent to S334 in the human IK channel) is the major phosphorylation site involved in channel inhibition (Neylon et al., 2004a). It is perhaps over-simplistic to consider this as the only phosphorylation site involved, as clearly other residues are also phosphorylated (Neylon et al., 2004a). Mutation of four residues in the calmodulin binding domain of the channel is sufficient to abolish the inhibitory effect of PKA (Neylon et al., 2004a), but whether mutation of S332 alone is sufficient was not tested. The finding that S332 and the other phosphorylation sites are located within the sequence that comprises the calmodulin binding domain raises the possibility that phosphorylation counteracts the conformational changes induced by calmodulin that leads to channel opening. PKA inhibits the channel in the continued presence of high Ca2+ concentration indicating that phosphorylation can close the channel even when calmodulin is promoting the activated state. This indicates that even in the open, calmodulin bound, state, the target phosphorylation site(s) remain accessible to the kinase. Recent information on the structure of the SK channel/calmodulin complex indicates that the region equivalent to S332 in the IK channel lies in the loop region between two a-helices (Schumacher et al., 2001). Based on these structural predictions, the loop region appears to be accessible to kinase regulation. Phosphorylation of this loop residue, and perhaps one or more of the other potential phosphorylation sites identified previously (Neylon et al., 2004a) leads to conformational changes that close the channel. The relative
contribution of each potential phosphorylation site, and the molecular mechanisms involved, remain to be determined. In other cases where PKA has been shown to modulate ion channels, PKA is bound to the channel complex through interaction of the regulatory subunit with anchor proteins (DeSouza et al., 2002; Gray et al., 1998). However, the catalytic subunit can also localize independently of the regulatory subunit and become bound in complexes via its variable amino terminus (Sastri et al., 2005). This region of PKA is subject to post-translational modification which may influence protein targeting (Sastri et al., 2005). As exposure of the channel to purified catalytic subunit dramatically inhibits channel current, it is suggested that the kinase is not normally associated with the channel complex but targets the channel in response to activation of the adenylate cyclase second messenger cascade. Such a mechanism would allow cell surface receptors to regulate neuronal firing via activation of PKA which modulates the phosphorylation status of the sAHP channel. In contrast, there is a reported additional effect of PKA on IK channels that appears unrelated to the inhibitory mechanism discussed above. IK channel currents in inside –out patches of cell membranes are markedly enhanced by Mg2+/ATP and this effect is inhibited, at least in some cells, by blockers of PKA (Gerlach et al., 2000, 2001; Hayashi et al., 2004). The effect of Mg2+/ATP is to increase open probability rather than alter the kinetics of Ca2+ activation (Gerlach et al., 2000). PKA has been shown to activate other types of potassium channels (Koh et al., 1996), including BK channels (Nara et al., 1998; Tian et al., 2003, 2001). The mechanism for activation of IK channels by PKA is distinct from the inhibitory mechanism as it is not abolished by mutation of the PKA consensus site S332 (Gerlach et al., 2000; Schrøder et al., 2000 ; von Hahn et al., 2001) or any of the other phosphorylation sites; T101, S176, and T327 (Wulf and Schwab, 2002). Instead, activation of the channel by Mg2+/ATP relies on a sequence of 14 amino acids (R353 – M366) located some 21 amino acids Cterminal to the S332 phosphorylation site (Gerlach et al., 2001). These findings raise the possibility that there are two sites of interaction of protein kinases with the IK channel; one involved in channel inhibition and the other in channel activation. The activation mechanism appears to involve a kinase action that is closely-associated with the channel as direct exposure of cell patches to Mg2+/ATP leads to activation of the current. Ion channels are thought to exist as macromolecular complexes consisting of kinases and phosphatases (Chung et al., 1991; DeSouza et al., 2002; Mason et al., 2002; Reinhart et al., 1991; Reinhart and Levitan, 1995; Sun et al., 2000). If PKA is resident within the IK channel macromolecular complex it implies that the regulator in this sense may not be cAMP itself but perhaps ATP. As no phosphorylation sites on the channel itself have been identified to be involved in this channel activation, it is likely that PKA phosphorylates other proteins in the complex to influence open probability. In this respect, it
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has been shown that protein kinase CK2 which is coassembled with the SK2 channel regulates Ca2+ sensitivity by phosphorylating calmodulin (Bildl et al., 2004). The mechanism for IK channels is likely to be different because the SK2 channel is not modulated by ATP and the Ca2+ sensitivity of the IK channel is not affected, however it raises the intriguing possibility that phosphorylation of calmodulin, or another channel-associated protein, leads to the Mg2+/ ATP-dependent increase in IK channel open probability. The consequence in neurons would be an increase in IK current, further hyperpolarization, and a reduction in neuronal activity. There have not been any reports of PKA inhibiting neuronal firing properties and thus the physiological relevance of this stimulatory effect is unknown. Phosphorylation pathways are paralleled by mechanisms that dephosphorylate the channel and these two systems usually act in concert to regulate channel activity. The slow AHP in hippocampal CA1 pyramidal neurons is modulated by blockade of protein phosphatases suggesting the presence of a phosphorylation/dephosphorylation cycle regulating channel function (Muller et al., 1992; Pedarzani et al., 1998; Pedarzani and Storm, 1993). Phosphatases PP1 or PP2A appear to be involved in this effect (Pedarzani et al., 1998). In enteric neurons, evidence suggests an involvement of Ca2+-dependent phosphatases (PP2B) in channel activation. A component of the postspike afterhyperpolarization in enteric neurons is mediated by at least two phosphatases, a Ca2+-dependent calcineurin (PP2B) and a non-Ca2+-dependent phosphatase inhibited by okadaic acid (PP1 and PP2A inhibitor) (Vogalis et al., 2004). Because the IK channel is activated by an increase in cytoplasmic Ca2+, calcineurin is likely to be involved, however how these proteins regulate the channel and whether they are part of the ion channel signalling complex is not known.
2. Conclusion PKA modulation of the sAHP provides a high level of regulation of neuronal function. In general, KCa channels that are opened to generate the sAHP are inhibited by PKAmediated phosphorylation that is linked to a number of neurotransmitter receptors. In some neurons, IK channels are the conductive elements for the sAHP current and we summarize evidence that PKA phosphorylates the IK channel directly. There is also evidence for a Mg2+/ATPdependent, but indirect, phosphorylation event that opens the channel. Calmodulin, which is closely associated with the channel is also susceptible to modulation by kinases. Finally, there is evidence that the IK channel has associated phosphatases that are themselves modified by the intracellular environment. Thus IK channels exist in molecular complexes whose regulation is finely tuned to set the excitability and firing patterns of neurons. We speculate that this level of regulation of the IK channel provides unique
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advantages over the control of cellular activity by the SK family of channels.
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Review
Regulation of the slow afterhyperpolarization in enteric neurons by protein kinase A Craig B. Neylon *, Christopher J. Fowler, John B. Furness Department of Anatomy and Cell Biology, University of Melbourne, Parkville 3010, Australia Received 18 January 2006; accepted 27 February 2006
Abstract The slow after-hyperpolarization (sAHP) following the action potential is an important determinant of the firing patterns of enteric neurons. The channel responsible for the sAHP thus serves as a critical control point at which neurotransmitters and inflammatory mediators modulate gut motility. Many of these receptor-evoked pathways are known to inhibit the sAHP and, thus, excite enteric neurons. They act through protein kinase A (PKA) which is a strong inhibitor of the sAHP current while protein phosphatases enhance the current. Increasing evidence suggests that the sAHP is mediated by the opening of intermediate-conductance Ca-activated potassium (IK) channels. This neuronal IK channel, previously known to be expressed in a variety of non-excitable cells, is strongly influenced by protein kinases. Investigation of the molecular basis for the modulation of IK channels by protein phosphorylation indicates that there are multiple mechanisms of channel control. Inhibition of channel activity by PKA involves phosphorylation sites located within the calmodulin-binding domain of the channel. The localization of these sites within the region involved in Ca2 + activation suggests that PKA-mediated phosphorylation of the channel opposes the conformational changes caused by binding of Ca/calmodulin, which would otherwise lead to opening of the channel. We suggest that the channel exists as a macromolecular complex involving calmodulin, protein kinases, protein phosphatase and possibly other proteins. The regulation of the channel through kinases and phophatases results in exquisite control of neuronal firing and subsequent modulation of enteric reflexes. D 2006 Published by Elsevier B.V. Keywords: Enteric nervous system; Afterhyperpolarization; Calcium-activated potassium channels; Protein kinase A; Phosphorylation
Contents 1. Introduction. . 2. Conclusion . . Acknowledgements References . . . . .
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1. Introduction Neurotransmitters modulate the firing properties of many neurons by stimulating second messenger cascades that phosphorylate ion channels. Phosphorylation exerts imme-
* Corresponding author. E-mail address: [email protected] (C.B. Neylon). 1566-0702/$ - see front matter D 2006 Published by Elsevier B.V. doi:10.1016/j.autneu.2006.02.028
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diate and/or long term changes in channel properties, leading to effects on the electrical activity of the neurons and downstream effects on physiological function. Regulation of neuronal ion channels by phosphorylation can also be mediated by hormones and cytokines and these may contribute to the changes in excitability of enteric neurons in pathological conditions (Linden et al., 2003). It is therefore important to understand the mechanisms by which phosphorylation modulates neuronal ion channels as these
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mechanisms are likely to underlie changes in neuronal function in disease. Ca2+-activated potassium channels (KCa channels) are important targets for phosphorylation because they regulate the firing properties of neurons. KCa channels are responsible for afterhyperpolarizing potentials (AHP) that follow the action potentials. Because they change the amplitude and duration of the AHP; they can have a dramatic effect on the frequency of action potential generation. Phosphorylation is known to modulate the AHP channel in enteric neurons, however it is only recently that we are beginning to understand the molecular mechanisms at the single channel level. This new data is defining the roles of protein kinases and phosphatases in channel regulation and the structural domains within the channels that are targeted by these signaling molecules. In order to understand the roles of KCa channel phosphorylation in regulating neuronal firing, it is important to describe the different classes of channels involved in generating the AHP. Three distinct KCa channel types are involved which vary in their electrophysiological and pharmacological properties. Large-conductance (BK) Ca2+activated K+ channels contribute to fast AHPs, that last only a few msec and are continuous with the repolarizing phase of the action potential. These voltage-dependent channels are activated by the depolarization during the action potential and by Ca2+ entry, which is through N-type Ca2+ channels in enteric neurons. BK channels are involved mainly in action potential repolarization and close rapidly when membrane potential returns to resting levels. A medium AHP (mAHP), that is observed in some sympathetic and CNS neurons, is generated by a voltageinsensitive, Ca2+-dependent current, that is blocked by apamin. Apamin-sensitive currents are generated by the small-conductance (SK) Ca2+-activated K+ channel family that are products of three closely-related genes; SK1, SK2 and SK3. The predominant contributor to the mAHP current in hippocampal CA1 neurons is the SK2 channel (Bond et al., 2004). A third type of AHP current, the slow AHP (sAHP), lasts in the order of seconds and is not sensitive to apamin or low concentrations of TEA. The sAHP is found in CA1 pyramidal cells of the hippocampus (Alger and Nicoll, 1980; Hotson and Prince, 1980; Lancaster and Adams, 1986; Pedarzani et al., 1998; Sah, 1996; Sah and Clements, 1999; Schwartzkroin and Stafstrom, 1980), sympathetic neurons of the celiac ganglion (Cassell and McLachlan, 1987), cortical neurons (Schwindt et al., 1988), amygdala neurons (Faber and Sah, 2002) and AH neurons in the enteric nervous system (Hirst et al., 1974, 1985; North and Tokimasa, 1987). Identification of the channel(s) responsible for the sAHP has been elusive. For example, there is no conclusive evidence for involvement of any of the SK channel proteins in generating the sAHP in the hippocampus (Bond et al., 2004; Sah and Faber, 2002; Villalobos et al., 2004). The sAHP in the enteric nervous system is expressed predominantly in intrinsic primary
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afferent neurons (IPANs, also known as AH neurons in the guinea-pig). Because AH neurons are the first neurons in the enteric circuit, receiving information from the mucosa and connecting to other neurons in the intestinal wall, the sAHP sets the timing of intrinsic intestinal reflexes (Furness et al., 2004a). By regulating action potential generation, sAHP channels control the activity of the enteric network and its inhibition severely impacts on intestinal motility. Thus, regulation of the sAHP channel by phosphorylation is an important mechanism in the overall control of intestinal function. While the identity of the channels underlying the sAHP in central neurons is unknown, the electrophysiological properties of the sAHP channel in enteric neurons are similar to those of the intermediate-conductance potassium (IK) channel, that is expressed in many non-excitable cells. The current in AH neurons is insensitive to apamin and low concentrations of TEA, is independent of membrane potential, is potently activated by Ca2+, and has a single channel conductance in the IK range (Vogalis et al., 2002). Compelling evidence that the IK channel is responsible was obtained by development of IK channel-specific antisera which showed localization of positive immunoreactivity to the same neurons (Dogiel Type II) that exhibit the pronounced sAHP and identification of a protein on Western blots that is similar in molecular weight to the IK channel monomer found in other tissues (Furness et al., 2004b, 2003; Neylon et al., 2004b). The current is blocked by the IK channel blocker, clotrimazole (Neylon et al., 2004b; Nguyen et al., 2005). However there are some notable differences including the kinetics of the Ca2+ dependence of the channel (Sah and Clements, 1999; Sah and Davies, 2000; Vogalis et al., 2002) which is not consistent with cloned IK channels expressed in heterologous systems (Ishii et al., 1997; Neylon et al., 1999). The reasons for these differences are not known, however they may be due to differences in the properties of native and exogenously expressed channels, the presence of different channel isoforms, or influence of partner proteins. In all neurons in which the sAHP is expressed, phosphorylation plays a critical role in its regulation. Modulation of the sAHP by phosphorylation is a primary function for many metabotropic neurotransmitters including acetylcholine, noradrenaline, serotonin, histamine, dopamine (Pedarzani and Storm, 1995) and the PKA pathway appears to be a major protein kinase signaling mechanism involved. Many neurotransmitter receptors are coupled to adenylate cyclase which increases neuronal cAMP levels (Zafirov et al., 1985) and suppresses the sAHP current (Andrade and Nicoll, 1987; Madison and Nicoll, 1986; Nicoll, 1988; Pedarzani and Storm, 1993, 1995). The elevation of intraneuronal cAMP levels (Bertrand and Galligan, 1995; Nemeth et al., 1986; Palmer et al., 1986; Zafirov et al., 1993, 1985) and activation of PKA (Erdemli et al., 1998; Lancaster and Batchelor, 2000; Pedarzani and Storm, 1993, 1995; Torres et al., 1995) is responsible for
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suppression of the AHP current. In myenteric AH neurons, several neurotransmitters including histamine (Xia et al., 1996), 5-hydroxytryptamine (Xia et al., 1994) and substance P suppress Ca2+-activated K+ conductance (Palmer et al., 1987; Xia et al., 1997). The activation of PKA by these receptors presumably leads to phosphorylation of the Ca2+activated K+ channel itself or key proteins involved in its regulation. This activity appears to be unique to the sAHP channel since the channels that underlie the mAHP do not appear to be modulated (Sah and McLachlan, 1993; Schwindt et al., 1988). It should be noted that the mAHP is not readily revealed or is absent in enteric AH neurons. It has been suggested that PKA phosphorylates the channel protein directly (Nicoll, 1988; Shuster et al., 1985), but this remains to be determined experimentally. It is clear that PKA can act directly on proteins that are integral to or anchored to the intracellular face of the membrane because a reduction in channel openings is produced by exposure of the inside – out patch of enteric AH neurons to the catalytic subunit of PKA (Vogalis et al., 2003). The first evidence for a direct modulation of the channel by PKA was obtained using recombinant IK channels heterologously expressed in Xenopus oocytes (Neylon et al., 2004a). PKA induces a rapid inhibition of macroscopic IK channel current when applied to the cytoplasmic side of the patch and this effect is abolished by mutation of a series of PKA phosphorylation sites (Neylon et al., 2004a). Experiments using recombinant partial sequences of the rat IK channel suggest that serine332 (equivalent to S334 in the human IK channel) is the major phosphorylation site involved in channel inhibition (Neylon et al., 2004a). It is perhaps over-simplistic to consider this as the only phosphorylation site involved, as clearly other residues are also phosphorylated (Neylon et al., 2004a). Mutation of four residues in the calmodulin binding domain of the channel is sufficient to abolish the inhibitory effect of PKA (Neylon et al., 2004a), but whether mutation of S332 alone is sufficient was not tested. The finding that S332 and the other phosphorylation sites are located within the sequence that comprises the calmodulin binding domain raises the possibility that phosphorylation counteracts the conformational changes induced by calmodulin that leads to channel opening. PKA inhibits the channel in the continued presence of high Ca2+ concentration indicating that phosphorylation can close the channel even when calmodulin is promoting the activated state. This indicates that even in the open, calmodulin bound, state, the target phosphorylation site(s) remain accessible to the kinase. Recent information on the structure of the SK channel/calmodulin complex indicates that the region equivalent to S332 in the IK channel lies in the loop region between two a-helices (Schumacher et al., 2001). Based on these structural predictions, the loop region appears to be accessible to kinase regulation. Phosphorylation of this loop residue, and perhaps one or more of the other potential phosphorylation sites identified previously (Neylon et al., 2004a) leads to conformational changes that close the channel. The relative
contribution of each potential phosphorylation site, and the molecular mechanisms involved, remain to be determined. In other cases where PKA has been shown to modulate ion channels, PKA is bound to the channel complex through interaction of the regulatory subunit with anchor proteins (DeSouza et al., 2002; Gray et al., 1998). However, the catalytic subunit can also localize independently of the regulatory subunit and become bound in complexes via its variable amino terminus (Sastri et al., 2005). This region of PKA is subject to post-translational modification which may influence protein targeting (Sastri et al., 2005). As exposure of the channel to purified catalytic subunit dramatically inhibits channel current, it is suggested that the kinase is not normally associated with the channel complex but targets the channel in response to activation of the adenylate cyclase second messenger cascade. Such a mechanism would allow cell surface receptors to regulate neuronal firing via activation of PKA which modulates the phosphorylation status of the sAHP channel. In contrast, there is a reported additional effect of PKA on IK channels that appears unrelated to the inhibitory mechanism discussed above. IK channel currents in inside –out patches of cell membranes are markedly enhanced by Mg2+/ATP and this effect is inhibited, at least in some cells, by blockers of PKA (Gerlach et al., 2000, 2001; Hayashi et al., 2004). The effect of Mg2+/ATP is to increase open probability rather than alter the kinetics of Ca2+ activation (Gerlach et al., 2000). PKA has been shown to activate other types of potassium channels (Koh et al., 1996), including BK channels (Nara et al., 1998; Tian et al., 2003, 2001). The mechanism for activation of IK channels by PKA is distinct from the inhibitory mechanism as it is not abolished by mutation of the PKA consensus site S332 (Gerlach et al., 2000; Schrøder et al., 2000 ; von Hahn et al., 2001) or any of the other phosphorylation sites; T101, S176, and T327 (Wulf and Schwab, 2002). Instead, activation of the channel by Mg2+/ATP relies on a sequence of 14 amino acids (R353 – M366) located some 21 amino acids Cterminal to the S332 phosphorylation site (Gerlach et al., 2001). These findings raise the possibility that there are two sites of interaction of protein kinases with the IK channel; one involved in channel inhibition and the other in channel activation. The activation mechanism appears to involve a kinase action that is closely-associated with the channel as direct exposure of cell patches to Mg2+/ATP leads to activation of the current. Ion channels are thought to exist as macromolecular complexes consisting of kinases and phosphatases (Chung et al., 1991; DeSouza et al., 2002; Mason et al., 2002; Reinhart et al., 1991; Reinhart and Levitan, 1995; Sun et al., 2000). If PKA is resident within the IK channel macromolecular complex it implies that the regulator in this sense may not be cAMP itself but perhaps ATP. As no phosphorylation sites on the channel itself have been identified to be involved in this channel activation, it is likely that PKA phosphorylates other proteins in the complex to influence open probability. In this respect, it
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has been shown that protein kinase CK2 which is coassembled with the SK2 channel regulates Ca2+ sensitivity by phosphorylating calmodulin (Bildl et al., 2004). The mechanism for IK channels is likely to be different because the SK2 channel is not modulated by ATP and the Ca2+ sensitivity of the IK channel is not affected, however it raises the intriguing possibility that phosphorylation of calmodulin, or another channel-associated protein, leads to the Mg2+/ ATP-dependent increase in IK channel open probability. The consequence in neurons would be an increase in IK current, further hyperpolarization, and a reduction in neuronal activity. There have not been any reports of PKA inhibiting neuronal firing properties and thus the physiological relevance of this stimulatory effect is unknown. Phosphorylation pathways are paralleled by mechanisms that dephosphorylate the channel and these two systems usually act in concert to regulate channel activity. The slow AHP in hippocampal CA1 pyramidal neurons is modulated by blockade of protein phosphatases suggesting the presence of a phosphorylation/dephosphorylation cycle regulating channel function (Muller et al., 1992; Pedarzani et al., 1998; Pedarzani and Storm, 1993). Phosphatases PP1 or PP2A appear to be involved in this effect (Pedarzani et al., 1998). In enteric neurons, evidence suggests an involvement of Ca2+-dependent phosphatases (PP2B) in channel activation. A component of the postspike afterhyperpolarization in enteric neurons is mediated by at least two phosphatases, a Ca2+-dependent calcineurin (PP2B) and a non-Ca2+-dependent phosphatase inhibited by okadaic acid (PP1 and PP2A inhibitor) (Vogalis et al., 2004). Because the IK channel is activated by an increase in cytoplasmic Ca2+, calcineurin is likely to be involved, however how these proteins regulate the channel and whether they are part of the ion channel signalling complex is not known.
2. Conclusion PKA modulation of the sAHP provides a high level of regulation of neuronal function. In general, KCa channels that are opened to generate the sAHP are inhibited by PKAmediated phosphorylation that is linked to a number of neurotransmitter receptors. In some neurons, IK channels are the conductive elements for the sAHP current and we summarize evidence that PKA phosphorylates the IK channel directly. There is also evidence for a Mg2+/ATPdependent, but indirect, phosphorylation event that opens the channel. Calmodulin, which is closely associated with the channel is also susceptible to modulation by kinases. Finally, there is evidence that the IK channel has associated phosphatases that are themselves modified by the intracellular environment. Thus IK channels exist in molecular complexes whose regulation is finely tuned to set the excitability and firing patterns of neurons. We speculate that this level of regulation of the IK channel provides unique
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advantages over the control of cellular activity by the SK family of channels.
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