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1Department of Neurobiology, McKnight Brain Institute, 2Civitan International Research Center, and the 3Vision Science Graduate Program, University of Alabama at Birmingham, Birmingham, Alabama
Submitted 4 October 2005; accepted in final form 11 October 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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). Even though it is generally accepted that receptor desensitization can influence synaptic amplitude and time course (Jones and Westbrook 1996), the physiological significance of desensitization of central nicotinic acetylcholine receptors (nAChRs) remains unresolved largely because of a lack of knowledge concerning their activation by endogenously released transmitter (Brown 2000; Giniatullin et al. 2005). Conversely, the pathological relevance of desensitization of nAChRs has been recognized for many years, particularly with respect to chronic nicotine (Collins et al. 1990; Dani and Heinemann 1996) and likely becomes an important factor during treatment of Alzheimer's disease with cholinesterase inhibitors (Paterson and Nordberg 2000).
In the absence of a comprehensive understanding of the function of nAChRs, however, arguments in favor of examining desensitization can be made. In multistate systems of which ligand-gated ion channels are members (Katz and Thesleff 1957), the fractional distribution of receptors between those states becomes relevant for predicting how the system will behave under a given set of circumstances. Specifically, the balance between activatable and desensitized states will set the maximum response amplitude and thus any alteration of this balance will confer plasticity to the system (Changeux et al. 1998). Interestingly many receptors have a built-in mechanism for performing this task—by allosteric modulation of the kinetics of receptor desensitization using Ca2+ and/or protein phosphorylation (Huganir and Greengard 1990). Taken to one extreme, such modulation acts as a time-dependent shuttle between functional and nonfunctional receptor pools.
The impact of this form of modulation becomes clearer if information about cholinergic–nicotinic signaling in the CNS is formalized. Cholinergic synaptic contacts in many parts of the brain are randomly distributed and, in the hippocampus, <10% are in apposition to postsynaptic sites (Descarries et al. 1997). Even assuming the transmitter is released at a high concentration, it will be in the low micromolar range after only a few microns of diffusion (Clements 1996), providing an explanation for lack of fast synaptic responses in many areas of the brain, including the medial habenula (Edwards et al. 1992), despite the strong expression of nAChRs in this region (Lester and Dani 1994; McCormick and Prince 1987; Mulle and Changeux 1990). Moreover, desensitization becomes important if postsynaptic receptors are required to detect low levels of ambient transmitter because the concentration "window" over which they remain sensitive is determined by the concentration dependency of both activation and desensitization (Steinbach 1990). Desensitization dominates at lower doses of agonist, but there will be a narrow concentration range over which receptors will start to activate before becoming fully desensitized by higher steady-state levels of transmitter (Lester 2004; Lester and Dani 1995). Under these conditions, any change in the sensitivity of conformational states to agonist will alter the responsiveness of the cell to available transmitter.
There is little information describing the modulation of desensitization of nAChRs in the CNS (Quick and Lester 2002). Results from peripheral tissue and heterologous expression systems indicate that desensitization is regulated by Ca2+, although the effect of Ca2+ may depend on the subtype of nAChR (Quick and Lester 2002). For example, in putative 
3
4*-subunit–containing receptors on chromaffin cells, an elevation in intracellular Ca2+ suppressed recovery from desensitization (Khiroug et al. 1997, 1998), whereas the opposite is true for 42 nAChRs (Fenster et al. 1999). Receptors in the peripheral nervous system (PNS) and MHb cells are considered to be primarily of the 34* subtype (McGehee and Role 1995), which gives rise to the prediction that their Ca2+ modulation should be similar. However, the precise physiological and pharmacological characteristics are different for the two groups of receptors. MHb nAChRs have a different rank order of agonist efficacy (Covernton et al. 1994; Mulle and Changeux 1990) as well as single-channel conductance (Mulle and Changeux 1990; Sivilotti et al. 1997) than nAChRs in the PNS, implying that their underlying subunit composition is not exactly the same and, as such, they may exhibit differential regulation of desensitization. Thus here we have addressed 1) whether Ca2+ can modulate recovery from desensitization of nAChRs in the MHb and 2) whether this resembles regulation of a similar nAChR subtype in the PNS. Results from these studies may reveal whether there is a universal mechanism for the regulation of desensitization of nAChRs by Ca2+. In addition, because many nAChRs are present on nerve terminals, including MHb axons (Marks et al. 1998), we consider whether Ca2+ entry by voltage-gated Ca2+ channels can influence nAChR function.
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METHODS |
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All experiments were performed on neurons isolated from medial habenula nuclei of 10- to 20-day-old rats using methods described previously (Quick et al. 1999). Methods for electrophysiology, drug application, and intracellular Ca2+ measurements were as described in the companion paper (Guo and Lester 2007). Deviations from standard solutions are described in the text.
Experimental protocols and data fitting
All experiments were designed to assess recovery from agonist-induced desensitization. Thus a standard paired-pulse protocol was used throughout and the interval between different trials was 3 min. The protocol consisted of an initial brief (2- to 10-s) desensitizing application of a high (100 µM) concentration of nicotine followed by a second pulse of nicotine (100 µM) at various interpulse intervals (250 ms to 10 s). Recovery from desensitization was assessed as the ratio of the peak amplitude of the second response (I2) with respect to the peak amplitude of the first response (I1). The time course of recovery was quantified from the time constants (
) and relative amplitudes (A) of single- and double-exponential components fitted to the data. In all cases recovery was constrained to be complete (i.e., I2/I1 = 1) using the equation
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Statistical analysis
The significance of the linear regression and comparisons among multiple groups were tested with one-way ANOVA using the program SPSS 1.0 (SPSS, Chicago, IL). In addition, paired and unpaired Student's t-tests were used. Significance was taken as P < 0.05 (*) and P < 0.01 (**). All data are presented as means ± SE.
Receptor nomenclature
Subtypes of nAChRs are referred to by their putative subunit composition with an asterisk to represent likely inclusion of additional subunits (Lukas et al. 1999).
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RESULTS |
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The goals of the study were to assess the influence of intracellular Ca2+ on recovery from desensitization of nAChRs in MHb neurons. Even though many different types of nAChR subunit mRNAs are expressed in the MHb region (Sheffield et al. 2000), previous studies showed that MHb neurons possess a reasonably homogeneous population of functional receptors with a minimal subunit composition of 3 and 4 subunits (Mulle et al. 1991; Quick et al. 1999; Sheffield et al. 2000). Extensive desensitization of nAChRs in MHb, similar to that of all types of nAChRs (Quick and Lester 2002), can be produced by quite brief applications of high concentrations of agonist (Lester and Dani 1995). In the current experiments, 5-s pulses of nicotine (100 µM) were used to induce desensitization and recovery from desensitization was measured with a second application at a number of different interpulse intervals. Recovery was quantified as the ratio of the peak of the second current to the peak of the first desensitizing current. Under standard intracellular Ca2+ buffering conditions (10 mM EGTA), recovery from this type of desensitization proceeded with two components, quantified by fast (f) and slow (s) exponential time constants having relative amplitudes of 39 ± 5 and 61 ± 5%, respectively (Fig. 1, A and B; Table 1). It was previously argued that the biphasic nature of desensitization in MHb nAChRs arises from a single receptor rather than from two separate receptor populations with fast and slow kinetics (Lester and Dani 1995).
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). Irrespective of the nature of the chelator, recovery from desensitization proceeded biexponentially (Fig. 1C). However, in the presence of BAPTA, recovery was more complete at longer interpulse intervals. Quantitatively this was accounted for by a greater contribution of the fast phase of recovery (71 ± 7% compared with 39 ± 5%) when BAPTA was used (Table 1). The process of desensitization in nicotinic receptors is usually described with a two–desensitized-states model, which posits that nAChRs may exist in both fast (shallow) and slowly accessible (deep) desensitized conformations (Boyd 1987; Feltz and Trautmann 1982). In terms of this model, our results with the Ca2+ chelators imply that intracellular Ca2+ acts to stabilize nAChRs in the deep desensitized conformation because there is less accumulation of receptors in the deep state (i.e., more receptors recover with fast kinetics) when Ca2+ is rapidly buffered. In further support of this idea, the fraction of nondesensitized receptors, estimated from the steady-state/peak ratios at the end of the first 5-s pulse of 100 µM nicotine was unaffected by the nature of the chelator. These ratios were 0.13 ± 0.02 (n = 8) and 0.13 ± 0.02 (n = 8) for EGTA and BAPTA, respectively. Although this pseudosteady-state parameter depends on both the forward and backward transitions between active and desensitized states (Dilger and Liu 1992), it will likely be dominated by the large rate constant that determines entry into the shallow desensitized conformation.
Moreover, because EGTA and BAPTA have similar Kd values for Ca2+ but different rates of binding, it is most probable that the change in [Ca2+]i, rather than any effect of the chelators on resting Ca2+, accounts for their differential effects on the relative amplitude of the two components. The most likely source of the Ca2+ increase is direct influx through the nAChR (Mulle et al. 1992; Vernino et al. 1992). If so, then any differences in the recovery profiles recorded using either EGTA or BAPTA should be eliminated in the nominal absence of extracellular Ca2+. This result was confirmed (Fig. 1D) and, in addition, the percentage recovery of the fast phase in EGTA/Ca2+-free conditions was increased and resembled that observed with BAPTA/extracellular Ca2+ (Table 1). These data imply that BAPTA effectively buffers Ca2+ influx and the differential effects of EGTA and BAPTA on the slow phase of recovery in the presence of extracellular Ca2+ are the direct result of their differential Ca2+-chelating properties.
To confirm that the fast phase of desensitization is relatively insensitive to Ca2+, recovery from desensitization was assessed after a shorter (2-s) desensitizing application of nicotine (100 µM). During very brief exposure to agonist, there is less time for equilibration with slowly accessible deep desensitized states and recovery will largely occur from the fast shallow desensitized state (Fenster et al. 1999; Lester and Dani 1995). With 10 mM EGTA in the recording pipette, recovery was similar in both the absence and presence of extracellular Ca2+ and could be reasonably well described by a single-exponential function with time constants of a magnitude similar to those for the fast component (f) of recovery during longer agonist exposure (Fig. 1E; Table 1). Double-exponential functions revealed a minor slow component (roughly 17%) that was not well defined (Table 1). Provided that nAChRs are the major contributor to intracellular Ca2+ transients under these conditions (see following text), these findings suggest that Ca2+ has little or no effect on the fast phase of recovery from desensitization.
Relationship between intracellular Ca2+ and recovery from desensitization
We have measured the fractional current carried by Ca2+ through nAChRs in MHb neurons to be around 3–4% in physiological conditions (Guo and Lester 2007), a percentage sufficient to cause a measurable rise in intracellular [Ca2+]. Thus MHb neurons are a suitable model to study the relationship between Ca2+ influx through nAChRs and the recovery from desensitization in detail. It is difficult, however, to maintain normal Ca2+ homeostasis in intact cells once a whole cell configuration is formed because cytoplasmic components are inevitably washed out. Fortunately, with few exceptions (Roberts 1993), Ca2+ buffers are resistant to diffusion in most cells studied (Neher 1995). Thus so as not to confound physiological buffering capacity and to allow Ca2+ detection by indo-1 (40 µM), a low concentration of EGTA (0.6 mM) was used in these experiments.
Simultaneous measurements of intracellular [Ca2+] and membrane currents were used to assess the relationship between recovery from desensitization and intracellular [Ca2+]. Recovery was assessed from the relative amplitude of the second response in a paired-pulse protocol (5 s; 100 µM nicotine) and compared with the instantaneous intracellular [Ca2+] immediately before the second pulse (Fig. 2 A). Interpulse intervals were set between 2 and 10 s because our previous experiments (see Fig. 1, C and E) indicated that the major effect of Ca2+ was on the slow component of recovery. Not surprisingly, as the interpulse interval was increased, recovery from desensitization was facilitated and intracellular [Ca2+] moved closer to resting levels (Fig. 2B). However, although these results per se do not imply modulation of desensitization by Ca2+, an inverse relationship between recovery and [Ca2+] could be extracted from the variability in individual cells. Example traces from cells with high and low intracellular [Ca2+] are illustrated in Fig. 2C. A plot of the instantaneous [Ca2+]i versus recovery at the 2-s interval revealed a significantly greater recovery at lower [Ca2+]i (Fig. 2D; the linear regression is significant with a correlation coefficient, R = 0.59; n = 37; P < 0.001, ANOVA).
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). It was therefore necessary to test whether CICR affected recovery in our experiments. This question was addressed by depleting intracellular Ca2+ stores using bath application of 1 µM thapsigargin. Recovery from desensitization was similar before (0.6 ± 0.05) and after (0.61 ± 0.07; n = 7; P > 0.05) thapsigargin treatment, suggesting that CICR does not contribute to the effect of intracellular Ca2+ transients on recovery. Mitochondria constitute another important intracellular Ca2+ store, which can effectively sequester Ca2+ during a rapid rise of intracellular Ca2+ (Pivovarova et al. 2002). However, because mitochondria buffering does not affect the nicotinic Ca2+ response in a physiological (2 mM external Ca2+) solution (Guo and Lester 2007), it is unlikely that these organelles would contribute to the modulation of nAChR desensitization by Ca2+. Voltage-dependent Ca2+ influx and recovery from desensitization
Many nAChRs exist presynaptically (Wonnacott 1997) where they may readily interact with voltage-gated Ca2+ channels to synergistically facilitate the release of neurotransmitter (Kulak et al. 2001; Tredway et al. 1999). Thus Ca2+ entering through voltage-gated Ca2+ channels during depolarization induced by nAChR activation or otherwise could act to modulate nAChR function. Although spontaneous Ca2+ transients or those induced by membrane depolarization have no direct role in promoting nAChR desensitization (Khiroug et al. 1998), it is suggested here that voltage-dependent Ca2+ influx may act as an additional means of attenuating recovery from desensitization.
Simultaneous monitoring of membrane current and [Ca2+]i during a 1-s depolarizing step from –50 to 0 mV demonstrates the existence of voltage-gated Ca2+ channels in MHb neurons (Fig. 3 A, top traces). The inward current (possibly a mixture of voltage-gated Na+ and Ca2+ currents) and its associated [Ca2+]i transient could both be blocked with 200 µM Cd2+ (Fig. 3A, bottom traces). In addition, 200 µM Cd2+ by itself had no effect on either the nicotinic current or recovery from desensitization. The peak current amplitude induced by 100 µM nicotine was 4,376 ± 910 pA in control and 4,161 ± 783 pA in the presence of extracellular Cd2+ (n = 7; P > 0.05), and the fractional recoveries (at the 6-s interpulse interval) were 0.68 ± 0.03 and 0.66 ± 0.02 in the absence and presence of Cd2+, respectively (n = 7; P > 0.05). Although the anatomical relationship between Ca2+ channels and nAChRs is unknown, it was reasoned that appropriately timed voltage-dependent Ca2+ entry should retard nAChR recovery from desensitization. To study this interaction, a paired-pulse protocol (5 s; 100 µM nicotine) at a fixed interpulse interval of 4 s was applied with or without a voltage step (1 s; –50 to 0 mV) 1 s after the first pulse. Examples of currents and intracellular [Ca2+] transients are shown in Fig. 3B. Intracellular [Ca2+] was significantly higher at the start of the second pulse when it was preceded by a voltage-step that produced a clear additional Ca2+ transient (Fig. 3C). Furthermore, the additional rise in intracellular [Ca2+] was associated with less recovery from desensitization (Fig. 3D). In a separate series of experiments, Cd2+ was shown to selectively block Ca2+ entry through voltage-gated channels (Fig. 3B, right traces) and restore recovery from desensitization to control levels (Fig. 3D), thus confirming that Ca2+ entry through voltage-gated calcium channels was responsible for the change in nAChR function.
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). As a result, the intracellular Ca2+ would likely be constantly oscillating. To mimic this type of activity, cells were challenged with a series of brief membrane depolarizations during the recovery phase (Fig. 4). Here, the paired-pulse protocol constituted a long desensitizing pulse of 10 s and a test pulse of 1 s separated by an interpulse interval of 8 s. A 7-s train of depolarizing pulses (from –50 to 0 mV; 100 ms) at 2.5 or 5 Hz was applied 500 ms after the end of the first pulse (Fig. 4, A and B). The relative changes in [Ca2+]i (
[Ca2+]i, calculated by subtracting the resting level from instantaneous [Ca2+]i at the start of the second pulse of nicotine) under control, and at 2.5- and 5-Hz depolarization were 0.04 ± 0.02, 0.09 ± 0.03, and 0.11 ± 0.02 (n = 7), respectively (Fig. 4C). The associated fractional recoveries from desensitization were 0.86 ± 0.03, 0.71 ± 0.04, and 0.58 ± 0.04, respectively (Fig. 4D), further indicating that recovery was inversely correlated with intracellular [Ca2+]. To test the possible involvement of Ca2+-induced Ca2+ release during the prolonged Ca2+ influx, intracellular Ca2+ stores were again depleted by bath application of 1 µM thapsigargin. In this case, the 10-s desensitizing agonist application was followed by 2-s depolarization at 5 Hz. Fractional recoveries assessed at an interpulse interval of 3 s before and after thapsigargin treatment were 0.51 ± 0.05 and 0.55 ± 0.04 (n = 7; P > 0.05; data not shown), suggesting the effects of depolarization on recovery were solely attributable to Ca2+ influx through voltage-gated calcium channels.
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; Hicks et al. 2000; Neely and Lingle 1986). As predicted during coapplication of nicotine (30 µM) and chlorisondamine (1 µM) the measured currents are progressively attenuated (Fig. 6E). Normalization of the responses to a control application of nicotine revealed no differences in either the rate or extent of block by chlorisondamine (Fig. 6F).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Mechanism of Ca2+ action
Autoregulation of recovery from desensitization arising from Ca2+ influx through nAChR channels was previously reported in brain. The mechanism appears to be universal because it is common to all major types of nAChRs, putative homomeric 7 receptors (Khiroug et al. 2003), and heteromeric 34* receptors in both the peripheral (Khiroug et al. 1997, 1998) and central (this study) nervous system. For 42 receptors, expressed in Xenopus oocytes, Ca2+ also affects recovery from desensitization, but in the opposite direction (Fenster et al. 1999). Although Ca2+ may have a direct influence on nAChR desensitization (Cachelin and Colquhoun 1989; Miledi 1980), it is more likely that its effects are mediated through protein kinases and phosphatases, as reported for peripheral nAChRs (Khiroug et al. 1998), and the disparate effects of Ca2+ may have resulted from a differential interaction with downstream second messengers in different systems.
If Ca2+ influx through nAChRs, which accounts for 3–4% of total charge influx (Guo and Lester 2007), is sufficient to affect recovery from desensitization, other means of increasing intracellular [Ca2+] in the vicinity of nAChRs should also influence the process. Indeed, Ca2+ entry through voltage-gated calcium channels is sufficient to attenuate recovery. It should be noted that, although our data suggest Ca2+-induced Ca2+ release does not affect the recovery, other means of releasing Ca2+ from stores, such as activation of the IP3 pathway, may be more effective.
It is now generally accepted that there are two major desensitized conformations of the nAChR (Quick and Lester 2002). Moreover, when regulation of these channels exists, it appears to largely but not exclusively affect the transitions between the short-lived shallow and long-lived deep desensitized states. This is the case for protein kinase A (PKA) modulation of muscle type nAChRs (Paradiso and Brehm 1998), the marked temperature dependency of receptor desensitization in PC12 cells (Boyd 1987), and the protein kinase C (PKC)–dependent regulation of both expressed (Fenster et al. 1999) and native 42 nAChRs (Marszalec et al. 2005). In Markov models of desensitization (e.g., see Fenster et al. 1999), accumulation of receptors in any conformation will depend on the rate constants governing the movement into and out of that particular state. Thus in simple terms, Ca2+ could act either by promoting entry into or by restricting exit from the "deep" desensitized state. The data in the current investigation do not permit us to fully distinguish between these two options. On the one hand, our findings seem to imply that the rate into the deep desensitization is most likely the target of Ca2+ because the return transition from deep to shallow states, as indicated by the time constant determining recovery from the slow phase of desensitization (s), was not affected by Ca2+ (see Table 1). Conversely, recovery from desensitization is slowed when Ca2+ is available only during the recovery phase (see Fig. 5), which is more consistent with regulation of the rate out of the deep desensitized conformation. Irrespective of the precise mechanism, our findings argue that high intracellular [Ca2+] promotes stabilization of the deep desensitized conformation. Such a negative-feedback mechanism could serve to further dampen nAChR activation during a prolonged period of stimulation.
Activation of central nAChRs by ambient levels of transmitter
The exact relevance of nAChR desensitization is unknown, particularly with respect to how it relates to synaptic function (Giniatullin et al. 2005). Typically fast ligand-gated channels are transiently stimulated only by transmitter and agonist is not present sufficiently long for desensitization to become physiologically relevant (Colquhoun and Ogden 1988; Colquhoun and Sakmann 1998). However, despite the widespread presence of nAChRs, there are relatively few examples of fast nicotinic-cholinergic transmission in the brain (Alkondon et al. 1998; Frazier et al. 1998; Futami et al. 1995; Matsubayashi et al. 2004). Specifically, with respect to the current study, there is no evidence for conventional synaptic activation of nAChRs in the MHb even given strong neurochemical and anatomical support (Brown 2000; Edwards et al. 1992). To produce a fast synaptic response, pre- and postsynaptic structures have to be in close proximity so that a high concentration of neurotransmitter can activate postsynaptic receptors. However, in other parts of the brain, extensive studies have revealed that only about 10% of the cholinergic terminals form morphologically defined synapses, whereas the majority are distributed randomly in the neuropil (Descarries et al. 1997). In addition, contrary to the well-studied neuromuscular junction, nAChRs in the CNS may localize outside synaptic structures (Hill et al. 1993). Therefore the transmitter acetylcholine (ACh) may have to diffuse some distance and will likely be in the nanomolar to micromolar range in the vicinity of nAChRs (Descarries et al. 1997; Zoli et al. 1999). Prolonged activation of receptors in this low-concentration range will favor slowly accessible high-affinity desensitized states (Changeux et al. 1984). Thus if nAChRs are capable of detecting low levels of ambient transmitter, agonists must be within the concentration "window" over which receptors start to activate even though desensitization is not complete (Steinbach 1990). Within such a "window," activatable and desensitized nAChRs are in a dynamic equilibrium, such that agonist could persistently open a fraction of nAChRs (Lester 2004). In the preceding scenario, modulation of desensitization becomes an important mechanism for adjusting the "window" and determining the level of activation at a give concentration of ACh. Higher intracellular [Ca2+] would "trap" more nAChRs in deep desensitized conformations and decrease the total number of activatable receptors. In terms of a concentration–response interaction, the desensitization curve would shift to the left and the "window" concentration range would become narrower (Lester 2004), thereby dampening the response to ambient/diffuse levels of agonist.
State-dependent modulation and coincidence detection
Our data show that Ca2+ entry through voltage-gated calcium channels slowed the process of recovery, but only when the nAChRs were already desensitized. Similar experiments in chromaffin cells showed that spontaneous intracellular Ca2+ transients do not affect nAChR current amplitude or desensitization onset (Khiroug et al. 1998). Likewise, intracellular perfusion of MHb cells with elevated [Ca2+] did not change the channel open probability—a measure of the fractional activation of nAChRs. Together these results imply that the modulation of nAChRs by intracellular Ca2+ is state dependent: The effects of Ca2+ alter transitions only between shallow and deep desensitized states. One possibility is that conformational changes on desensitization (Unwin et al. 1988) reveal sites for either Ca2+ binding or phosphorylation. Similar conformation-dependent effects are important in voltage-gated sodium channels, where large structural rearrangements are responsible for local anesthetic inhibition after depolarization-induced inactivation (Catterall 1999). Alternatively, the binding of Ca2+/phosphorylation may not occur in a state-dependent manner, but merely allow alteration of transition probabilities between certain states.
One consequence of this type of regulation is that transient activation of nAChRs, such as fast synaptic transmission, would proceed independently of the level of intracellular Ca2+. Only when receptors become saturated with agonist for prolonged periods of time would the effects of Ca2+ become realized. Moreover, true state-dependent tagging could serve as a signal for receptor desensitization/internalization, as occurs with G-protein–coupled receptors (Pierce and Lofkowitz 2001). Indeed, after prolonged desensitization of muscle nAChRs recovery is often incomplete (Katz and Thesleff 1957). A similar mechanism could occur under conditions of prolonged exposure to nicotine, when many nAChRs are presumed to be desensitized (Quick and Lester 2002). If this were to operate at presynaptic locations, the state-dependent modulation may serve as a coincidence-detection mechanism: Ca2+ influx through voltage-gated calcium channels during sustained action potential firing could trap nAChRs in deep desensitized states, even in the absence of Ca2+ influx through the nAChR channel.
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Address for reprint requests and other correspondence: R.A.J. Lester, Department of Neurobiology, SHEL1006, University of Alabama at Birmingham, 1825 University Boulevard, Birmingham, AL 35294-0021 (E-mail: rlester{at}nrc.uab.edu)
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