Modulation of a calcium-sensitive nonspecific cation channel by closely associated protein kinase and phosphatase activities

Abstract

Regulation of nonspecific cation channels often underlies neuronal bursting and other prolonged changes in neuronal activity. In bag cell neurons of Aplysia, it recently has been suggested that an intracellular messengerinduced increase in the activity of a nonspecific cation channel may underlie the onset of a 30-min period of spontaneous action potentials referred to as the ''afterdischarge.'' In patch clamp studies of the channel, we show that the open probability of the channel can be increased by an average of 10.7-fold by application of ATP to the cytoplasmic side of patches. Duration histograms indicate that the increase is primarily a result of a reduction in the duration and percentage of channel closures described by the slowest time constant. The increase in open probability was not observed using 5-adenylylimidodiphosphate, a nonhydrolyzable ATP analog, and was blocked in the presence of H7 or the more specific calcium͞ phospholipid-dependent protein kinase C (PKC) inhibitor peptide (19 -36) . Because the increase in activity observed in response to ATP occurred without application of protein kinase, our results indicate that a kinase endogenous to excised patches mediates the effect. The effect of ATP could be reversed by exogenously applied protein phosphatase 1 or by a microcystin-sensitive phosphatase also endogenous to excised patches. These results, together with work demonstrating the presence of a protein tyrosine phosphatase in these patches, suggest that the cation channel is part of a regulatory complex including at least three enzymes. This complex may act as a molecular switch to activate the cation channel and, thereby, trigger the afterdischarge.Modulation of ion channel activity by intracellular messengers underlies many prolonged changes in animal behavior (1). One dramatic example is found in Aplysia in which an increase in the excitability of bag cell neurons initiates behaviors that culminate in egg-laying (2). Specifically, brief stimulation of the pleuroabdominal connective nerve or application of reproductive tract peptides triggers a Ϸ30-min period of spontaneous repetitive action potentials known as the afterdischarge (3, 4). This is followed by a refractory period lasting 18 hr or more during which additional afterdischarges cannot be elicited (3, 5). Recent evidence suggests that activation of bag cell neuron nonspecific cation channels may provide the depolarizing drive underlying the afterdischarge (6, 7). As might be expected from the complex regulation of bag cell neuron activity, the regulation of cation channel activity also is complex. Cation channels are voltage-and Ca 2ϩ -sensitive and, upon patch excision, can be found in several distinguishable modes of gating. The most dramatic distinction occurs between the bursting and the continuously active gating modes. Bursting, a term that often is applied to the grouping of action potentials, here refers to groupings of channels openings. These groupings are separated by an added population of long-lived channel...

Association/Dissociation of a Channel–Kinase Complex Underlies State-Dependent Modulation

“…It was possible to identify these channels based on their conductance (25-30 pS; ϳ2 pA at Ϫ60 mV), voltage dependence of opening (an increase in P O with depolarization), and absence of voltage-dependent inactivation. In previous work, we showed that application of ATP to the cytoplasmic face of cation channel-containing patches produced an increase in channel P O (Wilson et al, 1998;Magoski et al, 2002). We found that this increase in P O was attributable to the activity of a closely associated PKC-like kinase phosphorylating the cation channel or some adjacent protein ( Fig.…”

“…Protein kinase C (PKC) is closely associated with the cation channel. This interaction persists in excised inside-out patches and serves to enhance activity through phosphorylation of the channel (Wilson et al, 1998;Magoski et al, 2002). Following the afterdischarge, the neurons enter an ϳ18 h refractory period, during which a second afterdischarge and ensuing second bout of reproductive behaviors cannot be initiated.…”

“…To orchestrate this regulation, kinases can be targeted directly to ion channels (Holmes et al, 1996;Tsunoda et al, 1997;Yu et al, 1997;Tibbs et al, 1998;Davare et al, 2001) in an association that amounts to a physical link, such that kinase-dependent regulation of the channel can be observed even in cell-free patches or bilayers (Rehm et al, 1989;Chung et al, 1991;Bielefeldt and Jackson, 1994;Wang and Kotlikoff, 1996;Wilson et al, 1998. However, the extent to which these associations are regulated and the physiological consequence of any change in such arrangements remains poorly understood.…”

Kinases Bicker Over an Ion Channel

“…Previous studies had shown that application of ATP alone to the cytoplasmic side of these patches causes an increase in open probability (P O ), suggesting that a kinase might be intimately associated with the channel. PKC inhibitor peptides blocked ATP-induced increase in P O , consistent with a PKC-like kinase (Wilson et al, 1998). Magoski and Kaczmarek (2005) reported that less than one-half of the patches containing these cation channels showed an increased channel P O in the presence of ATP, whereas the rest showed no change or a decrease in P O [Magoski and Kaczmarek (2005), their The authors investigated the molecular mechanism underlying this variable response.…”

“…This channel is regulated by a variety of kinases and phosphatases including protein kinase C (PKC). PKC is colocalized with the cation channel in excised patches and increases channel activity via phosphorylation (Wilson et al, 1998;Magoski et al, 2002). In their recent article in The Journal of Neuroscience, Magoski and Kaczmarek (2005) showed that the state of PKC-channel interaction reflects the state of the bag cell neuron: PKC is coupled to the cation channel during the afterdischarge and uncoupled during the refractory state.…”