Down-regulation of Voltage-gated Ca2+ Channels by Neuronal Calcium Sensor-1 Is beta  Subunit-specific*

Matthieu RoussetDagger §, Thierry CensDagger , Sophie GavariniDagger , Andreas Jeromin, and Pierre CharnetDagger ||

From the Dagger  Centre de Recherche de Biochimie Macromoléculaire, CNRS Unité Propre de Recherche 1086, Institut Federatif de Recherche 24, 1919 Route de Mende, 34293 Montpellier, France and  Mt. Sinai Hospital, SLRI-860, Toronto, Ontario M5G 1X5, Canada

Received for publication, September 17, 2002, and in revised form, December 16, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Neuronal Ca2+ sensor protein-1 (NCS-1) is a member of the Ca2+ binding protein family, with three functional Ca2+ binding EF-hands and an N-terminal myristoylation site. NCS-1 is expressed in brain and heart during embryonic and postnatal development. In neurons, NCS-1 facilitates neurotransmitter release, but both inhibition and facilitation of the Ca2+ current amplitude have been reported. In heart, NCS-1 co-immunoprecipitates with K+ channels and modulates their activity, but the potential effects of NCS-1 on cardiac Ca2+ channels have not been investigated. To directly assess the effect of NCS-1 on the various types of Ca2+ channels we have co-expressed NCS-1 in Xenopus oocytes, with CaV1.2, CaV2.1, and CaV2.2 Ca2+ channels, using various subunit combinations. The major effect of NCS-1 was to decrease Ca2+ current amplitude, recorded with the three different types of alpha 1 subunit. When expressed with CaV2.1, the depression of Ca2+ current amplitude induced by NCS-1 was dependent upon the identity of the beta  subunit expressed, with no block recorded without beta  subunit or with the beta 3 subunit. Current-voltage and inactivation curves were also slightly modified and displayed a different specificity toward the beta  subunits. Taken together, these data suggest that NCS-1 is able to modulate cardiac and neuronal voltage-gated Ca2+ channels in a beta  subunit specific manner.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ca2+ entry through voltage-gated Ca2+ channels is essential for various cellular processes that include muscle contraction, pacemaker activity, synaptic transmission, or gene expression. Several types of Ca2+ channels have been characterized (T, L, N, P/Q, and R) that appear to play a specific role in each of these functions. These channels share a common architecture composed of a major alpha 1 subunit (for which ten genes are known) tightly associated with regulatory subunits alpha 2-delta (four different genes), beta  (four genes), and possibly gamma  (eight genes) in a functional multimeric complex (1-4). This molecular diversity, further expanded by the existence of several splice variants for each of these genes, produces a large number of possible Ca2+ channel subunit combinations with different pharmacological and biophysical properties and specific cellular and subcellular localization (5). The precise regulation of the Ca2+ influx in response to various physiological situations is further controlled by several regulatory mechanisms, working at different levels, including channel expression, localization, or activity, via additional interactions with modulatory proteins. Several Ca2+-dependent feedback mechanisms sense incoming Ca2+ ions to finely tune channel activity to the cellular Ca2+ demands and prevent cytotoxic Ca2+ overload (6). These mechanisms use Ca2+-sensing proteins and are specific of a given type of Ca2+ channel. It has been shown, for example, that Ca2+-dependent inactivation of the L-type Ca2+ channel (encoded by the CaV1.2 alpha 1 subunit) is governed by a Ca2+-driven interaction between calmodulin and the C-terminal tail of the channel alpha 1 subunit (7-10). A similar functional interaction also appears to exist on the P/Q-type Ca2+ channel (encoded by the CaV2.1 subunit), the major channel type involved in synaptic transmission in the mammalian central nervous system (11-13).

The neuronal Ca2+ sensor protein-1 (NCS-1),1 the mammalian homologue of frequenin, belongs to a group of small Ca2+-binding proteins comprising four EF-hand motifs, three of which are able to bind Ca2+ in the micromolar (EF-2) or submicromolar (EF-3,4) range (14-16). NCS-1 also contains an N-terminal myristoylation site (17). NCS-1 has been shown to facilitate synapse formation, spontaneous and/or evoked neurotransmitter release, paired-pulse facilitation, and exocytosis in several cell types (18-22). NCS-1 interacts directly with phosphatidylinositol 4-kinase in yeast (23), COS-7 (24), and chromaffin cells (25), leading to the hypothesis that part of the effects mediated by NCS-1 could involve modifications in cellular trafficking through regulation of the phosphatidylinositol signaling pathway, thereby affecting vesicular transport and recycling, as well as inositol 1,4,5-trisphosphate-sensitive Ca2+ stores (24, 25).

However, a possible involvement of NCS-1 in expression and regulation of voltage-gated Ca2+ channels has also been proposed. Overexpression of a dominant-negative mutant of NCS-1, which displays impaired Ca2+-dependent conformational changes (19), or direct loading of presynaptic nerve terminals with NCS-1 suggested that voltage-independent inhibition, as well as activity-dependent facilitation of P/Q-type Ca2+ channels (CaV2.1), could be controlled by NCS-1, possibly via direct protein-protein interactions (18). Effects on N-type Ca2+ channel (CaV2.2) properties have also been reported (20), and the expression of NCS-1 in mammalian cardiac myocytes and subsequent effect on K+ channel expression (26) gave rise to the possibility that NCS-1 may regulate multiple types of Ca2+ channels and other voltage-dependent ion channels, not only in neurons (27).

In a first step to explore this possibility, we have co-expressed NCS-1 with three different types of Ca2+ channel, CaV1.2, CaV2.1, and CaV2.2, associated with different combinations of auxiliary beta  subunit, and measured the resulting Ba2+ and Ca2+ currents. These combinations are likely to be expressed in different cell types where they represent potential targets for NCS-1 effects. Our goal was to explore the effect of NCS-1 on both Ca2+ channel expression and properties and to provide a first description of the molecular requirements necessary for NCS-1 effects on Ca2+ channel that may help in the understanding of the precise mode of action of this Ca2+-binding protein. We have, however, focused this study on the P/Q-type (CaV2.1) Ca2+ channels, which seem to be a primary target in various cell types (28). Our results show that NCS-1 down-regulates expression of L-, N-, and P/Q-type Ca2+ channels in a beta  subunit-specific manner and induces minor modifications of the electrophysiological properties of the channel. We provide evidence of direct functional effects of NCS-1, in addition to modifications in the expression level and/or trafficking of the channels to the membrane.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Oocyte Preparation-- The following cDNA were used, and the GenBankTM accession number is provided: CaV1.2 (alpha 1C), M67515; CaV2.1 (alpha 1A), M64373; CaV2.2 (alpha 1B), D14157; NCS-1, L27421; beta 1b, X61394; beta 2a, M80545; beta 3, M88751; beta 4, L02315; and alpha 2-delta 1, M86621. Mutations NCS-1E120Q and NCS-1G2A have been described previously (29). Ca2+ channel subunits were subcloned into the pmt2 vector, whereas NCS-1 and its mutants were subcloned into pcDNA3 (Invitrogen).

Xenopus laevis oocyte preparation and injection were performed as described previously (30). Each oocyte was injected with 5-10 nl of a cDNA mixture containing the alpha 1+alpha 2delta +beta +NCS-1 cDNAs at ~0.3 ng/nl with a ratio of 1:2:3:1. When one or more of these cDNAs was omitted, cDNA concentrations were kept constant by addition of the appropriate volume of deionized water. Oocytes were kept for 2 to 4 days before recordings at 18 °C and under gentle agitation.

Electrophysiology-- Whole-cell Ba2+ currents were recorded under two-electrode voltage clamp using the GeneClamp 500 amplifier (Axon Instruments, Union City, CA). Current and voltage electrodes (less than 1 megohm) were filled with 3 M KCl, pH 7.2, with KOH. Ba2+ and Ca2+ current recordings were performed after injection of BAPTA (~50 nl of the following (in mM): BAPTA-free acid (Sigma), 100; CsOH, 10; HEPES, 10; pH 7.2 with CsOH) using the following bathing solution (in mM): BaOH/CaOH, 10; TEAOH, 20; NMDG, 50; CsOH, 2; HEPES, 10; pH 7.2, with methanesulfonic acid. Currents were filtered and digitized using a DMA-Tecmar Labmaster and subsequently stored on a Pentium-based personal computer using the pClamp software (version 6.02; Axon Instruments). Ba2+ or Ca2+ currents were recorded during a 400-ms test pulse from -80 to +10 mV. Current amplitudes were measured at the peak of the current. Comparisons of averaged amplitudes between batches were always made with amplitudes measured the same day after injection. Comparisons between similar experiments were made by normalizing all averaged amplitudes with respect to the control current amplitude set as 100%. Isochronal steady-state inactivation curves (2.5 s of conditioning voltage followed by a 400-ms test pulse to +10 mV) were fitted using the equation, I/Imax = Rin + (1 - Rin)/(1 + exp((V - Vin)/k)), where I is the current amplitude measured during the test pulse at +10 mV for conditioning voltages varying from -80 to +50 mV, Imax is the current amplitude measured during the test pulse for a conditioning voltage to -80 mV, Vin is the potential for half-inactivation, V is the voltage, k is the slope factor, and Rin is the proportion of non-inactivating current. Current to voltage curves were fitted using the equation, I/Imax = G*(V - Erev)/(1 + exp((V - Vact)/k)), where I is the current amplitude measured during voltage steps varying from -80 to +50 mV, Imax is the peak current amplitude measured at the minimum of the current-voltage curve, G is the normalized macroscopic conductance, Erev is the apparent reversal potential, Vact is the potential for half-activation, V is the value of the voltage step, and k is a slope factor.

Inactivation kinetics were estimated by fitting Ba2+ current decay with two exponential components using the equation, I(t) A1e(-t/tau 1) + A2e(-t/tau 2) + C, where I is the current amplitude, t is the time, tau 1, tau 2, A1, and A2 represent the time constants and amplitudes of the two components, and C is a constant. The proportion of the slow time constant (%tau 2) is the ratio A2/(A1+A2).

Several independent experiments (N, number of batches of oocytes injected) were performed, always including a control group without NCS-1 expressed. These experiments always gave the same statistical result, and the total number of recordings (from n oocytes of these N experiments) are thus presented. All values are presented as mean ± S.E., and comparison between groups of oocytes were evaluated using a Student's t test, with a statistical significance set at the p value <0.05.

Biochemistry-- Selected oocytes were first homogenized in the lysis buffer (5 µl/oocyte of 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 50 mM NaF, 10 mM beta -glycerophosphate, and 5 mM Na4P2O7) and centrifuged at 4 °C and 14000 rpm for 5 min. The upper aqueous phase was collected and subjected to 10% (see Fig. 2C) or 15% SDS-polyacrylamide gel electrophoresis (loaded with ~3 oocytes/lane). Proteins were electrotransferred to nitrocellulose filters. For immunological detection, the blots were first blocked for 1 h with 8% non-fat milk powder in TBST (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.1% Tween 20), incubated overnight at 4 °C with anti-NCS-1 (Zymol at 1/1000 dilution in 0.1% bovine serum albumin in TBST) or anti-CaV2.1 antibodies (Alomone Laboratories Jerusalem, Israel), and after five washes, incubated with an anti-rabbit antibody coupled to horseradish peroxidase. Antibody binding was detected by chemiluminescence (PerkinElmer Life Sciences; 1/10000 in 0.1% bovine serum albumin, 0.8% nonfat milk powder in TBST). Correct expression of NCS-1 was always checked after electrophysiological recordings.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of NCS-1 in XenopusOocytes-- Previous works on chromaffin, human embryonic kidney 293, and COS-7 cells have shown that NCS-1 was endogenously expressed at non-negligible levels (24, 28).2 In non-injected X. laevis oocytes, the level of expression of the endogenous NCS-1 was barely detectable in Western-blots and much lower than in human embryonic kidney 293 and COS-7 cells (see Fig. 1A). Thus X. laevis oocytes are a system of choice to study the functional effect of NCS-1 on voltage-gated Ca2+ channels. In these oocytes, injection of the cDNA coding for rat NCS-1 led, as expected, to a massive expression of a protein of a molecular mass of ~23 kDa, in accordance with the theoretical molecular mass of NCS-1 (21.9 kDa; see Fig. 1B). In the following experiments, the oocytes used for current recordings were collected after recordings and submitted to a similar Western blot analysis to ensure that the NCS-1 protein was properly expressed.


View larger version (47K):
[in this window]
[in a new window]
 
Fig. 1.   Non-injected oocytes do not express detectable levels of NCS-1. A, Western blot showing the low level of endogenous NCS-1 protein expressed in non-injected oocytes (lane 1) compared with COS-7 or human embryonic kidney 293 cells (lanes 2 and 3, respectively). B, injection of pcDNA3.1 plasmid encoding NCS-1 led, in 2 to 4 days, to the apparition of a single NCS-1 immunoreactive band of ~23 kDa of molecular mass corresponding to overexpression of NCS-1 (lane 1). Lane 2, control, non-injected oocytes. Each lane was loaded with ~3 oocytes. Numbers on the right show the positions of the molecular mass markers.

Down-regulation of CaV1.2, CaV2.1, and CaV2.2 Expression by NCS-1-- In a first set of experiments, the effects of NCS-1 were tested on Ba2+ currents flowing through L-, N-, and P/Q-type Ca2+ channels. For each Ca2+ channel type, this was done by co-injecting a mixture of cDNA containing beta 2, alpha 2-delta , and the appropriate alpha 1 Ca2+ channel subunits (CaV1.2, CaV2.2, or CaV2.1, respectively) with either NCS-1 cDNA or water into two different batches of oocytes. After 2 to 4 days of incubation, Ba2+ current amplitudes were recorded from the two batches of oocytes injected the same day, during a single 400-ms-long depolarizing step to +10 mV from a holding potential of -80 mV and compared. Under these conditions, a clear decrease in the averaged Ba2+ current amplitude was seen upon co-expression of NCS-1 (see Fig. 2A). This effect was most pronounced in oocytes expressing the CaV2.2 Ca2+ channel, where the averaged Ba2+ current amplitude recorded in the batch of oocytes co-injected with rat NCS-1 cDNA (N = 1 experiment, n = 38 oocytes) was only 10% of the control current amplitude recorded from oocytes co-injected with H2O instead of the NCS-1 cDNA (N = 1, n = 35). However, a similar effect was also found when NCS-1 was co-injected with CaV1.2 (N = 3, n = 71; 52% of the control amplitude n = 70) or CaV2.1 Ca2+ channel subunits (N = 7, n = 206; 46% of the control amplitude n = 162).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of NCS-1 on CaV1.2, CaV2.1, and CaV2.2 current amplitudes. A, NCS-1 reduced Ba2+ currents through L-, P/Q-, and N-type Ca2+ channels. Histogram showing the effects of NCS-1 on Ba2+ current amplitude recorded in oocytes injected with the alpha 2-delta , the beta 2, and CaV1.2, CaV2.1, or CaV2.2 Ca2+ channel subunits, with or without NCS-1 cDNA, are shown. Peak Ba2+ currents were recorded 2-4 days after injection during a test pulse to +10 mV from a holding potential of -80 mV and expressed as % of control (averaged current amplitude recorded with the same subunit combination but without NCS-1 expressed). *, significantly different from control (p < 0.05); n.s., not significantly different. B, histogram showing the effects of NCS-1 on Ba2+ current amplitude recorded on oocytes injected with the CaV2.1, the alpha 2-delta , and the beta 1, beta 2, beta 3, beta 4, or no beta  Ca2+ channel subunits, with or without NCS-1 cDNA. Peak Ba2+ currents were recorded 2-5 days after injection during a test pulse to +10 mV from a holding potential of -80 mV. *, significantly different from control (p < 0.05); n.s., not significantly different. In A and B, Western blot analyses of the expression of NCS-1 in the oocytes used for the recordings are shown at the bottom. Each lane was loaded with ~3 oocytes. C, Western blot of total proteins obtained from oocytes injected with, from left to right, the CaV2.1+alpha 2-delta +beta 2 cDNAs, the CaV2.1+alpha 2-delta +beta 2+NCS-1 cDNAs, or water, probed with an anti-CaV2.1 antibody. Each lane was loaded with ~ 3oocytes. Note the presence of a specific band at 250 kDa of similar density in oocytes injected with the CaV2.1 subunit with or without NCS-1. The cross-reactive band at ~160 kDa, also found in non-injected oocytes, was used as a control of the loading charge.

The Ca2+ channel beta  subunit is an important determinant of the final Ca2+ current amplitude observed at the oocyte surface membrane and regulates many of its electrophysiological properties (4, 31, 32). Hence, we tested the role of the beta  subunit on the effects of NCS-1. Using the same experimental approach, we co-expressed NCS-1 with the CaV2.1 Ca2+ channel either with the alpha 2-delta subunit alone or with the alpha 2-delta and one of the four beta  subunits (beta 1-beta 4). Again, in each case, the resulting current amplitude was compared with the current amplitude recorded in oocytes injected with the same combination of Ca2+ channel subunits but without NCS-1. Interestingly, when NCS-1 was co-expressed with CaV2.1 without beta  subunit, almost no effect on current amplitude was observed (N = 2, n = 32 and 26 for control). A similar result was also found upon co-expression of the beta 3 subunit (N = 12, n = 198, with control n = 198), whereas co-expression of NCS-1 with the CaV2.1 and beta 1, beta 2, or beta 4 subunit decreased the expressed Ba2+ current amplitude to between 25 and 45% of their respective control values (see Fig. 2B, N = 3, 7, 2 and n = 100, 206, 71 and n = 65, 162, 84 for controls, respectively). The lack of effect of NCS-1 on the CaV2.1 Ca2+ channel subunit, expressed alone or with the beta 3 subunit, could not be attributed to a deficit of NCS-1 protein in these oocytes, because a robust expression of NCS-1 was also detected in these oocytes by Western blot (see bottom of Fig. 2B). On the other hand, to ensure that the decrease of the Ba2+ current amplitude obtained upon co-expression of NCS-1 with the beta 1, beta 2, or beta 4 subunit was not because of a deleterious effect on the expression of any cloned protein, we analyzed the level of expression of the CaV2.1 protein in these conditions by Western blot. Fig. 2C demonstrates that a clear band of an approximate molecular mass of 250 kDa, matching of the theoretical molecular mass of the CaV2.1 subunit (251 kDa), could be specifically detected only in oocytes injected with the CaV2.1 subunit cDNA (+alpha 2-delta +beta 2 subunits; see Fig. 2C, left lane). In oocytes co-injected with NCS-1 cDNA and the same combinations of Ca2+ channel subunits, this CaV2.1 immunoreactive band was also present at similar intensity (Fig. 2C, middle lane, each lane approximately loaded with three oocytes), whereas it was absent in non-injected oocytes (Fig. 2C, right lane). These results therefore revealed a true, beta  subunit-specific effect of NCS-1 on the Ba2+ current amplitude that cannot be attributed to nonspecific down-regulation of protein expression.

Effect of NCS-1 on CaV2.1 Properties-- We next investigated whether co-expression of NCS-1 with the CaV2.1 subunit might have other functional consequences on Ca2+ channel properties. Current-voltage curves and isochronal inactivation curves were constructed from currents recorded in oocytes expressing the CaV2.1 subunit with the alpha 2-delta alone or with the beta 1, beta 2, or beta 3 subunits, in the presence or absence of NCS-1. These recordings were performed using either Ba2+ or Ca2+ ions as extracellular permeant cations to track any Ca2+-specific modulation.

As presented in Table I, in the presence of Ca2+ ions and in the absence of beta  subunit (alpha 1A+alpha 2-delta subunits), no modifications in the activation and inactivation parameters were observed upon co-expression of NCS-1. This lack of effect was also noted upon co-expression of the beta 1 or beta 3 subunit (see Fig. 3 and Table I). Interestingly, when co-expressed with CaV2.1 and the beta 2a subunit, NCS-1 significantly depolarized the current-voltage curve (Vact -5.0 and -0.8 mV without and with NCS-1 respectively, p < 0.05) and reduced inactivation (Rin = 69 and 56% respectively, p < 0.05). Similar effects were also found in the presence of extracellular Ba2+ (Table II) and thus were not Ca2+-dependent.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Effects of NCS-1 on Cav2.1 Ca2+ currents
Current-voltage and inactivation curve parameters calculated from oocytes expressing the indicated combination of subunits, in the presence of 10 mM external Ca2+. See "Experimental Procedures" for details.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of NCS-1 on the functional properties of CaV2.1. Normalized current-voltage and inactivation curves recorded in oocytes injected with the CaV2.1 alpha 1A+alpha 2delta +beta 1 (A) or alpha 1A+alpha 2delta +beta 2 (B) Ca2+ channel subunits with (open circle) or without (open square) NCS-1 are shown. Recordings were made using 10 mM extracellular Ca2+. NCS-1 induced a significant depolarization of the voltage for activation (marked by an asterisk on the bottom of the current-voltage curve; see "Experimental Procedures" and Table I) and decreased the residual current (marked by an asterisk on the inactivation curve). This effect was only seen with the beta 2 subunit.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Effects of NCS-1 on Cav2.1 Ba2+ currents
Current-voltage and inactivation curve parameters calculated from oocytes expressing the indicated combination of subunits, in the presence of 10 mM external Ba2+. See "Experimental Procedures" for details.

We then analyzed the effects of NCS-1 on Ba2+ current kinetics. CaV2.1 current inactivation could be approximated by an exponential decaying phase, best described using a fast (tau 1) and a slow (tau 2) component. None of these components appeared to be significantly affected by expression of the Ca2+-binding protein NCS-1 (Fig. 4). This lack of effect was found for channels co-expressed with the beta 1 or the beta 2 subunit and in the presence of either extracellular Ba2+ or Ca2+. Neither the time constants (tau 1 and tau 2) nor their respective amplitude (%tau 2) were changed at all voltages examined (see Fig. 4). Moreover, no effect on channel activation and reactivation were observed upon co-expression of NCS-1, whether the CaV2.1 subunit was expressed with the alpha 2-delta alone or with any of the four beta  subunits (not shown). Therefore, although both channel expression and channel properties seemed to be regulated by NCS-1 in a beta  subunit-specific manner, they appear to require different subunit arrangements, i.e. expression was modified when beta 1, beta 2, or beta 4 subunits were expressed, whereas modifications in channel properties were only recorded in the presence of the beta 2 subunit.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Inactivation kinetics of CaV2.1 are marginally modified by NCS-1. Voltage dependence of the fast (tau 1) and slow (tau 2) time constants of inactivation and proportion of the slow time constant (%tau 2) of CaV2.1 channels co-expressed with alpha 2-delta and beta 1 (A) or beta 2 (B) subunits with (open circle) or without NCS-1 (open square) is shown. Currents were recorded in Ba2+, during depolarizations of 2.5 s. NCS-1 did not statistically modify (p > 0.05) Ba2+ current kinetics at all voltages tested. Current traces are shown on top.

To get some insight into the possible molecular determinants involved in NCS-1 effects, the same experiments, with the beta 2a subunit, were conducted using two mutants of NCS-1. NCS-1E120Q, with its third EF-hand disrupted, showed impaired Ca2+-dependent conformational changes (19) but was still able to bind cellular proteins. NCS-1G2A, a myristoylation-deficient mutant of NCS-1, relocalized NCS-1 from the perinuclear region to the cytosol (29).

Co-expression of either NCS-1E120Q or NCS-1G2A had the same effect as wild-type NCS-1 on the current-voltage curve of the CaV2.1+alpha 2-delta +beta 2 Ca2+ channel (i.e. a small but significant positive shift of ~5 mV; see Fig. 5B and Table III). They differed, however, in their effects on the inactivation curve. Although the NCS-1E120Q completely suppressed the effect of NCS-1 on the residual current (Rin; see Fig. 5 and Table III), co-expression of NCS-1G2A left this parameter unchanged but suppressed the shift in Vin induced by wild-type NCS-1. These two mutants also had different actions on current amplitudes. Indeed, whereas NCS-1E120Q reduced the Ba2+ current amplitude when compared with control currents, recorded the same day in oocytes not injected with NCS-1, NCS-1G2A had no consistent effect, although a small, albeit not significant, reduction could be noted (see Fig. 5A). In the case of NCS-1E120Q, however, this effect was less marked than when recorded in oocytes co-injected with the wild-type NCS-1, suggesting that both Ca2+-dependent conformational changes, and possibly myristoylation, participated to the observed effects. It should be noted that none of these mutants had any effect on the functional properties of the CaV2.1+alpha 2-delta Ca2+ channels, expressed alone or with the beta 1 or beta 3 subunits, whereas the effects on current amplitude were reproduced with the beta 1 subunit (data not shown).


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of NCS-1E120Q and NCS-1G2A on CaV2.1 expression and properties. A, effects of wild-type NCS-1, NCS-1E120Q, and NCS-1G2A mutants on CaV2.1 channel expression. Oocytes were injected with CaV2.1+alpha 2-delta +beta 2a subunit cDNAs with or without the appropriate NCS-1 mutants, and currents were measured in 10 mM Ba2+ during a test depolarization to +10 mV, as described for Fig. 2. Note that NCS-1 and NCS-1E120Q both decreased current amplitude significantly (p < 0.05). B, oocytes were injected with CaV2.1+alpha 2-delta +beta 2a subunit cDNAs with (open circle) or without (open square; Control) the appropriate NCS-1 mutants, NCS-1E120Q in the left panel and NCS-1G2A in the right panel. Currents were measured in 10 mM extracellular Ba2+ as described for Fig. 3. Both mutations slightly shifted the current-voltage curve in the positive direction, but only NCS-1E120Q seemed to be able to reverse the decrease in the residual current (Rin; see Table III for Vact, Vin, and Rin values).

                              
View this table:
[in this window]
[in a new window]
 
Table III
Effects of mutated NCS-1 on Cav2.1 Ba2+ currents
Current-voltage and inactivation curve parameters calculated from oocytes expressing the indicated combination of subunits, in the presence of 10 mM external Ba2+. See "Experimental Procedures" for details.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recent studies have extended the area of expression of NCS-1 from the nervous system to neuroendocrine cells and even cardiac myocytes (21, 26, 33). In these cell types, NCS-1 modulates synaptic transmission (14, 22, 34), secretion (21, 25), or cellular excitability (18, 20) via complex processes that include regulation of key enzymes for membrane transport (15, 17, 23, 24, 35) and specific regulation of various ion channels (16, 18-20, 28). K+ channels were the first ion channel target to be characterized in expression systems (26, 36). Co-expression studies described an up-regulation of channel expression and activity, specifically recorded with the KV4 K+ channel family (26, 36). Modifications in the expression and properties of other ion channels have also been reported. In endocrine cells and neurons P/Q-type and N-type Ca2+ channels are clearly affected (18-20, 28), but no effect on L-type channels has been reported so far (20). These results suggest the existence of specific effects among the different voltage-activated Ca2+ channel types or among different tissues. However, the molecular basis of this specificity, and in particular the role of the subunit composition of the channel, in the observed effects remains unknown.

In the present work, using heterologous expression of defined Ca2+ channel subunits, we show that NCS-1 has two major effects on high voltage-activated Ca2+ channels: (1) a decrease in the current amplitude that is beta  subunit-specific but observed with CaV1.2, CaV2.1, and CaV2.2 Ca2+ channels; and (2) a small modification in the activation and inactivation parameters of the CaV2.1, only seen when the alpha 1 subunit is expressed with the beta 2a subunit.

Effect of NCS-1 on Ca2+ Current Amplitude-- Effects of NCS-1 on P/Q- and N-type Ca2+ channel current amplitudes have been documented over the past years but always based on experiments using either dominant negative mutants or overexpression of wild-type NCS-1 in native cells constitutively expressing the protein (18-20). Our study is therefore the first to analyze the effects of NCS-1 on multiple types of Ca2+ channel in the same environment, using heterologous expression of defined Ca2+ channel subunits. In neurons, NCS-1 has been shown to clearly increase N-type Ca2+ channel expression (20), with significant modifications of the electrophysiological parameters. The opposite effects have been found on P/Q-type Ca2+ channels in bovine chromaffin cells, where a dominant negative mutant of NCS-1 increased Ca2+ current amplitude (19) without effect on L-type Ca2+ channels. Using cDNA injection, we show that all three channel types are down-regulated by co-expression of NCS-1. Our experimental conditions and our analysis of the expression level of the CaV2.1 subunit (Fig. 2C) suggest that this effect must take place after protein synthesis and could involve modifications in the correct folding and trafficking of the channel complex to the plasma membrane and/or regulation of the channel activity per se, taking place after insertion of the channel into the plasma membrane. Membrane expression of high voltage-activated Ca2+ channels is known to rely on a short sequence located within the intracellular loop connecting the homologous domains I and II of the alpha 1 subunit (37). This sequence, located close to the beta  subunit binding site (AID), acts as a retention signal in the endoplasmic reticulum and thus reduces trafficking of the alpha 1 subunit to the membrane, just like a surface expression brake. This brake is usually removed by the alpha 1/beta subunit association, which occurs in the reticulum. The fact that the decrease in current amplitude induced by NCS-1 was only observed when beta 1, beta 2, or beta 4 subunits were expressed suggests that NCS-1 may interfere with this mechanism. A similar effect on channel expression has been recently reported (38) for the small GTPase Kir/gem, which binds directly to the beta  subunit, thus preventing the alpha 1/beta association and restoring the expression brake imposed by the retention signal. Direct specific binding of NCS-1 to this site and/or to the beta  subunit is thus an attractive hypothesis to explain the reduction of the current amplitude but needs to be further explored by additional experiments designed to directly test the biochemical interactions between these subunits. Such a mechanism, however, may not be exclusive, and other pathways, acting directly or indirectly on channel activity, such as Src-dependent inhibition (28), or direct modulation of the G-protein-coupled receptor pathways (19), may also exist. Although in our recording conditions, the Src-dependent and G-protein pathways could be discarded (the Src kinase inhibitor, PP1, had no effect; data not shown), it is worth noting that G-protein beta gamma subunits and the Ca2+ channel beta  subunit possess very close binding sites on the main alpha 1 Ca2+ channel subunit (39-41), leaving open the possibility that interactions between the alpha 1 subunit on one hand, and G-protein, Ca2+ channel beta  subunit, and NCS-1 on the other hand, could be mutually exclusive and/or under the control of tissue-specific conditions. The I-II loop would therefore acts as a cross-road for cell-specific regulations.

Interestingly, NCS-1 had no effect on CaV2.1 Ca2+ channel co-expressed with the beta 3 subunit, the beta  subunit that displays the lowest affinity for the AID (42), and which also shows the weakest potency to increase current amplitude in expression systems (43). In neurons, where the N-type Ca2+ channel is predominantly associated with the beta 3 subunit (44) the increase in vesicular transport and membrane trafficking induced by NCS-1, acting through the activation of the phosphatidylinositol 4-kinase beta  (45), may overcome the effect on the alpha 1 retention signal, poorly masked/removed by the beta 3 subunit, and lead to the observed overexpression of N-type Ca2+ channels (20). In this scenario, the tissue specificity of the effects of NCS-1 on Ca2+ channels should thus be critically dependent on the subunit composition of the channel. Whether NCS-1 acts directly or indirectly on the Ca2+ channel alpha 1 or beta  subunit requires further investigation.

The fact that mutants that prevented Ca2+ binding (NCS-1E120Q) and protein myristoylation (NCS-1G2A) both decreased the effects of NCS-1 on channel expression underlines the requirement for a fully functional NCS-1 protein to record these effects. Mutation of glutamate 120 to glutamine (NCS-1E120Q mutant) disrupts a high affinity Ca2+ binding site and impairs Ca2+-dependent conformational changes (19). This mutant acts as a dominant negative mutant for the regulation of the P/Q channel in chromaffin cells (19). In Xenopus oocytes the NCS-1E120Q mutant appeared also less potent than wild-type NCS-1. However, a clear and significant decrease in current amplitude was nevertheless observed, suggesting that the mutation did not completely suppress NCS-1 activity. These differential effects constitute another argument in favor of the presence of different mechanisms working in chromaffin cells or Xenopus oocytes to regulate Ca2+ channels, i.e. removal of a voltage-independent inhibition versus down-regulation of channel trafficking, and suggest that Ca2+-dependent changes are absolutely necessary only for the removal of the voltage-independent inhibition of the P/Q channels (19, 28). Preserved interactions of NCS-1E120Q with cellular proteins can be an argument for the dominant negative effect of the mutant, as suggested (19), but can also constitute an interesting area of investigation to identify preserved interactions that may be still functional and involved in a Ca2+-independent down-regulation of the channel activity.

Mutation of the myristoylation site, NCS-1G2A, is known to affect subcellular localization of NCS-1 (29), with minor modifications in the protein structure (46). The tendency of the NCS-1G2A mutation to decrease Ca2+ current amplitude was not found statistically significant. This may simply reflect a small decrease in the availability of the NCS-1G2A mutant at early stages of protein assembly, because of the loss of the perinuclear localization (29) or because of more indirect effects, related to mutation-induced modifications in the degree of cooperativity in Ca2+ binding (46). The construction and testing of the double mutant NCS-1G2A/E120Q should help to solve this issue.

Effect of NCS-1 on Ca2+ Current Properties-- Beyond the decrease in current amplitude, we also noted that NCS-1 could specifically affect the electrophysiological properties of the CaV2.1 channel, but only when co-expressed with the beta 2a subunit. Increased inactivation and a positive shift of the current-voltage curve were the most significant changes. These effects were seen in the presence of extracellular Ba2+ or Ca2+ and therefore, in our conditions, did not seem to be Ca2+-dependent. The fact that the decrease in current amplitude and the modifications in the channel properties did not have the same beta  subunit specificity suggested different underlying mechanisms. Direct interactions between Ca2+ channels and NCS-1 at the plasma membrane have not been reported so far, but the presence of NCS-1 in synaptic-like microvesicles and its co-localization in presynaptic terminals with the proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex, VAMP (45) and syntaxin (18), also known to interact with P/Q Ca2+ channels (1), suggest that NCS-1 is appropriately located for these potential functional interactions. Indeed, a previously published work (18) reported an enhancement of the facilitation of the P/Q-type Ca2+ channel in the Calyx of Held, occurring via an acceleration of current activation. A similar frequency-dependent facilitation was also apparent in oocytes (data not shown), but the large membrane capacitance of the oocytes did not allow a precise analysis of the modifications in the activation kinetics. Interestingly, in the Calyx of Held, these effects were recorded during acute perfusion of NCS-1 and could be occluded by perfusion of a C-terminal peptide of NCS-1, suggesting direct interactions with the channel (18). A large hydrophobic crevice in this region has been proposed as a potential site for target recognition (15).

Alternatively, it has also been proposed that NCS-1 could activate the same targets as calmodulin (35), a regulatory element of the CaV1.2 and CaV2.1 Ca2+ channels, responsible for the Ca2+ sensitivity of inactivation and facilitation (10, 12, 13, 47, 48). The binding site for calmodulin on these channels is positioned on their intracellular C-terminal tails and is constituted of multiple, Ca2+-dependent, or constitutive microsites. The same sites can accommodate CABP1, another Ca2+-binding protein, which is able to displace calmodulin from its sites, to induce a Ca2+-independent positive shift of the current-voltage curve accompanied by an acceleration of the inactivation kinetics (11). These effects are reminiscent of the action of NCS-1 on Ca2+ channels in Xenopus oocytes. However, several questions need to be answered before one can speculate that NCS-1 and CABP1 share the same regulatory pathway. For example, CABP1 has only been tested on CaV2.1 channels containing the beta 2a subunit, and we have no information, for the moment, on its beta  subunit specificity.

In conclusion, we demonstrate here that NCS-1-dependent regulation of high threshold Ca2+ channels includes modifications in the current amplitude and the electrophysiological properties. These modifications are specifically modulated by the beta  subunit of the Ca2+ channel. Our results, together with previously published work on NCS-1, CABP1, calmodulin, and Ca2+ channels, suggest that these effects could take place at two sites on the alpha 1 subunit known for their regulatory role, the I-II loop and the C-terminal tail of the channel. These sites also interact with the Ca2+ channel beta  subunit and the Gbeta gamma subunits of the G-protein suggesting that regulation via G-protein-coupled receptors may also be affected. These results provide a possible molecular explanation for the tissue and Ca2+ channel type specificity of NCS-1, which can be tested biochemically and pharmacologically. They also suggest that down-regulation of Ca2+ channel activity may not be restricted to neuronal or neuroendocrine cells but may also take place in cardiac myocytes, where NCS-1 and the L-type Ca2+ channels are expressed (26).

    ACKNOWLEDGEMENTS

We thank Drs. T. P. Snutch, Y. Mori, and E. Perez-Reyes for Ca2+ channel cDNAs, Dr. I. Lefevre, M. Franco, E. Mandart, S. Estrach, and T. Lorca for helpful discussion during the course of these experiments, and J. M. Donnay and A. Bernet for help with the preparation of oocytes.

    FOOTNOTES

* This work was supported by Association pour la Recherche contre le Cancer, the Association Française contre les Myopathies, and Ligue Nationale contre le Cancer (Comité Pyrénées Orientales).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of a doctoral fellowship from the French Ministry for Research and Education.

|| To whom correspondence should be addressed. Tel.: 33-467-613-352; Fax: 33-467-521-559; E-mail: charnet@crbm.cnrs-mop.fr.

Published, JBC Papers in Press, December 19, 2002, DOI 10.1074/jbc.M209537200

2 A. Jeromin, unpublished observations.

    ABBREVIATIONS

The abbreviation used is: NCS-1, neuronal Ca2+ sensor protein-1.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Catterall, W. A. (2000) Annu. Rev. Cell Dev. Biol. 16, 521-555[CrossRef][Medline] [Order article via Infotrieve]
2. Hanlon, M. R., and Wallace, B. A. (2002) Biochemistry 41, 2886-2894[CrossRef][Medline] [Order article via Infotrieve]
3. Hofmann, F., Lacinova, L., and Klugbauer, N. (1999) Rev. Physiol Biochem. Pharmacol. 139, 33-87[Medline] [Order article via Infotrieve]
4. Birnbaumer, L., Qin, N., Olcese, R., Tareilus, E., Platano, D., Costantin, J., and Stefani, E. (1998) J. Bioenerg. Biomembr. 30, 357-375[CrossRef][Medline] [Order article via Infotrieve]
5. Felix, R. (1999) Receptors. Channels 6, 351-362[Medline] [Order article via Infotrieve]
6. Budde, T., Meuth, S., and Pape, H. C. (2002) Nat. Rev. Neurosci 3, 873-883[CrossRef][Medline] [Order article via Infotrieve]
7. Peterson, B. Z., Lee, J. S., Mulle, J. G., Wang, Y., de Leon, M., and Yue, D. T. (2000) Biophys. J. 78, 1906-1920[Abstract/Free Full Text]
8. Romanin, C., Gamsjaeger, R., Kahr, H., Schaufler, D., Carlson, O., Abernethy, D. R., and Soldatov, N. M. (2000) FEBS Lett. 487, 301-306[CrossRef][Medline] [Order article via Infotrieve]
9. Peterson, B. Z., DeMaria, C. D., Adelman, J. P., and Yue, D. T. (1999) Neuron 22, 549-558[Medline] [Order article via Infotrieve]
10. Zuhlke, R. D., Pitt, G. S., Deisseroth, K., Tsien, R. W., and Reuter, H. (1999) Nature 399, 159-162[CrossRef][Medline] [Order article via Infotrieve]
11. Lee, A., Westenbroek, R. E., Haeseleer, F., Palczewski, K., Scheuer, T., and Catterall, W. A. (2002) Nat. Neurosci 5, 210-217[CrossRef][Medline] [Order article via Infotrieve]
12. DeMaria, C. D., Soong, T. W., Alseikhan, B. A., Alvania, R. S., and Yue, D. T. (2001) Nature 411, 484-489[CrossRef][Medline] [Order article via Infotrieve]
13. Lee, A., Wong, S. T., Gallagher, D., Li, B., Storm, D. R., Scheuer, T., and Catterall, W. A. (1999) Nature 399, 155-159[CrossRef][Medline] [Order article via Infotrieve]
14. Olafsson, P., Wang, T., and Lu, B. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8001-8005[Abstract]
15. Bourne, Y., Dannenberg, J., Pollmann, V., Marchot, P., and Pongs, O. (2001) J. Biol. Chem. 276, 11949-11955[Abstract/Free Full Text]
16. Burgoyne, R. D., and Weiss, J. L. (2001) Biochem. J. 353, 1-12[CrossRef][Medline] [Order article via Infotrieve]
17. McFerran, B. W., Weiss, J. L., and Burgoyne, R. D. (1999) J. Biol. Chem. 274, 30258-30265[Abstract/Free Full Text]
18. Tsujimoto, T., Jeromin, A., Saitoh, N., Roder, J. C., and Takahashi, T. (2002) Science 295, 2276-2279[Abstract/Free Full Text]
19. Weiss, J. L., Archer, D. A., and Burgoyne, R. D. (2000) J. Biol. Chem. 275, 40082-40087[Abstract/Free Full Text]
20. Wang, C. Y., Yang, F., He, X., Chow, A., Du, J., Russell, J. T., and Lu, B. (2001) Neuron 32, 99-112[Medline] [Order article via Infotrieve]
21. McFerran, B. W., Graham, M. E., and Burgoyne, R. D. (1998) J. Biol. Chem. 273, 22768-22772[Abstract/Free Full Text]
22. Chen, X. L., Zhong, Z. G., Yokoyama, S., Bark, C., Meister, B., Berggren, P. O., Roder, J., Higashida, H., and Jeromin, A. (2001) J. Physiol 532, 649-659[Abstract/Free Full Text]
23. Hendricks, K. B., Wang, B. Q., Schnieders, E. A., and Thorner, J. (1999) Nat. Cell Biol. 1, 234-241[CrossRef][Medline] [Order article via Infotrieve]
24. Zhao, X., Varnai, P., Tuymetova, G., Balla, A., Toth, Z. E., Oker-Blom, C., Roder, J., Jeromin, A., and Balla, T. (2001) J. Biol. Chem. 276, 40183-40189[Abstract/Free Full Text]
25. Pan, C. Y., Jeromin, A., Lundstrom, K., Yoo, S. H., Roder, J., and Fox, A. P. (2002) J. Neurosci 22, 2427-2433[Abstract/Free Full Text]
26. Guo, W., Malin, S. A., Johns, D. C., Jeromin, A., and Nerbonne, J. M. (2002) J. Biol. Chem. 277, 26436-26443[Abstract/Free Full Text]
27. Weiss, J. L., and Burgoyne, R. D. (2002) Trends Neurosci. 25, 489-491[CrossRef][Medline] [Order article via Infotrieve]
28. Weiss, J. L., and Burgoyne, R. D. (2001) J. Biol. Chem. 276, 44804-44811[Abstract/Free Full Text]
29. O'Callaghan, D. W., Ivings, L., Weiss, J. L., Ashby, M. C., Tepikin, A. V., and Burgoyne, R. D. (2002) J. Biol. Chem. 277, 14227-14237[Abstract/Free Full Text]
30. Cens, T., Mangoni, M. E., Richard, S., Nargeot, J., and Charnet, P. (1996) Pflugers Arch. 431, 771-774[CrossRef][Medline] [Order article via Infotrieve]
31. Perez-Reyes, E., Castellano, A., Kim, H. S., Bertrand, P., Baggstrom, E., Lacerda, A. E., Wei, X. Y., and Birnbaumer, L. (1992) J. Biol. Chem. 267, 1792-1797[Abstract/Free Full Text]
32. Singer, D., Biel, M., Lotan, I., Flockerzi, V., Hofmann, F., and Dascal, N. (1991) Science 253, 1553-1557[Medline] [Order article via Infotrieve]
33. Paterlini, M., Revilla, V., Grant, A. L., and Wisden, W. (2000) Neuroscience 99, 205-216[CrossRef][Medline] [Order article via Infotrieve]
34. Pongs, O., Lindemeier, J., Zhu, X. R., Theil, T., Engelkamp, D., Krah-Jentgens, I., Lambrecht, H. G., Koch, K. W., Schwemer, J., and Rivosecchi, R. (1993) Neuron 11, 15-28[Medline] [Order article via Infotrieve]
35. Schaad, N. C., De, Castro, E., Nef, S., Hegi, S., Hinrichsen, R., Martone, M. E., Ellisman, M. H., Sikkink, R., Rusnak, F., Sygush, J., and Nef, P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 9253-9258[Abstract/Free Full Text]
36. Nakamura, T. Y., Pountney, D. J., Ozaita, A., Nandi, S., Ueda, S., Rudy, B., and Coetzee, W. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 12808-12813[Abstract/Free Full Text]
37. Bichet, D., Cornet, V., Geib, S., Carlier, E., Volsen, S., Hoshi, T., Mori, Y., and De Waard, M. (2000) Neuron 25, 177-190[Medline] [Order article via Infotrieve]
38. Beguin, P., Nagashima, K., Gonoi, T., Shibasaki, T., Takahashi, K., Kashima, Y., Ozaki, N., Geering, K., Iwanaga, T., and Seino, S. (2001) Nature 411, 701-706[CrossRef][Medline] [Order article via Infotrieve]
39. De Waard, M., Liu, H., Walker, D., Scott, V. E., Gurnett, C. A., and Campbell, K. P. (1997) Nature 385, 446-450[CrossRef][Medline] [Order article via Infotrieve]
40. Pragnell, M., De, Waard, M., Mori, Y., Tanabe, T., Snutch, T. P., and Campbell, K. P. (1994) Nature 368, 67-70[CrossRef][Medline] [Order article via Infotrieve]
41. Zamponi, G. W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T. P. (1997) Nature 385, 442-446[CrossRef][Medline] [Order article via Infotrieve]
42. De Waard, M., Witcher, D. R., Pragnell, M., Liu, H., and Campbell, K. P. (1995) J. Biol. Chem. 270, 12056-12064[Abstract/Free Full Text]
43. De Waard, M., Pragnell, M., and Campbell, K. P. (1994) Neuron 13, 495-503[Medline] [Order article via Infotrieve]
44. Scott, V. E., De, Waard, M., Liu, H., Gurnett, C. A., Venzke, D. P., Lennon, V. A., and Campbell, K. P. (1996) J. Biol. Chem. 271, 3207-3212[Abstract/Free Full Text]
45. Scalettar, B. A., Rosa, P., Taverna, E., Francolini, M., Tsuboi, T., Terakawa, S., Koizumi, S., Roder, J., and Jeromin, A. (2002) J. Cell Sci. 115, 2399-2412[Abstract/Free Full Text]
46. Ames, J. B., Hendricks, K. B., Strahl, T., Huttner, I. G., Hamasaki, N., and Thorner, J. (2000) Biochemistry 39, 12149-12161[CrossRef][Medline] [Order article via Infotrieve]
47. Lee, A., Scheuer, T., and Catterall, W. A. (2000) J. Neurosci. 20, 6830-6838[Abstract/Free Full Text]
48. Zuhlke, R. D., Pitt, G. S., Tsien, R. W., and Reuter, H. (2000) J. Biol. Chem. 275, 21121-21129[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.