点击显示 收起
【关键词】 Neurokinin 1
Neurokinin (NK) 1 receptors and CaV2.3 calcium channels are both expressed in nociceptive neurons, and mice lacking either protein display altered responses to noxious stimuli. Here, we examined modulation of CaV2.3 through NK1 receptors expressed in human embryonic kidney 293 cells. We find that NK1 receptors generate complex modulation of CaV2.3. In particular, weak activation of these receptors evokes mainly stimulation of CaV2.3, whereas strong receptor activation elicits profound inhibition that overlaps with channel stimulation. Unlike R-type channels encoded by CaV2.3, L-type (CaV1.3), N-type (CaV2.2), and P/Q-type (CaV2.1) channels are inhibited, but not stimulated, through NK1 receptors. Pharmacological experiments show that protein kinase C (PKC) mediates stimulation of CaV2.3 through NK1 receptors. The signaling mechanisms underlying inhibition were explored by expressing proteins that buffer either Gq/11 (regulator of G protein signaling protein 3T and carboxyl-terminal region of phospholipase C-1) or G subunits (transducin and the carboxyl-terminal region of bovine G-protein-coupled receptor kinase). A fast component of inhibition was attenuated by buffering G, whereas a slow component of inhibition was reduced by buffering Gq/11. When both G and Gq/11 were simultaneously buffered in the same cells, inhibition was virtually eliminated, but receptor activation still triggered substantial stimulation of CaV2.3. We also report that NK1 receptors accelerate the inactivation kinetics of CaV2.3 currents. Altogether, our results indicate that NK1 receptors modulate CaV2.3 using three different signaling mechanisms: a fast inhibition mediated by G, a slow inhibition mediated by Gq/11, and a slow stimulation mediated by PKC. This new information concerning R-type calcium channels and NK1 receptors may help in understanding nociception, synaptic plasticity, and other physiological processes.
R-type Ca2+ currents have been pharmacologically defined by their resistance to blockers of L-type, N-type, and P/Q-type Ca2+ channels (Zhang et al., 1993) and by their sensitivity to SNX-482, a peptide component of spider venom (Newcomb et al., 1998). Considerable evidence indicates that native R-type currents are mediated by CaV2.3 subunits (Piedras-Rentería and Tsien, 1998; Tottene et al., 2000; Lee et al., 2002; Sochivko et al., 2002). Recent studies have begun to elucidate the physiological roles of both R-type currents and CaV2.3. For example, R-type currents have been shown to participate in neurosecretion and in the formation of after depolarizations, plateau potentials, and bursting activity in hippocampal CA1 pyramidal neurons (Wu et al., 1998; Gasparini et al., 2001; Kuzmiski et al., 2005; Metz et al., 2005; Tai et al., 2006). Studies also indicate that native R-type channels mediate Ca2+ entry into dendritic spines (Sabatini and Svoboda, 2000) and that R-type channels play important roles in synaptic plasticity (Yasuda et al., 2003; Humeau et al., 2005). Likewise, CaV2.3 has been demonstrated to be involved in cerebellar function, morphine tolerance, and synaptic plasticity (Kubota et al., 2001; Breustedt et al., 2003; Dietrich et al., 2003; Yokoyama et al., 2004; Osanai et al., 2006). It is noteworthy that CaV2.3 is also implicated in nociception, as evidenced by the fact that CaV2.3 knockout mice exhibit functional deficits in pain perception (Saegusa et al., 2000) and intrathecal injection of SNX-482 produces analgesia (Murakami et al., 2004).
Several previous studies demonstrated that CaV2.3 and native R-type currents are modulated by signaling mechanisms linked to G protein-coupled receptors (Wu et al., 1998; Yu and Shinnick-Gallagher, 1998; Meza et al., 1999; Melliti et al., 2000; Sabatini and Svoboda, 2000; Bannister et al., 2004; Kohlmeier and Leonard, 2006; Tai et al., 2006). In our previous experiments, we showed that CaV2.3 is both inhibited and stimulated through Gq/11-coupled muscarinic acetylcholine receptors (Meza et al., 1999; Melliti et al., 2000; Bannister et al., 2004). The neurokinin (NK) 1 receptor is a Gq/11-coupled receptor with a well established role in nociception (Macdonald et al., 1996; Duffy, 2004). Responses to noxious stimuli are dramatically altered in NK1 receptorknockout mice (De Felipe et al., 1998). NK1 receptors are expressed with CaV2.3 in primary sensory neurons, in projection neurons within the dorsal horn of the spinal cord, and in various regions of mammalian brain, including amygdala, hippocampus, and striatum (Niidome et al., 1992; Saegusa et al., 2000; Lee et al., 2002; Duffy, 2004; Murakami et al., 2004). Thus, NK1 receptors may modulate CaV2.3 in vivo, and such modulation may be important in anxiety, nociception, synaptic plasticity, and other neurological processes.
In the present study, we analyzed modulation of CaV2.3 by NK1 receptors expressed in human embryonic kidney (HEK) 293 cells. We show that NK1 receptors produce a robust and complex modulation of CaV2.3 that involves three distinct signaling mechanisms: a fast inhibition mediated by G, a slow inhibition mediated by Gq/11, and a slow stimulation mediated by protein kinase C (PKC). To our knowledge, this is the first demonstration that CaV2.3 is inhibited through a Gq/11-dependent signaling mechanism. Our results indicate that NK1 receptors predominantly stimulate CaV2.3 in the presence of low agonist concentrations. By contrast, these receptors trigger profound inhibition that overlaps with stimulation of CaV2.3 in the presence of high agonist concentrations. Our findings provide new information about modulation of CaV2.3 that may be relevant to understanding pain transduction, neuronal excitability, and synaptic plasticity.
Cell Culture and Transfection. HEK293 cells (American Type Culture Collection, Manassas, VA) were propagated in culture medium containing 90% Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA), 10% defined fetal bovine serum (Hyclone Laboratories, Logan, UT) and 50 µg/ml gentamicin. Cells of low passage number (<20) were trypsinized weekly and replated onto 60-mm culture dishes at 20% confluence. Cells were transfected by CaPO4 precipitation within 3 to 5 days of plating. The transfection mixture contained a mixture of expression plasmids encoding CaV2.3 (or in selected experiments CaV1.3, CaV2.1, or CaV2.2) and ancillary Ca2+ channel 2 and 3 subunits (each at 1.25 µg/dish), the human NK1 receptor (0.29 µg/dish), and enhanced green fluorescent protein (EGFP; 0.125 µg/dish). The plasmid encoding unfused EGFP was omitted when peptides fused in-frame to EGFP were expressed (see below). In experiments with CaV2.1, the 1a subunit was cotransfected in place of 3 to enhance expression of P/Q-type current. In selected experiments, the transfection mixture included plasmids encoding human rod transducin (Gt) or the carboxyl-terminal region of bovine G protein-coupled receptor kinase 3 (GRK3ct) fused to a membrane-associating myristic acid attachment sequence (MAS-GRK3ct). We also expressed the following proteins fused in-frame to the carboxyl terminus of EGFP: human regulator of G protein signaling protein 3T (RGS3T), the carboxyl-terminal region (T903-L1216) of rat phospholipase C-1 (PLC1ct), and the RGS domain (Q45-Q170) of human p115 RhoGEF (p115RGS), each at 1.25 µg/dish. The day after transfection, cells were briefly trypsinized, replated onto 12-mm round glass coverslips, and incubated at 37°C. Electrophysiological recordings were made 24 to 32 h later. Successfully transfected cells were visually identified under UV illumination. Only isolated green cells were used for experiments.
Expression Plasmids. Rabbit brain CaV2.3 (GenBank accession number X67856) was in pcDNA3.1+ (Invitrogen). Rat brain 2 (GenBank accession number M86621) was in pMT2 (Genetics Institute, Cambridge, MA). Rabbit brain 3 (GenBank accession number X64300) was in pcDNA3 (Invitrogen). Rabbit skeletal muscle 1a (GenBank accession number M25817) was in pKCRH2. Human NK1 receptor (GenBank accession number NM015727) was in pCI (Promega, Madison, WI). Jellyfish EGFP (GenBank accession number U55763) was in pEGFP-C3 (Clontech, Cambridge, UK). Human Gt (GenBank accession number X63749) was in pcDNA3.1+. Human RGS3T (GenBank accession number U27655) was in pEGFP-C3. Rat PLC1ct (GenBank accession number M20636) was in pEGFP-C1. Bovine MAS-GRK3ct (GenBank accession number NM_174500) was in pcDNA3.1+. Human p115RGS (GenBank accession number U64105) was in pEGFP-C3 and pcDNA3.1-.
Patch-Clamp Recordings. Large-bore patch pipettes were pulled from 100-µl borosilicate glass micropipettes and filled with internal solution containing 155 mM CsCl, 10 mM Cs2-EGTA, 4 mM Mg-ATP, 0.32 mM Li-GTP, and 10 mM HEPES, with pH adjusted to 7.4 using CsOH. In one experiment, Li-GTP was replaced by 1 mM Li-GDPS. Aliquots of pipette solutions were stored at -80°C, kept on ice after thawing, and filtered at 0.22 µm immediately before use. The bath solution contained 145 mM NaCl, 40 or (where indicated) 5 mM CaCl2, 2 mM KCl, and 10 mM HEPES, with pH adjusted to 7.4 using NaOH. When filled with internal solution, pipettes had resistances of 1.0 to 1.5 M. After forming a G seal in the cell-attached configuration, residual pipette capacitance was compensated using the negative capacitance circuit of the amplifier. Ca2+ currents were recorded in whole-cell, ruptured-patch mode. The resistance of the whole-cell configuration was routinely >1G. The steady holding potential was -90 mV. No corrections were made for liquid junction potentials. Currents were filtered at 2 to 10 kHz using the built-in Bessel filter (four-pole low-pass) of an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA) and sampled at 10 to 50 kHz using a Digidata 1200 or 1320A analog-to-digital board installed in a Gateway Pentium computer. The pCLAMP software programs Clampex and Clampfit (version 8.0; Molecular Devices) were used for data acquisition and analysis, respectively. Figures were made, and statistical analyses were performed using Origin (version 6.0 or 7.5; OriginLab Corp., Northampton, MA). Linear cell capacitance (C) was determined by integrating the area under the whole-cell capacity transient, evoked by a voltage-clamp step from -90 to -80 mV. Voltage errors (typically <5 mV) were minimized using the analog series-resistance compensation circuit of the amplifier to reduce the time constant for decay of the whole-cell capacity transient as much as possible. Voltage error was calculated as the product of maximal inward Ca2+ current and series access resistance (RS). RS was calculated as the time constant for the whole-cell capacity transient x (1/C). All currents were corrected on-line for linear capacitance and leakage currents using -P/4 subtraction. Ca2+ current amplitudes were measured at the time of peak inward current. All experiments were conducted at room temperatures (20-24°C). Results are reported as mean ± S.E.M. Data were analyzed using unpaired or paired t tests. The significance level was set at 0.05.
In all experiments, the recording chamber (RC-12; Warner Instruments, Hamden, CT) was continuously perfused at the rate of 2to 3 ml/min. Agonist application and washout were achieved by bulk exchange of the bath solution, and they were 90% complete within 11 and 27 s, respectively, as judged by block and unblock of CaV2.3 current by 0.1 mM Cd2+. Temperature was continuously monitored using a small thermocouple placed in the bath.
Reagents. Neurokinin A (NKA), substance P, pertussis toxin (PTX), cholera toxin (CTX), staurosporine, bisindolylmaleimide (Bis) I, and Bis V were purchased from EMD Biosciences (San Diego, CA). NKA and substance P were dissolved in 5% acetic acid to make 1 mM stock solutions, aliquoted, and stored at -80°C. Staurosporine, Bis I, Bis V, and PP2 were dissolved in dimethyl sulfoxide to make 1 mM stock solutions, aliquoted, and stored at 4°C. The final concentrations of acetic acid and dimethyl sulfoxide were always <0.005 and <0.2%, respectively, which did not significantly alter basal properties of CaV2.3 or its modulation through NK1 receptors.
Strongly Activated NK1 Receptors Inhibited the Amplitude, Altered the Voltage Dependence of Activation, and Accelerated Inactivation of CaV2.3 Currents. Figure 1A shows families of whole-cell Ca2+ currents recorded from an HEK293 cell that expressed both NK1 receptors and CaV2.3 channels. Currents were recorded from the same cell before and during application of 1 µM NKA. This concentration of NKA caused profound inhibition of CaV2.3 current amplitudes. On average, currents evoked at a test potential of +20 mV were inhibited by 57 ± 3% (n = 30). Figure 1B plots the averaged, normalized current-voltage (I-V) relationships for control and inhibited currents. Similar magnitudes of inhibition were observed at different test potentials (Fig. 1B), suggesting that NK1 receptors trigger a mostly voltage-independent inhibition of CaV2.3. In support of this idea, inhibited currents failed to exhibit prepulse facilitation (data not shown). However, as illustrated in Fig. 1B, the scaled I-V relationship for inhibited currents was shifted to negative potentials relative to control currents. This voltage shift was statistically significant (p < 0.05) over a narrow range of test potentials (-10 to 0 mV). These results show that strongly activated NK1 receptors inhibit CaV2.3 current amplitudes and shift the voltage dependence of activation to slightly more negative potentials.
Fig. 1. Strongly activated NK1 receptors inhibit the amplitudes, alter the voltage dependence of activation, and accelerate inactivation of CaV2.3 currents. A, current families recorded from the same cell before and during exposure to 1 µM NKA. Currents were evoked at 0.2 Hz by depolarizations from -90 to -40 through +75 mV in 5-mV increments. C = 26 pF; RS = 2.5 M. B, NKA shifts the voltage dependence of activation to slightly more negative potentials over a limited range of test potentials. I-V relationships were determined for six cells before () and during () inhibition by 1 µM NKA. Voltage protocol is as described in A. I-V relationships were normalized to the maximal control current in each cell, typically recorded at +15 or +20 mV. To facilitate comparison with control currents, the average I-V relationship for inhibited currents () was scaled by 1.765 (the ratio of 0.955, which was the maximum normalized average of control currents evoked at +15 mV, to 0.541, the corresponding maximum normalized average current evoked at +15 mV during inhibition by NKA). In these recordings, the average RS was 2.3 ± 0.2 M, the average maximal current amplitude was 1708 ± 412 pA, and the average maximal voltage-error was 3.8 ± 0.9 mV (n = 6). C, average dose-response relationship for inhibition. Currents were evoked at 0.5 Hz by step depolarizations from -90 to +30 mV. Each cell was exposed to a single concentration of NKA. Symbols represent mean ± S.E.M. of 5 to 30 cells. The smooth curve represents the fit of the average data to the Hill equation: Ca2+ current (% of initial) = (100 - D)/[1 + ()/EC50)nH], where D, the percentage of Ca2+ current that could not be inhibited, is 44.1%. The EC50 was 9.8 nM, and the Hill coefficient (nH) was 0.93. D, time course of NKA-mediated inhibition and washout. Currents were evoked at 0.5 Hz by steps from -90 to +30 mV. Current amplitudes are plotted as a function of time. Application of 1 µM NKA is indicated by a thick horizontal line. Inset, Ca2+ currents recorded at the indicated times. These currents show that inactivation of CaV2.3 is significantly accelerated during and after modulation through NK1 receptors. C = 13 pF; RS = 5.1 M.
The dose dependence of inhibition was determined by exposing cells to various concentrations of NKA. The resultant data were combined, and a dose-inhibition curve was constructed (Fig. 1C). The averaged, normalized data were fit with a Hill equation, yielding an EC50 of 9.8 nM and a Hill coefficient of 0.93. A significant fraction of current (44%) was apparently not inhibited by the highest concentration of NKA (10 µM). This large residual current probably reflects the fact that NK1 receptors also trigger stimulation of CaV2.3 that overlaps with inhibition (see below).
As shown in Fig. 1D, the onset of inhibition seemed to contain both fast and slow components (see also Fig. 3, A and B). We did not attempt to quantify these components because application of NKA was relatively slow in our experiments (see Materials and Methods). Upon washout of NKA, current amplitudes slowly returned to near control levels and in some cells over-recovered, consistent with the presence of overlapping channel stimulation (see below). The complete recovery of current amplitudes typically required 1 to 2 min of washout.
Fig. 3. NK1 receptors couple to PTX- and CTX-insensitive G proteins and inhibit CaV2.3 independently of protein kinases. A, inhibition of CaV2.3 in response to 1 µM NKA is insensitive to PTX. Currents were evoked at 0.5 Hz by 10-ms steps from -90 to +30 mV. Current amplitudes are plotted versus time for a cell pretreated with 1 µg/ml PTX. C = 20 pF; RS = 2.1 M. B, inhibition of CaV2.3 in response to 1 µM NKA is insensitive to CTX. Currents were evoked at 0.5 Hz by 12.5-ms depolarizations from -90 to +30 mV. Current amplitudes are plotted for a cell pretreated with 1 µg/ml CTX. C = 15 pF; RS = 2.7 M. C, Histograms summarize percent inhibition of CaV2.3 currents in response to 1 µM NKA. Inhibitors were used at 100 nM (staurosporine), 500 nM (Bis I), 50 µM (genistein), 1 µM (PP2), and 1 mM (GDPS). Asterisks (*) indicate significant difference (p < 0.05) from controls.
Individual Ca2+ currents, recorded at the indicated times, are illustrated in Fig. 1D. These currents are shown to introduce an additional aspect of modulation, namely, that exposure to NKA significantly accelerated inactivation of CaV2.3 currents. This kinetic effect is described in greater detail below (Fig. 5).
Fig. 5. NK1 receptors significantly accelerate the inactivation kinetics of CaV2.3. A, activation. Currents were recorded from the same cell before (light trace) and 130 s after (bold trace) continuous exposure to 1 µM NKA. The inhibited current is scaled for comparison. C = 28 pF; RS = 2.4 M.B, inactivation. Currents were recorded from the same cell before (light trace) and 100 s after (bold trace) application of 1 µM NKA. The inhibited current is scaled for comparison. C = 29 pF; RS = 3.8 M. C, on average, activation of CaV2.3 is not significantly accelerated by NKA. Average time constants of activation ( activation) are plotted as a function of test potential. Symbols represent mean ± S.E.M. of four to six cells. Data from currents evoked at 0.5 Hz by 10-ms depolarizations from -90 to -5 through +40 mV in 5-mV increments. Current families were evoked before application of NKA () and during maximal inhibition by 1 µM NKA (). D, inactivation is significantly accelerated at all test potentials except -5 mV. Average time constants of inactivation ( inactivation) are plotted as a function of test potential. Symbols represent mean ± S.E.M. of five to six cells. Currents evoked at 0.5 Hz by 300-ms depolarizations from -90 to -5 through +50 mV in 5-mV increments. Current families were evoked before application of NKA () and during maximal inhibition by 1 µM NKA (). E, comparison of the time courses for inhibition of current amplitudes and acceleration of inactivation. C = 53 pF; RS = 2.9 M. Currents were evoked at 0.33 Hz by 300-ms steps from -90 to +20 mV.
Because substance P is an important natural agonist for the NK1 receptor, we also evaluated the effects of this neuropeptide. Inhibition of CaV2.3 by 1 µM substance P was quantitatively similar (52 ± 2%; n = 3) to that produced by 1 µM NKA. However, the effects of substance P were not readily reversible, and this agonist was thus deemed unsuitable for the present purposes. By contrast, the effects of NKA were reliably reversed upon washout, allowing us to confirm recovery of current amplitudes and to thereby distinguish channel modulation from channel rundown.
Neither NKA nor substance P evoked any modulation of CaV2.3 in cells that had not been transfected with NK1 receptors, suggesting that HEK293 cells lack endogenous tachykinin receptors. Furthermore, in cells transfected with NK1 receptors and ancillary Ca2+ channel 2-b and 3 subunits, but no CaV2.3 subunit, whole-cell currents (typically <20 pA) were unaffected by NKA (data not shown). It is therefore unlikely that modulation of endogenous cation channels (e.g., TRP family channels) contributed to our results.
CaV2.3 Was Primarily Stimulated during Weak Activation of NK1 Receptors. While performing the abovementioned dose-response experiments, we noticed that very low concentrations of NKA triggered considerable stimulation of CaV2.3 but little inhibition. This concentration-dependent effect of NKA is documented in Fig. 2A. On average, a low concentration of NKA (1 nM) evoked 39 ± 3% stimulation but only 7 ± 1% inhibition of CaV2.3 current amplitudes (n = 30 cells). Similar concentration-dependent modulation of CaV2.3 was seen in experiments where the bath contained a more physiological concentration of Ca2+ (Fig. 2C). For example, with 5 mM external Ca2+, 1 nM NKA stimulated current amplitudes by 73 ± 10% and inhibited current amplitudes by 16 ± 3% (n = 12). A high concentration of NKA (1 µM) evoked similar inhibition with either 5 mM Ca2+ (65 ± 3%; n = 12) or 40 mM Ca2+ (57 ± 3%; n = 30) in the bath. Both the onset and recovery from stimulation (in response to 1 nM NKA) exhibited slow kinetics (Fig. 2, A and C), consistent with our previous reports (Meza et al., 1999; Melliti et al., 2000; Bannister et al., 2004).
Fig. 2. CaV2.3 is primarily stimulated during weak activation of NK1 receptors. A, a low concentration (1 nM) of NKA triggers substantial stimulation of CaV2.3 but very little inhibition. Currents were evoked by 12.5-ms steps to +20 mV delivered at 0.5 Hz. The bath contained 40 mM Ca2+. C = 28 pF; RS = 2.4 M. B, individual currents recorded at the times indicated in A. C, experiment performed with 5 mM Ca2+ in the bath. C = 43 pF; RS = 2.4 M. D, individual currents recorded at the times indicated in C. E, NKA evokes inhibition but no stimulation of CaV2.3 currents in the presence of the PKC inhibitor Bis I. F, histograms summarize percentage of stimulation of CaV2.3 currents in response to 1 nM NKA. Asterisks (*) indicate significant difference (p < 0.05) from controls. Inhibitors were used at 100 nM (staurosporine), 500 nM (Bis I), 500 nM (Bis V), and 1 µM (PP2).
Stimulation Was Mediated by PKC. Previous studies have shown that CaV2.3 is stimulated by muscarinic acetylcholine and metabotropic glutamate receptors acting through PKC (Stea et al., 1995; Meza et al., 1999; Melliti et al., 2000; Kamatchi et al., 2003; Bannister et al., 2004). As shown in Fig. 2E, stimulation of CaV2.3 through NK1 receptors was blocked by 500 nM Bis I, a specific inhibitor of PKC. In contrast, stimulation was not blocked by 500 nM Bis V, an inactive control compound for Bis I (Fig. 2F). We also tested 1 µM PP2, a Src inhibitor, because NK1 receptors were previously reported to form scaffolding complexes with Src tyrosine kinases (DeFea et al., 2000). PP2 had no effect on stimulation (Fig. 2F). However, staurosporine, which also blocks PKC, eliminated stimulation. These results indicate that PKC mediates stimulation of CaV2.3 through NK1 receptors.
Because NK1 receptors trigger both stimulation and inhibition of CaV2.3 currents, the true magnitude of inhibition is probably underestimated in the above-mentioned data summaries. Consistent with this possibility, inhibition evoked by 1 nM NKA was substantially larger (35 ± 4%; n = 9) in cells exposed to 500 nM Bis I than in control cells (7 ± 1%; n = 30).
L-, N-, and P/Q-Type Channels Were Inhibited, but Not Stimulated, by NK1 Receptors. We next asked whether NK1 receptors could modulate Ca2+ channel types other than CaV2.3. To this end, HEK293 cells were transfected with NK1 receptors and either CaV1.3 (L-type), CaV2.1 (P/Q-type), or CaV2.2 (N-type) plus ancillary 2- and subunits. The response of CaV1.3 to the low concentration (1 nM) of NKA could not be reliably distinguished from rundown, but CaV2.1 and CaV2.2 were clearly inhibited, by 24 ± 6% (n = 6) and 43 ± 5% (n = 8), respectively. In each of the above-mentioned experiments, 1 nM NKA failed to evoke discernible stimulation of CaV1.3, CaV2.1, or CaV2.2. The high concentration (1 µM) of NKA also failed to evoke stimulation and instead produced 74 ± 5% (n = 6), 61 ± 4% (n = 6), and 79 ± 3% (n = 7) inhibition of these channel types, respectively. We did not attempt to determine whether inhibition of these non-R-type channels was voltage-dependent or -independent, because this issue seems complex (Kammermeier et al., 2000). In summary, our data show that CaV1.3, CaV2.1, and CaV2.2 were inhibited, but not stimulated, through NK1 receptors. Inhibition of CaV1.3 required strongly activated NK1 receptors, whereas even weakly activated NK1 receptors produced significant inhibition of CaV2.1 and CaV2.2. In our experiments, CaV2.3 was the only channel that was stimulated through NK1 receptors.
NK1 Receptors Coupled to PTX- and CTX-Insensitive G Proteins and Inhibited CaV2.3 Independently of Protein Kinases. NK1 receptors couple primarily to Gq/11 in native cells (Macdonald et al., 1996). However, when overexpressed in non-native cells, these receptors may also couple to Gs and Go (Roush and Kwatra, 1998). To test for potential involvement of Gs and Go in modulating CaV2.3, we pretreated cells with 1 µg/ml PTX for 4 h before electrophysiological experiments. In our hands, this concentration and duration of PTX exposure completely blocks inhibition of N-type (CaV2.2) calcium channels through Gi-coupled -opioid receptors (data not shown). In similar experiments, cells were exposed to freshly prepared CTX (1 µg/ml) for 17 to 22 h before patch-clamp recordings. As summarized in Fig. 3, both PTX and CTX failed to reduce inhibition of CaV2.3 currents in response to 1 µM NKA. However, inhibition was significantly reduced (to 27 ± 4%; n = 10) in cells dialyzed for at least 5 min with a pipette solution containing 1 mM GDPS (Fig. 3C). These data suggest that a toxin-insensitive G protein (presumably Gq/11) mediated modulation of CaV2.3 in our experiments.
As also shown in Fig. 3C, inhibition was not decreased by either 500 nM Bis I or 100 nM staurosporine. In fact, the magnitude of inhibition was significantly larger in cells exposed to these compounds (p < 0.05). Staurosporine is a broad-spectrum inhibitor of serine/threonine kinases (including PKC). The larger inhibition in the presence of these kinase inhibitors may partly reflect the fact that PKC-mediated stimulation of CaV2.3 prevents accurate measurement of inhibition. In addition, PKC-dependent phosphorylation probably interferes with inhibition of CaV2.3 by G subunits (Bannister et al., 2004). We also tested 1 µM PP2 and 50 µM genistein, the latter being a broad-spectrum inhibitor of tyrosine kinases. Neither PP2 nor genistein reduced inhibition of CaV2.3 in response to 1 µM NKA (Fig. 3C). These data suggest that inhibition of CaV2.3 through NK1 receptors did not involve serine/threonine kinases or tyrosine kinases.
Gq/11 and G Both Mediated Inhibition of CaV2.3. We next explored the molecular mechanism(s) of inhibition by expressing proteins that sequester either Gq/11 or G subunits. Figure 4A illustrates a representative experiment for a cell transfected with rod transducin (Gt), which effectively buffers G dimers but is not expected to couple to receptors other than rhodopsin (Kammermeier et al., 2000). In Gt-transfected cells, 1 µM NKA inhibited current amplitudes by only 29 ± 3% (n = 6), significantly less than in control cells (Fig. 4E). Furthermore, the onset of inhibition seemed qualitatively slower in Gt-expressing cells (Fig. 4A). Similar results were obtained in cells transfected with MAS-GRK3ct, which has also been shown to buffer G dimers (Kammermeier et al., 2000). The reduced magnitude and slowed onset of inhibition in Gt- and MAS-GRK3ct-expressing cells is consistent with the idea that G mediates a fast component of inhibition. It would seem that the remaining slow component of inhibition is mediated by Gq/11.Toexamine this possibility, we expressed RGS3T, which previous studies have shown to be a particularly effective antagonist of Gq/11-mediated signaling (Scheschonka et al., 2000; Bannister et al., 2002). In RGS3T-expressing cells, application of 1 µM NKA triggered rapid inhibition of current amplitude followed by a relaxation or "sag" to a lower, plateau level of inhibition. This sag corresponds to the onset of PKC-dependent channel stimulation (see below). All together, 1 µM NKA inhibited current amplitudes by 44 ± 3% (n = 17) in RGS3T-expressing cells, significantly less than inhibition (60 ± 5%; n = 6) in matched control cells (Fig. 4E). Inhibition was similarly attenuated (41 ± 3%; n = 14) by expression of PLC1ct (Fig. 4E), which also antagonizes Gq/11 signaling (Kammermeier et al., 2000). The above-mentioned results suggest that G mediated a fast component, whereas Gq/11 mediated a slow component of inhibition. However, we note that G might have additionally contributed to the slow component and Gq/11 to the fast component of inhibition if these G protein subunits were incompletely buffered in our experiments (see Discussion).
Fig. 4. Gq/11 and G both inhibit CaV2.3. A, expression of Gt, which buffers G, blocks a fast component of inhibition. Currents were evoked at 0.5 Hz by 12.5-ms depolarizations to +25 mV. C = 17 pF; RS = 2.5 M. B, expression of RGS3T, which buffers Gq/11, blocks a slow component of inhibition. Currents were evoked at 0.5 Hz by 12.5-ms depolarizations to +30 mV. C = 14 pF; RS = 4.9 M. C, coexpression of Gt and RGS3T in the same cell greatly reduces inhibition but not stimulation. Currents were evoked at 0.5 Hz by 12.5-ms depolarizations to +20 mV. C = 30 pF; RS = 3.0 M. D, stimulation is blocked by Bis I (500 nM) in cells coexpressing both Gt and RGS3T. C = 29 pF; RS = 4.1 M. E, histograms summarize percentage of inhibition in response to 1 µM NKA. Asterisks (*) indicate significant difference (p < 0.05) from controls.
As shown in Fig. 4C, inhibition in response to 1 µM NKA was greatly diminished when both Gt and RGS3T were expressed in the same cell. Under these conditions, 1 µM NKA triggered substantial stimulation (46 ± 7%; n = 9) but very little inhibition (7 ± 3%; n = 8) of CaV2.3 currents. This residual stimulation was completely blocked by Bis I (Fig. 4D) but not by Bis V (data not shown). On average, the high concentration of NKA (1 µM) inhibited current amplitudes by 18 ± 4% (n = 9) in cells that expressed both Gt and RGS3T and exposed to 500 nM Bis I (Fig. 4E). These data clearly show that Gq/11 and G both contribute to inhibition of CaV2.3 through NK1 receptors. This seems to be the first report that CaV2.3 is inhibited through a Gq/11-dependent signaling mechanism.
The residual modulation of CaV2.3 observed in cells transfected with both Gt and RGS3T (Fig. 4, C-E) was somewhat unexpected, because we had previously found that Gq/11-mediated stimulation of CaV2.3 was strongly blocked by expression of either regulator of G protein signaling protein 2 or PLC1ct (Melliti et al., 2000). Furthermore, we previously observed that Gq/11-mediated inhibition of the L-type channel CaV1.2c was virtually eliminated by expression of RGS3T (Bannister et al., 2002). The residual modulation might be explained if Gq/11 and G were incompletely buffered by Gt and RGS3T in our present experiments. On the other hand, the residual modulation might indicate the involvement of another toxin-insensitive G protein (e.g., G12/13) in addition to Gq/11. To explore the latter possibility, we expressed the RGS domain of p115RhoGEF (p115RGS), previously shown to function as a GTPase-activating protein for G12 and G13 (Kozasa et al., 1998). In cells transfected with p115RGS in pcDNA3.1-, application of 1 nM NKA evoked robust stimulation (43 ± 8%; n = 7), whereas 1 µM NKA evoked substantial inhibition (66 ± 5%; n = 8) of CaV2.3 currents (data not shown). These magnitudes of stimulation and inhibition are indistinguishable from those obtained in control cells. To ensure that we recorded exclusively from cells expressing p115RGS, we fused it to the carboxyl terminus of EGFP. In response to 1 nM NKA, CaV2.3 currents were stimulated by 42 ± 3% (n = 9) in cells expressing EGFP-p115RGS, compared with 36 ± 15% (n = 5) stimulation in matched controls (p > 0.05). In response to the high concentration (1 µM) of NKA, currents were inhibited by 61 ± 3% (n = 6) in EGFP-p115RGS cells and by 67 ± 8% (n = 4) in matched controls (p > 0.05). Although these experiments are not exhaustive, they suggest that G12/13 did not play a significant role in modulating CaV2.3 through NK1 receptors in our system.
NK1 Receptors Accelerated Inactivation of CaV2.3. As first mentioned in relation to Fig. 1D, the inactivation kinetics of CaV2.3 currents were noticeably accelerated after exposure to NKA. This effect was quantified by fitting individual Ca2+ currents with a single exponential function to obtain time constants for macroscopic activation and inactivation. As shown in Fig. 5B, inactivation of CaV2.3 currents was clearly accelerated by NKA. For example, currents evoked at +20 mV inactivated with an average time constant (inact.) of 33 ± 2ms(n = 6) before and 18 ± 2ms(n = 5) after a continuous 2-min exposure to 1 µM NKA (p < 0.001). Figure 5D shows that inactivation was significantly accelerated at most test potentials. The acceleration of inactivation was not due to increased bath temperature during NKA application, because temperature was continuously monitored both before and during NKA applications and did not vary by more than ±0.5°C. Furthermore, acceleration was not attributable to decreasing RS, because membrane charging speed was monitored before and after NKA application and RS either remained constant or increased during the recordings.
By contrast, the activation kinetics of CaV2.3 did not seem to be significantly accelerated by NKA (Fig. 5A). Thus, at a test potential of +20 mV, currents activated with an average time constant (act.) of 1.5 ± 0.3 ms before and 1.2 ± 0.3 ms (n = 6) after a continuous 2-min exposure to 1 µM NKA; this difference was not significant (p > 0.05). Figure 5C plots average values of act. at various test potentials. Activation was significantly accelerated only at a test potential of +10 mV (act. = 1.8 ± 0.3 ms before and 1.2 ± 0.2 ms after NKA exposure; n = 6; p = 0.03; paired t test).
Inhibition of current amplitudes consistently attained stable levels before acceleration of inactivation had became complete. We quantified this relationship by measuring times required for inhibition and acceleration to near completion. Measuring from the beginning of NKA application, inhibition of current amplitude was 90% complete in 15 ± 1 s, whereas acceleration of inactivation required 28 ± 3s(n = 5) to attain 90% completion. As illustrated in Fig. 5E, current amplitudes also recovered more quickly than inactivation rate after NKA washout. This difference in recovery was consistently observed (four of five cells). We did not extensively pursue the mechanism underlying acceleration of inactivation. However, it may be worth mentioning that inactivation was significantly accelerated in cells exposed to 100 nM Bis I [inact. at 20 mV was 36 ± 1 ms before and 23 ± 1ms(n = 7) after a continuous 1- to 2-min application of 1 µM NKA], suggesting that PKC is not involved.
This work shows that NK1 receptors triggered complex modulation of Ca 2.3 (R-type) Ca2+V channels. We have identified three distinct signaling mechanisms involved in this modulation: a fast inhibition mediated by G, a slow inhibition mediated by Gq/11, and a slow stimulation mediated by PKC (Fig. 6). We note that the stimulatory effect predominated during low receptor occupancy, whereas the three forms of modulation produced overlapping influences on CaV2.3 during high receptor occupancy. We also find that NK1 receptors markedly accelerated inactivation of CaV2.3. The increased rate of inactivation further reduced R-type Ca2+ influx during modulation through NK1 receptors.
Fig. 6. Schematic summary of results. Solid arrows indicate well established signaling pathways and mechanisms of channel modulation. Dashed arrows indicate less well established signaling pathways that might modulate CaV2.3. For example, if G was incompletely buffered by Gt or MAS-GRK3ct in our experiments, it might contribute to slow inhibition of CaV2.3. Likewise, if Gq/11 was incompletely buffered by RGS3T or PLC1ct, it might contribute to fast inhibition of CaV2.3. Depletion of PIP2 has not been experimentally demonstrated to modulate CaV2.3. Finally, the diagram postulates that Gq/11 might modulate CaV2.3 through direct interaction.
We present evidence that Gq/11 as well as G mediated inhibition of CaV2.3 through NK1 receptors (Fig. 4). This finding is in general agreement with Kammermeier et al. (2000), who showed that Gq/11 and G are both required for voltage-independent inhibition of native N-type Ca2+ channels by NK1 receptors heterologously expressed in rat sympathetic neurons. In the present experiments, we found that separately buffering G or Gq/11 partially blocked inhibition of CaV2.3 and altered the time course of inhibition (Fig. 4). We also found that simultaneously buffering both G and Gq/11 in the same cells greatly reduced inhibition (Fig. 4C) but did not eliminate it (Fig. 4, D and E). It is intriguing that substantial stimulation of CaV2.3 current was observed in cells where G and Gq/11 were simultaneously buffered (Fig. 4C). Although we did not rigorously quantify the time course of channel modulation in our experiments, our results are consistent with the notion that G mediated a fast component of inhibition, whereas Gq/11 mediated a slow component (Fig. 6). However, our data do not exclude the possibility that G also contributed to slow inhibition or that Gq/11 contributed to fast inhibition.
Previous studies indicate that Gq/11 mediates slow inhibition of L-type (CaV1.2c) and N-type (CaV2.2) Ca2+ channels (Melliti et al., 2001; Bannister et al., 2002). Our present finding that Gq/11 triggered slow inhibition of CaV2.3 suggests that this form of inhibition is conserved among different classes of Ca2+ channels. We did not attempt to identify the molecular mechanism of slow inhibition here, beyond showing that Gq/11 was involved, because this would have required extensive additional experiments (Gamper et al., 2004). However, depletion of membrane phosphoinositol-4,5-bisphosphate (PIP2) is a strong candidate for this mechanism (Fig. 6), because PIP2 depletion seemed to underlie slow Gq/11-mediated inhibition of both N-type (see Gamper et al., 2004) and P/Q-type calcium channels (Wu et al., 2002). It is also theoretically possible that Gq/11 inhibited CaV2.3 directly (Fig. 6).
We found that NK1 receptors shifted the voltage dependence of CaV2.3 activation to slightly more negative potentials (Fig. 1B). This finding agrees with a recent demonstration by Tai et al. (2006) that the voltage dependence of activation of native R-type currents is shifted to negative potentials during modulation by muscarinic acetylcholine receptors. It is worth noting that the negative shift in voltage dependence reported here and by Tai et al. (2006) is opposite that typically observed for G protein-inhibited channels of the CaV2 family. Additional experiments are needed to elucidate the underlying mechanism. However, because application of exogenous PIP2 shifts the voltage dependence of activation of CaV2.1 (Wu et al., 2002) and CaV2.2 (Gamper et al., 2004) to more positive potentials, we hypothesize that depletion of endogenous PIP2 might underlie the negative shift in CaV2.3 activation (Fig. 6).
We find that CaV2.3 was preferentially stimulated during weak activation of NK1 receptors (Fig. 2). By contrast, CaV2.3 was both inhibited and stimulated during strong activation of NK1 receptors. This remarkable "bipolar" modulation of CaV2.3 might have important physiological consequences if NK1 receptors experience widely varying concentrations of agonist in vivo, as seems probable.
Preferential triggering of stimulation by weakly activated NK1 receptors probably reflects the presence of considerable biochemical amplification within the stimulatory pathway (Fig. 6). This pathway likely involved sequential activation of three enzymes: Gq/11, PLC, and PKC. By contrast, G-mediated inhibition probably reflected direct binding of G to CaV2.3 with little or no amplification. Depletion of membrane PIP2 would most simply involve sequential activation of only two enzymes, Gq/11 and PLC. These differences in biochemical amplification might help to explain our results with G- and Gq/11-buffering proteins. We found that buffering both G and Gq/11 in the same cells (by coexpressing Gt and RGS3T) blocked inhibition of CaV2.3 more effectively than it blocked stimulation (Fig. 4, C-E). In principle, it should be easier to block G- and Gq/11-mediated inhibition of CaV2.3 than Gq/11-mediated stimulation because of the greater theoretical amplification involved in stimulation. The apparent ineffectiveness of RGS3T in blocking stimulation of CaV2.3 (Fig. 4C) may be explained if only a few Gq/11 needed to escape buffering by RGS3T to activate PKC.
Stimulation of R-type currents has been demonstrated in native cells. For example, Yu and Shinnick-Gallagher (1998) showed that native R-type currents are stimulated through corticotropin-releasing factor receptors in central amygdala neurons. In addition, Tai et al. (2006) showed that native R-type currents are stimulated through muscarinic acetylcholine and metabotropic glutamate receptors in hippocampal CA1 neurons. Our present experiments indicate that PKC mediated stimulation of CaV2.3 through NK1 receptors (Fig. 2), consistent with previous observations that CaV2.3 is stimulated by phorbol esters or Gq/11-coupled muscarinic acetylcholine receptors or metabotropic glutamate receptors (Stea et al., 1995; Meza et al., 1999; Melliti et al., 2000; Kamatchi et al., 2003; Bannister et al., 2004). Tai et al. (2006) found that native hippocampal R-type currents are stimulated through a Ca2+-independent, diacylglycerol-dependent PKC isoform, in agreement with our previous study of cloned CaV2.3 channels (Bannister et al., 2004).
The ability of NK1 receptors to both inhibit and stimulate CaV2.3 suggests that native R-type currents might be modulated in a dynamic fashion during activation of Gq/11-coupled receptors. As one possibility, G-mediated inhibition of CaV2.3 could be selectively blocked by recruiting G-binding proteins (e.g., G protein-coupled receptor kinases) to receptor-channel complexes. Likewise, Gq/11-mediated inhibition of CaV2.3 could be selectively blocked by recruiting RGS proteins or downstream effectors of Gq/11 besides PLC. As suggested by our results (Fig. 4C), antagonizing both G and Gq/11 signaling at the same time could substantially reduce inhibition of CaV2.3, but leave stimulation relatively intact. Selective blockade of inhibition could thereby have significant consequences for physiological processes involving R-type Ca2+ influx.
Our present results may be relevant to understanding molecular mechanisms of nociception and other neurobiological processes. NK1 receptors are involved in diverse physiological functions, including nociception, aggression, anxiety, asthma, cough, depression, emesis, irritable bowel syndrome, and migraine (De Felipe et al., 1998; Duffy, 2004). Likewise, CaV2.3 has been shown to participate in a variety of processes, including nociception (Saegusa et al., 2000), neurosecretion (Wu et al., 1998; Gasparini et al., 2001), synaptic plasticity (Sabatini and Svoboda, 2000; Breustedt et al., 2003; Dietrich et al., 2003; Yasuda et al., 2003), electrical excitability of central neurons (Kuzmiski et al., 2005; Metz et al., 2005; Tai et al., 2006), cerebellar function (Osanai et al., 2006), morphine analgesia and tolerance (Yokoyama et al., 2004), and anxiety (Saegusa et al., 2000; Lee et al., 2002). NK1 receptors are expressed at high densities in the dorsal horn of the spinal cord and in the amygdala, hippocampus, and striatum (Duffy, 2004). CaV2.3 is similarly expressed in spinal cord dorsal horn (Saegusa et al., 2000), amygdala (Lee et al., 2002), and hippocampus and striatum (Niidome et al., 1992). Thus, these two proteins are expressed together in common regions of the nervous system and are implicated in some of the same neurological functions. It therefore seems plausible that NK1 receptors could modulate CaV2.3 within nociceptive pathways and other neurological circuits.
R-type currents are important in determining the electrical properties of hippocampal neurons. For example, R-type currents contribute to afterdepolarizations and bursting activity of hippocampal CA1 neurons (Metz et al., 2005), and receptor-mediated stimulation of R-type currents profoundly alters the dendritic integration and intrinsic resonance properties of these neurons by shifting the pattern of Ca2+ influx from L-, N-, and P/Q-type channels to R-type channels (Tai et al., 2006). R-type currents are also critical in shaping Ca2+ dynamics in dendritic spines of hippocampal CA1 neurons (Sabatini and Svoboda, 2000; Yasuda et al., 2003). In spines, native R-type currents can be inhibited through G proteincoupled receptors (Sabatini and Svoboda, 2000) or brief trains of action potentials (Yasuda et al., 2003); intriguingly, the latter form of inhibition blocks -burst-induced long-term potentiation (Yasuda et al., 2003). We speculate that stimulation, as well as inhibition, of R-type currents influences synaptic plasticity at dendritic spines. It also seems plausible that receptor-mediated stimulation of R-type currents contributes to plateau potentials and spiking in dorsal horn and other neurons.
The present study provides new information concerning R-type channels and NK1 receptors. This information may help in understanding the molecular basis of nociception, electrical excitabililty, synaptic plasticity and other physiological processes.
Acknowledgements
We thank Dr. Keith Elmslie (Penn State College of Medicine, Hershey, PA) and Dr. Katarina Stroffekova (Utah State University, Logan, UT) for helpful suggestions regarding organization and content of the manuscript. Dr. Karim Melliti made the original observation that CaV2.3 is inhibited through a slow Gq/11-mediated pathway.
Portions of this work were previously presented at the 2003 and 2006 annual meetings of the Biophysical Society (Biophys J Suppl 84:537a; Biophys J Suppl 90:396a).
U.M. and A.T. contributed equally to this work.
ABBREVIATIONS: NK, neurokinin; HEK, human embryonic kidney; PKC, protein kinase C; EGFP, enhanced green fluorescent protein; MAS-GRK3ct, carboxyl-terminal region of G protein-coupled receptor kinase 3; Gt, rod transducin; RGS3T, regulator of G protein signaling protein 3T; RGS2, regulator of G protein signaling protein 2; PLC1ct, carboxyl-terminal region (T903-L1216) of phospholipase C-1; C, cell capacitance; RS, series access resistance; NKA, neurokinin A; PTX, pertussis toxin; CTX, cholera toxin; Bis, bisindolylmaleimide; I-V, current-voltage; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine.
【参考文献】
Bannister RA, Melliti K, and Adams BA (2002) Reconstituted slow muscarinic inhibition of neuronal (CaV1.2c) L-type Ca2+ channels. Biophys J 83: 3256-3267.
Bannister RA, Melliti K, and Adams BA (2004) Differential modulation of CaV2.3 Ca2+ channels by Gq/11-coupled muscarinic receptors. Mol Pharmacol 65: 381-388.[Abstract/Free Full Text]
Breustedt J, Vogt KE, Miller RJ, Nicoll RA, and Schmitz D (2003) 1E-containing Ca2+ channels are involved in synaptic plasticity. Proc Natl Acad Sci USA 100: 12450-12455.[Abstract/Free Full Text]
DeFea KA, Vaughn ZD, O'Bryan EM, Nishijima D, Dery O, and Bunnett NW (2000) The proliferative and antiapoptotic effects of substance P are facilitated by formation of a -arrestin-dependent scaffolding complex. Proc Natl Acad Sci USA 97: 11086-11091.[Abstract/Free Full Text]
De Felipe C, Herrero JF, O'Brien JA, Palmer JA, Doyle CA, Smith AJ, Laird JM, Belmonte C, Cervero F, and Hunt SP (1998) Altered nociception, analgesia and aggression in mice lacking the receptor for substance P. Nature (Lond) 392: 394-397.
Dietrich D, Kirschstein T, Kukley M, Pereverez A, von der Brelie C, Schneider T, and Beck H (2003) Functional specialization of presynaptic CaV2.3 Ca2+ channels. Neuron 39: 483-496.
Duffy RA (2004) Potential therapeutic targets for neurokinin-1 receptor antagonists. Expert Opin Emerg Drugs 9: 9-21.
Gamper N, Reznikov V, Yamada Y, Yang J, and Shapiro MS (2004) Phosphatidylinositol 4,5-bisphosphate signals underlie receptor-specific Gq/11-mediated modulation of N-type Ca2+ channels. J Neurosci 24: 10980-10992.[Abstract/Free Full Text]
Gasparini S, Kasyanov AM, Pietrobon D, Voronin LL, and Cherubini E (2001) Presynaptic R-type calcium channels contribute to fast excitatory synaptic transmission in the rat hippocampus. J Neurosci 21: 8715-8721.[Abstract/Free Full Text]
Humeau Y, Herry C, Kemp N, Shaban H, Fourcaudot E, Bissiere S, and Luthi A (2005) Dendritic spine heterogeneity determines afferent-specific Hebbian plasticity in the amygdala. Neuron 45: 119-131.
Kamatchi GL, Tiwari SN, Chan CK, Chen D, Do SH, Durieux ME, and Lynch C III (2003) Distinct regulation of expressed calcium channels 2.3 in Xenopus oocytes by direct or indirect activation of protein kinase C. Brain Res 968: 227-237.
Kammermeier PJ, Ruiz-Velasco V, and Ikeda SR (2000) A voltage-independent calcium current inhibitory pathway activated by muscarinic agonists in rat sympathetic neurons requires both Gq/11 and G. J Neurosci 20: 5623-5629.[Abstract/Free Full Text]
Kohlmeier KA and Leonard CS (2006) Transmitter modulation of spike-evoked calcium transients in arousal related neurons: muscarinic inhibition of SNX-482-sensitive calcium influx. Eur J Neurosci 23: 1151-1162.
Kozasa T, Jiang X, Hart MJ, Sternweis PM, Singer WD, Gilman AG, Bollag G, and Sternweis PC (1998) p115 RhoGEF, a GTPase activating protein for G12 and G13. Science (Wash DC) 280: 2109-2111.[Abstract/Free Full Text]
Kubota M, Murakoshi T, Saegusa H, Kazuno A, Zong S, Hu Q, Noda T, and Tanabe T (2001) Intact LTP and fear memory but impaired spatial memory in mice lacking CaV2.3 (1E) channel. Biochem Biophys Res Commun 282: 242-248.
Kuzmiski JB, Barr W, Zamponi GW, and MacVicar BA (2005) Topiramate inhibits the initiation of plateau potentials in CA1 neurons by depressing R-type calcium channels. Epilepsia 46: 481-489.
Lee SC, Choi S, Lee T, Kim HL, Chin H, and Shin HS (2002) Molecular basis of R-type calcium channels in central amygdala neurons of the mouse. Proc Natl Acad Sci USA 99: 3276-3281.[Abstract/Free Full Text]
Macdonald SG, Dumas JJ, and Boyd ND (1996) Chemical cross-linking of the substance P (NK-1) receptor to the subunits of the G proteins Gq and G11. Biochemistry 35: 2909-2916.
Melliti K, Meza U, and Adams B (2000) Muscarinic stimulation of 1E Ca channels is selectively blocked by the effector antagonist function of RGS2 and phospholipase C-beta1. J Neurosci 20: 7167-7173.[Abstract/Free Full Text]
Melliti K, Meza U, and Adams B (2001) RGS2 blocks slow muscarinic inhibition of N-type Ca2+ channels reconstituted in a human cell line. J Physiol (Lond) 532: 337-347.[Abstract/Free Full Text]
Metz AE, Jarsky T, Martina M, and Spruston N (2005) R-type calcium channels contribute to afterdepolarization and bursting in hippocampal CA1 pyramidal neurons. J Neurosci 25: 5763-5773.[Abstract/Free Full Text]
Meza U, Bannister R, Melliti K, and Adams B (1999) Biphasic, opposing modulation of cloned neuronal 1E Ca channels by distinct signaling pathways coupled to M2 muscarinic acetylcholine receptors. J Neurosci 19: 6806-6817.[Abstract/Free Full Text]
Murakami M, Nakagawasai O, Suzuki T, Mobarakeh II, Sakurada Y, Murata A, Yamadera F, Miyoshi I, Yanai K, Tan-No K, et al. (2004) Antinociceptive effect of different types of calcium channel inhibitors and the distribution of various calcium channel alpha 1 subunits in the dorsal horn of spinal cord in mice. Brain Res 1024: 122-129.
Newcomb R, Szoke B, Palma A, Wang G, Chen X, Hopkins W, Cong R, Miller J, Urge L, Tarczy-Hornoch K, et al. (1998) Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas. Biochemistry 37: 15353-15362.
Niidome T, Kim MS, Friedrich T, and Mori Y (1992) Molecular cloning and characterization of a novel calcium channel from rabbit brain. FEBS Lett 308: 7-13.
Osanai M, Saegusa H, Kazuno AA, Nagayama S, Hu Q, Zong S, Murakoshi T, and Tanabe T (2006) Altered cerebellar function in mice lacking CaV2.3 Ca2+ channel. Biochem Biophys Res Commun 344: 920-925.
Piedras-Rentería ES and Tsien RW (1998) Antisense oligonucleotides against 1E reduce R-type calcium currents in cerebellar granule cells. Proc Natl Acad Sci USA 95: 7760-7765.[Abstract/Free Full Text]
Roush ED and Kwatra MM (1998) Human substance P receptor expressed in Chinese hamster ovary cells directly activates Gq/11, GS, and GO. FEBS Lett 428: 291-294.
Sabatini BL and Svoboda K (2000) Analysis of calcium channels in single spines using optical fluctuation analysis. Nature (Lond) 408: 589-593.
Saegusa H, Kurihara T, Zong S, Minowa O, Kazuno A, Han W, Matsuda Y, Yamanaka H, Osanai M, Noda T, et al. (2000) Altered pain responses in mice lacking 1E subunit of the voltage-dependent Ca2+ channel. Proc Natl Acad Sci USA 97: 6132-6137.[Abstract/Free Full Text]
Scheschonka A, Dessauer CW, Sinnarajah S, Chidiac P, Shi CS, and Kehrl JH (2000) RGS3 is a GTPase-activating protein for Gi and Gq and a potent inhibitor of signaling by GTPase-deficient forms of Gq and G11. Mol Pharmacol 58: 719-728.[Abstract/Free Full Text]
Sochivko D, Pereverzev A, Smyth N, Gissel C, Schneider T, and Beck H (2002) The CaV2.3 Ca2+ channel subunit contributes to R-type Ca2+ currents in murine hippocampal and neocortical neurons. J Physiol (Lond) 542: 699-710.[Abstract/Free Full Text]
Stea A, Soong TW, and Snutch TP (1995) Determinants of PKC-dependent modulation of a family of neuronal calcium channels. Neuron 15: 929-940.
Tai C, Kuzmiski JB, and MacVicar BA (2006) Muscarinic enhancement of R-type calcium currents in hippocampal CA1 pyramidal neurons. J Neurosci 26: 6249-6258.[Abstract/Free Full Text]
Tottene A, Volsen S, and Pietrobon D (2000) 1E subunits form the pore of three cerebellar R-type calcium channels with different pharmacological and permeation properties. J Neurosci 20: 171-178.[Abstract/Free Full Text]
Wu L, Bauer CS, Zhen XG, Xie C, and Yang J (2002) Dual regulation of voltage-gated calcium channels by PtdIns(4,5)P2. Nature (Lond) 419: 947-952.
Wu LG, Borst JG, and Sakmann B (1998) R-type Ca2+ currents evoke transmitter release at a rat central synapse. Proc Natl Acad Sci USA 95: 4720-4725.[Abstract/Free Full Text]
Yasuda R, Sabatini BL, and Svoboda K (2003) Plasticity of calcium channels in dendritic spines. Nat Neurosci 6: 948-955.
Yokoyama K, Kurihara T, Saegusa H, Zong S, Makita K, and Tanabe T (2004) Blocking the R-type (CaV2.3) Ca2+ channel enhanced morphine analgesia and reduced morphine tolerance. Eur J Neurosci 20: 3516-3519.
Yu B and Shinnick-Gallagher P (1998) Corticotropin-releasing factor increases dihydropyridine- and neurotoxin-resistant calcium currents in neurons of the central amygdala. J Pharmacol Exp Ther 284: 170-179.[Abstract/Free Full Text]
Zhang JF, Randall AD, Ellinor PT, Horne WA, Sather WA, Tanabe T, Schwarz TL, and Tsien RW (1993) Distinctive pharmacology and kinetics of cloned neuronal Ca2+ channels and their possible counterparts in mammalian CNS neurons. Neuropharmacology 32: 1075-1088.
作者单位：Department of Physiology and Pharmacology, Universidad Autónoma de San Luis Potosí, San Luis Potosí, México (U.M.); and Department of Biology, Utah State University, Logan, Utah (A.T., R.A.B., B.A.A.)