|
|
|
|
|
|
|
||
Address correspondence to David J. Adams School of Biomedical Sciences, The University of Queensland, Queensland 4072, Australia. Fax: (07) 3365-4933; email: dadams{at}uq.edu.au
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1 and 2
enhanced the maximum conductance of expressed channels at lower ß3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The ß3-induced current suppression was reversed at a HP of -120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba2+ (40 mM) as a charge carrier also largely diminished the effect of ß3 at -80 mV. Therefore, experimental conditions (HP, divalent cation concentration, and ß3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel ß3 effect. Steady-state inactivation curves revealed that N-type channels exhibited "closed-state" inactivation without ß3, and that ß3 caused an 
40-mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately -85 mV. The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of ß3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at -80 mV. Similar "ultra-slow" inactivation was observed for N-type channels without ß3. Thus, ß3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by causing a hyperpolarizing shift of the inactivation without affecting "ultra-slow" and "closed-state" inactivation properties.
Key Words: voltage-dependent calcium channel • Xenopus oocyte • ß3 auxiliary subunit • negative regulation • voltage-dependent inactivation
1 subunit interaction domain; HP, holding potential; HVI, high-voltage inactivation; LVI, low-voltage inactivation; VDCC, voltage-dependent calcium channel.
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). High-voltage–activated calcium channels are comprised of a pore-forming 1 subunit, auxiliary ß and 2 subunits, and, in some cases, an auxiliary 
subunit (for review see Catterall, 2000). Based on 1 gene similarity, they are divided into two families of L-type (Cav1.1–1.4) and non-L-type calcium channels that include P/Q- (Cav2.1), N- (Cav2.2) and R-types (Cav2.3). Conserved transmembrane and pore domains of the 1 subunits are 50% identical between the families and >80% identical within a family (see Ertel et al., 2000). VDCC auxiliary ß (Cavß1–4) and 2 (Cav21–4) subunits associate with the 1 functional subunits (for review see Walker and De Waard, 1998) and affect the biophysical properties of the 1 subunits as observed for the auxiliary subunits of voltage-dependent sodium and potassium channels (for review see Isom et al., 1994; Trimmer, 1998).
N-type calcium channels (Cav2.2) were pharmacologically characterized in chicken dorsal root ganglia neurons (Nowycky et al., 1985) and cloned together with their splice variants in mammalian central (Snutch et al., 1990; Dubel et al., 1992; Williams et al., 1992; Coppola et al., 1994; Lin et al., 1997; Kaneko et al., 2002) and peripheral neurons (Lin et al., 1997). Given that N-type channels, together with P/Q- and R-types, are distributed predominantly in presynaptic nerve terminals, they are involved in nerve-evoked release of neurotransmitter (for review see Waterman, 2000; Fisher and Bourque, 2001). Therefore, modulation of VDCC currents by altering functional channel expression levels and biophysical properties is critical for regulation of neurotransmission in the nervous system. In particular, the amplitude and duration of macroscopic VDCC current, and hence intracellular Ca2+ concentration during action potentials, has significant effect on neurotransmitter release as the release is proportional to [Ca2+]m, where m takes a value from 2.5 to 4 (for review see Wu and Saggau, 1997).
With regard to the modulation of VDCC currents, both ß and 2 subunits have been shown primarily to increase macroscopic current amplitude (Mori et al., 1991; Neely et al., 1993; Wakamori et al., 1993; Jones et al., 1998; Klugbauer et al., 1999). Coexpression of ß subunits enhanced the level of channel expression in the plasma membrane (Williams et al., 1992; Brust et al., 1993) by chaperoning the translocation of 1 subunits (Chien et al., 1995; Yamaguchi et al., 1998; Gao et al., 1999; Gerster et al., 1999) from ER where ß subunits antagonize the binding between 1 and an ER retention protein (Bichet et al., 2000). In addition, ß subunits also increased channel open probability without affecting single-channel conductance (Neely et al., 1993; Wakamori et al., 1993, 1999; Jones et al., 1998; Gerster et al., 1999; Hohaus et al., 2000). A hyperpolarizing shift of I-V relationships by ß subunits (Neely et al., 1993; Yamaguchi et al., 1998) also partially contributes to an increase in macroscopic current amplitude. The ß subunit has been shown to interact with 1 subunit interaction domain (AID) in the cytoplasmic I-II linker of 1 subunit (Pragnell et al., 1994; Witcher et al., 1995), and all four ß subunits interacted with AID of Cav2.21 in vitro with high affinity (Kd of 5 nM; Scott et al., 1996). The interaction between 1 and ß subunits through AID plays a critical role in channel trafficking from the ER to plasma membrane (Gerster et al., 1999) and therefore VDCC current potentiation by ß subunits (Pragnell et al., 1994; De Waard et al., 1995). On the other hand, coexpression of 2 subunits, like ß subunits, augmented ligand binding (Bmax) and 1 protein expression levels in the plasma membrane (Williams et al., 1992; Brust et al., 1993; Shistik et al., 1995) without increasing channel open probability and single-channel conductance (Bangalore et al., 1996; Jones et al., 1998; Wakamori et al., 1999) or shifting activation in a hyperpolarizing direction (Qin et al., 1998; Wakamori et al., 1999; Gao et al., 2000). The mechanism underlying 2-induced potentiation of 1 expression appears to be different from that of ß subunits. A domain of 2, which interacts with 1 subunit, was proposed to be located in an extracellular region (Gurnett et al., 1997), and therefore it is unlikely that 2 subunits antagonize an interaction between an ER retention protein and an intracellular AID of 1 subunits. Furthermore, lack of evidence for the 2-induced 1 subunit trafficking was shown using an immunohistochemical technique (Gao et al., 1999).
In contrast to VDCC current potentiation by auxiliary subunit, VDCCs are well known to be negatively regulated by G protein ß subunits (for review see Zamponi, 2001) and this effect is modulated by calcium channel ß subunits (Canti et al., 2000; Feng et al., 2001). Furthermore, syntaxin 1A, a key synaptic protein participating in neurotransmitter release, exerted an inhibitory effect on N- and Q-type calcium channels, thereby probably preventing Ca2+ overload or excessive neurotransmitter release (Bezprozvanny et al., 1995; Degtiar et al., 2000). Although VDCC auxiliary subunits positively regulate neurotransmitter release by enhancing neuronal (N-, P/Q- and R-type) calcium channel currents, there is less evidence for auxiliary subunit-induced negative regulation of these channels. Patil et al. (1998) have found pronounced channel inactivation during a train of action potential-like waveforms compared with a single square pulse for N-, P/Q- and R-type channels, and this was dependent on ß subunit isoforms. In the present study, we characterize previously overlooked large negative regulatory effect of high levels of the ß3 subunit on macroscopic currents carried by N- and R-type calcium channels. A preliminary report of these results, in part, has been presented in an abstract form (Yasuda et al., 2002).
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1, rat Cav2.21 (former name 1B) and rabbit Cav1.21 (1C); ß, rat ß3 and Xenopus ß3xo; and 2, rabbit 21, were synthesized using the mMessage mMachine in vitro transcription kit (Ambion). Stage V-VI oocytes were removed from anesthetized female Xenopus laevis and treated for 2-3 h with 2.5 mg/ml collagenase (Type I; Sigma-Aldrich) for defolliculation. The oocytes were then injected with either an 1 subunit alone, or in combination with an 2 and/or a ß subunit. Except for rat Cav2.31 (1E), of which cDNA was injected intranuclearly at a volume of 9.4 nl, 50 nl of cRNA mixtures were injected into the oocytes using an automatic microinjector (Drummond). The injected cells were incubated at 18°C in a ND96 solution (in mM): 96 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 5 HEPES, 5 pyruvic acid, and 50 µg/ml gentamicin, pH 7.5, before recording.
Electrophysiological Recordings
3–8 d after the cRNA/cDNA injection, whole-cell calcium channel currents were recorded from the oocytes using the two-electrode voltage-clamp technique with Axon GeneClamp500B (Axon Instruments, Inc.). Voltage-recording and current-passing microelectrodes were filled with 3 M KCl and typically had resistances of 0.5–1.5 M
and 0.3–0.7 M, respectively. Ionic currents were often of the order of several microamperes in amplitude, which resulted in a significant voltage error due to a voltage drop across resistance of a ground electrode. To minimize this error, an independent two-electrode virtual-ground circuit with a 3 M KCl agar bridge was used. Unless stated otherwise, all recordings were made with a bath solution containing 5 mM Ba2+ as a charge carrier of which composition was (in mM): 5 BaCl2, 85 tetraethylammonium hydroxide (TEAOH), 5 KCl, 5 HEPES, titrated to pH 7.4 with methansulfonic acid. When 5 mM CaCl2 was substituted for 5 mM BaCl2, other components of the bath solution remained the same. When 40 mM BaCl2 was used, TEAOH was reduced to 42.5 mM. Oocytes were perfused continuously at a rate of 1.5 ml/min at room temperature (22°C). Activation of Cl- currents was eliminated by injecting 25–50 nl of 50 mM 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) at least 15 min before recording. Most oocytes exhibited considerable run-down or run-up at the beginning of recording and experiment did not commence until fluctuation of peak currents with repeated depolarizing pulses reduced to less than ±2% within a 1-min period. During the study of inactivation kinetics, if oocytes exhibited >120% recovery from inactivation, experiments were repeated using the same oocytes.
Using pCLAMP 8 software (Axon Instruments, Inc.), membrane currents were acquired at 10 kHz after a low-pass filter at 1 kHz and an on-line leak-subtraction with a -P/4 pulse protocol. I-V relationships were obtained by step depolarization from -80 to +50 mV in 10-mV increments, from holding potentials (HPs) of either -80 or -120 mV, with a 4-min interval between I-V curves obtained at each HP. For steady-state inactivation, unless stated otherwise, the HP was changed from -120 to 0 mV in 5- or 10-mV increments, with each HP maintained for 3 min before each test pulse. Steady-state inactivation curves were derived from peak currents elicited by a test pulse to 0 mV given at the end of each HP. Normalized currents were calculated by dividing each peak current by that observed at a HP of -120 mV.
Data Analysis
Data were analyzed using Prism 3.0 (GraphPad). Each I-V relationship was fitted with smooth curves derived from a Boltzmann equation: I = Gmax(Vt - Erev)/{1 + exp[(V1/2, act - Vt)/kact]}, where Gmax is the maximum conductance, Vt is the test potential, Erev is the apparent reversal potential, V1/2, act is the midpoint of activation, and kact is the slope factor. Ratios of Gmax (Gmax at a HP of -120 mV/Gmax at a HP of -80 mV) were calculated as an index of voltage-dependent inhibition. For steady-state inactivation, each data was fitted by a single Boltzmann equation: I/I-120 = 1/[1 + exp((V1/2, inact - Vt)/kinact)], where V1/2, inact is the midpoint of inactivation, kinact is the slope factor, and I-120 is the control current amplitude at HP of -120 mV, or a dual Boltzmann equation: I/I-120 = ILVI{1/[1 + exp((V1/2, inact LVI - Vt)/kLVI)]} + IHVI{1/[1 + exp((V1/2, inact HVI - Vt)/kHVI)]}, where LVI and HVI are low- and high-voltage inactivation, respectively, and ILVI and IHVI are the normalized current amplitudes (ILVI + IHVI = 1). Unless stated otherwise, the goodness-of-fit of both equations was compared with F-test to achieve best fit.
Complimentary DNA Clones of Ca2+ Channel Subunits
The Cav2.21 (the central nerve splice variant 1B-d) and ß3 cDNAs were provided by Dr. D. Lipscombe (Brown University, Providence, RI); Cav1.21 and Cav2.31 cDNAs were provided by Dr. G. Zamponi (University of Calgary, Calgary, AL); 21 cDNA was provided by Dr. F. Hofmann and Dr. N. Klugbauer (Technische Universität München, Germany); and Xenopus ß3xo cDNA was provided by Dr. L. Birnbaumer (UCLA, Los Angeles, CA).
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
1 and 2 and 1.25 ng/cell of ß3, produced robust Ba2+ currents through open N-type calcium channels in response to a step depolarization to 0 mV from HPs of -60 and -80 mV (Fig. 1 A). The peak current amplitude was significantly reduced and inactivation kinetics slowed in a concentration-dependent manner by increasing concentrations of ß3 (Fig. 1, A and B). The effect was more pronounced at the more positive HP of -60 mV, where an 80% reduction in peak current amplitude was observed with injection of 12.5 ng ß3. Although a similar ß3-induced inhibitory effect was observed on R-type (Cav2.3) calcium channels, it was less pronounced than for N-type channels (Fig. 1, C and D). In contrast, injection of 25 ng ß3 subunit markedly enhanced L-type (Cav1.2) calcium channel currents at both HPs (Fig. 1, E and F).
|
1 and 2 subunits. As shown in Fig. 2, at a HP of -80 mV, ß3 subunit exhibited a biphasic, bell-shaped effect on both peak amplitude of I-V curves and the maximum conductance (Gmax); that is, concentration-dependent enhancement at low concentrations (0–1.25 ng) and inhibition at high concentrations (2.5–12.5 ng). Gmax was increased to a maximum by 1.25 ng ß3 and was 1.7-fold higher than that of the control group in which N-type calcium channels were expressed without ß3. The peak Gmax observed with 1.25 ng ß3 was reduced by 74% in the presence of 12.5 ng ß3, which corresponds to 56% inhibition compared with the control group (Fig. 2 C). To determine whether this inhibition by ß3 may be attributed to voltage-dependent channel inactivation or other voltage-independent mechanisms such as inhibition of functional channel expression, the oocyte was held at -120 mV to remove voltage-dependent inactivation. At a HP of -120 mV, ß3 enhanced the calcium channel current in a concentration-dependent manner, and the maximum current (2.8-fold enhancement compared with the control group) was obtained with 5 ng ß3. This result suggests that the inhibitory effect of ß3 observed at -80 mV was due to voltage-dependent channel inactivation. The difference in concentration-dependence observed for Gmax at -120 mV compared with that for Gmax ratio suggests that two distinct mechanisms may underlie the effect of ß3 subunit (Fig. 2 C).
|
), where the existence of two components in inactivation with V1/2, inact of approximately -70 and -40 mV was demonstrated with intranuclear injection of various concentrations of ß3 cDNA. The more positive V1/2, inact reported in their study may be due to a shorter prepulse of 25 s. The ß3-concentration dependence of Gmax and %LVI were compared (Fig. 3 C). The EC50 value for %LVI (1.91 ng) was significantly different from that obtained for Gmax (0.91 ng). The result likely explains the biphasic response of Gmax at a HP of -80 mV seen in Fig. 2 C. Subsequent kinetic experiments revealed that HPs of 3 min were of insufficient duration to induce maximal inactivation (see Fig. 8) and therefore more accurately reflect "pseudo-steady-state" inactivation.
|
1 subunit alone (Cav1.2, 2.2 or 2.31) in oocytes has been shown to be blocked by antisense injection for ß3xo (Tareilus et al., 1997; Canti et al., 2001). Therefore, it is possible that the Cav2.21 subunit could interact with the endogenous ß3xo, especially when expressed without heterologous rat ß3. If this is the case, the two different V1/2, inact observed could arise from different ß3 subunits combining with the 1 subunit such that HVI and LVI might originate from N-type calcium channels composed of Cav2.21/ß3xo/2 and Cav2.21/ß3/2, respectively. This hypothesis was tested by coexpressing various concentrations of ß3xo with Cav2.21 and 2. Similar to the rat ß3, exogenous ß3xo not only enhanced the N-type channel current amplitude at the HP of -120 mV (9.0-fold increase in Gmax), but also caused a leftward shift of the inactivation curve (Fig. 4). The maximum increase in Gmax (9.0-fold) caused by ß3xo (Fig. 4 B) was much more pronounced than that (2.8-fold) by rat ß3 (Fig. 2 C), although amino acid sequences for ß3 exhibit a high similarity (Tareilus et al., 1997). Importantly, the ß3xo-induced leftward shift of inactivation is inconsistent with the idea that the rat ß3-induced leftward shift of the inactivation curve was dependent on the presence of ß3xo. Thus, the two components of inactivation curves may be due to two different forms of the N-type calcium channel caused by interactions with different levels of coexpressed ß3 subunit. Although, at a HP of -80 mV, ß3 at higher concentrations inhibited Gmax, resulting in a biphasic effect (Fig. 2 C); ß3xo simply enhanced channel Gmax in a concentration-dependent manner (Fig. 4 B). This could be attributed to the difference in V1/2, inact of LVI obtained with ß3 and ß3xo which are approximately -85 and -70 mV, respectively.
|
The 2 Subunit Is Not Involved in the Down-regulation of N-type Calcium Channels
In the absence of 2, ß3 caused a biphasic response and a hyperpolarizing shift of inactivation also observed in the presence of 2 (Fig. 5). This result indicates that the ß3 subunit does not require the 2 subunit for its effects. Higher concentrations of ß3 (5 and 12.5 ng) inhibited Gmax even at a holding potential of -120 mV (Fig. 5 B). The underlying mechanism of this additional channel inhibition is unknown. To determine whether the inactivation is voltage dependent, further investigation with more negative HPs may be warranted, but it is unlikely to be attributed to competition of RNA translation or toxicity produced by high levels of RNA because the same concentration of ß3 did not exhibit this effect in the presence of 2 (Fig. 3).
|
2 is also known to enhance VDCC currents (Klugbauer et al., 1999). To confirm whether 2 exhibits a biphasic effect on the calcium channel current due to a voltage-dependent inactivation, the effects of 2 on Gmax and Gmax ratio were examined. In the presence of ß3 (2.5 ng), 2 enhanced the current by 4.1-fold at -80 mV, but had no significant effect on the Gmax ratio (Fig. 6, A and B). A similar result was obtained in the absence of ß3, where 2 enhanced currents by 3.5-fold at -80 mV but did not change the Gmax ratio (Fig. 6, C and D). In contrast to the ß3 subunit, the 2 subunit enhanced N-type calcium channel current amplitude without a significant negative regulatory effect.
|
|
) of 14 s and 8.8 min (Fig. 8, A and E). Test pulses with longer intervals of 10 min did not alter the kinetics (Fig. 8 A, closed diamonds), suggesting there is no accumulated inactivation caused by repetitive depolarization. The recovery from the inactivation was similarly very slow with time constants of 29 s and 21.8 min (Fig. 8, A and E). Similar slow kinetics for the onset and recovery from inactivation were observed for N-type channels expressed without ß3 (Fig. 8, B and E). N-type channels expressed with or without the high concentration (12.5 ng) of ß3 mainly exhibit LVI or HVI, respectively (see Fig. 3 B), and therefore the very slow closed-state inactivation observed independent of ß3 is a common property of two different states or compositions (LVI and HVI) of N-type channels. Thus, each inactivation component, LVI or HVI, has been shown to represent the net effect of a kinetically complex inactivation process with at least two exponential components. In other words, ß3 simply shifts the voltage dependence of the closed-state inactivation in a hyperpolarizing direction and does not substantially affect the inactivation kinetics. Similar results were obtained for R-type calcium channels (Fig. 8, C–E). Although inactivation kinetics of R-type channels were faster than for N-type channels, the time constant of the slow component was still in the order of minutes.
|
Effect of Divalent Cations on Voltage-dependent Inactivation of N-type Calcium Channels
It is well known that different species and concentration of divalent cations can affect the voltage dependence of ion channel gating (for review see Hille, 2001). To determine whether the ß3-induced current suppression is observed with the physiological divalent ion, Ca2+, the biophysical properties of N-type channels expressed in the absence and presence of ß3 (12.5 ng/cell) were examined with 5 mM Ca2+ as a charge carrier. Although Gmax was reduced by half with 5 mM Ca2+ compared with that obtained with 5 mM Ba2+ in the presence of ß3, the Gmax ratio of 5.1 obtained with 5 mM Ca2+ was close to that of 6.4 obtained with 5 mM Ba2+ (Fig. 9, A and B). The comparable Gmax ratios were attributed to similar V1/2, inact of -81.0 and -85.4 mV with 5 mM Ca2+ and 5 mM Ba2+, respectively (Fig. 9 C). The ß3-induced negative shifts in inactivation under both conditions were also comparable as the difference in V1/2, inact with or without ß3 was 41.9 mV with 5 mM Ca2+ and 38.1 mV with 5 mM Ba2+. In contrast, 40 mM Ba2+ obscured the ß3 effect by reducing the Gmax ratio to 1.6 and shifting V1/2, inact to -62.0 mV, which is 20 mV more positive than for 5 mM Ba2+ (Fig. 9) and the difference in V1/2, inact with or without ß3 was more than 10 mV smaller than for the others. Therefore, the ß3 subunit induced a negative shift in "closed-state" inactivation with 5 mM Ba2+ and was substantially unchanged with 5 mM Ca2+, whereas a higher concentration of Ba2+ masked the ß3 effect. The slight positive shift of V1/2, act and V1/2, inact with 5 mM Ca2+ compared with 5 mM Ba2+ may be due to the binding of Ca2+ to surface charged groups and/or a "specific regulatory site" (Zamponi and Snutch, 1996) in addition to charge screening, whereas Ba2+ only possesses a charge screening ability (see Hille, 2001).
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Sandoz et al., 2001), N- (Lin et al., 1997; Stephens et al., 2000; Canti et al., 2001), R- (Jones et al., 1998), and L-type (Castellano et al., 1993; Yamaguchi et al., 1998; Gerster et al., 1999; Xu and Lipscombe, 2001) calcium channels. Similarly, we found that Ba2+ currents through L-type channels were markedly enhanced by ß3. In contrast, high ß3 levels were found to significantly suppress N- and R-type channel currents at HPs of -60 and -80 mV (see Table I). This effect was most prominent using recording protocols referring to physiological conditions.
|
; Waterman, 2000) where the resting membrane potential varies from -55 to -80 mV (e.g., Adams and Harper, 1995; Shimizu et al., 1996; Fujimura et al., 1997; Hyllienmark and Brismar, 1999), we chose a HP of -60 or -80 mV to investigate the physiological characteristics of these calcium channels in the presence and absence of ß3. At these HPs, inhibitory effects of ß3 were prominent, whereas at a HP of -120 mV only the enhancement of the macroscopic current was observed. HPs more negative than -90 mV have commonly been used to remove steady-state inactivation and to investigate the effects of auxiliary subunits on VDCC properties, where ß3 enhancement of N- and R-type channel currents has been reported (Jones et al., 1998; Stephens et al., 2000; Canti et al., 2001). Thus, the ß3 inhibitory effect may have been overlooked due to the use of hyperpolarized HPs.
The extracellular divalent cation concentration used as a charged carrier was found to also dramatically influence the inhibitory effect of ß3. The physiological extracellular Ca2+ concentration is 2 mM; however, to resolve the VDCC currents, 10–40 mM Ba2+ is commonly used in the extracellular recording solution. Replacing 5 mM Ba2+ with 5 mM Ca2+ as the charge carrier, it was evident that the species of divalent cation (Ca2+ or Ba2+) was not so important for either V1/2, inact in the presence of 12.5 ng ß3 or the degree of ß3-induced negative shifts of inactivation, as the changes in these values by the replacement were <5 mV. However, the divalent cation concentration had a critical influence on the ß3-induced current inhibition as V1/2, inact of -85 mV with 5 mM Ba2+ was shifted to -62 mV in the presence of 40 mM Ba2+, resulting in obscuring the effect of ß3. Given that lowering the extracellular divalent cation concentration generally shifts steady-state voltage-dependent properties in a negative direction (Hille, 2001), it is expected that a physiological concentration of 2 mM Ca2+ will exhibit an even greater negative V1/2, inact and more pronounced inhibition by ß3. Similarly, a significant influence of low divalent cation concentrations (2–5 mM) on L-type (Cav1.3) calcium channels has been observed to produce a unique, hyperpolarized activation threshold (Xu and Lipscombe, 2001).
Under physiological condition, the mRNA or protein expression of 1, ß, and 2 subunits changes during neural development (Jones et al., 1997; Vance et al., 1998) and the expression of those subunits appears to be individually controlled (Vance et al., 1998). Therefore, it is important to characterize any concentration dependence of auxiliary subunit–induced modulation of VDCC properties. Usually, however, a fixed combination of 1 and ß subunit cRNA or cDNA is injected in a concentration ratio from 1:0.3 to 1:1. In agreement with previous reports in which only current enhancement was observed, when we examined a concentration ratio of 1:0.5 for the combination of Cav2.21 and ß3 subunits at a HP of -80 mV, ß3 increased N-type calcium channel current amplitude by 1.7- or 2.6-fold compared with Cav2.21 alone in the presence or absence of 2, respectively. Under similar experimental conditions, an 3-fold increase in current amplitude by ß3 was observed when Cav2.21 and ß3 were coexpressed at a ratio of 1:0.5; however, higher concentrations of ß3 were not examined (Lin et al., 1997). In a previous study, the concentration dependence of ß-induced modulation of N-type calcium channel properties revealed two components of inactivation (Canti et al., 2001). However, current suppression induced by higher concentrations of ß3 was not observed at a HP of -100 mV (Canti et al., 2001). It is difficult to confirm that expression levels of ß3 subunit in their and present studies are physiological. However, when the concentrations of Cav2.21 and ß3 were decreased while maintaining a cRNA concentration ratio of 1: ß3 of 1:5, a similar inhibitory effect of ß3 was observed (unpublished data). This suggests that a high concentration of ß3 is not necessary to observe this phenomenon, but a balance between 1 and ß3 subunits is more important. It is intriguing that a significant involvement of the ß3 subunit in N-type calcium channel currents in dorsal root ganglia neurons was demonstrated using ß3-deficient mice and voltage-dependent inactivation of calcium channel currents observed at a HP of -50 mV in wild-type mice was largely diminished in the ß3-deficient mice (Murakami et al., 2002). Our results are consistent with this observation and thereby suggest a role of ß3 inhibitory effect under physiological and pathophysiological conditions.
Together, by using experimental conditions (HP, divalent cation concentration, various subunit concentration) more closely approximating normal physiological conditions, we observe a profound effect of ß3 to inhibit N- and R-type calcium channel currents.
Mechanism of ß3-induced Current Inhibition
At a HP of -120 mV, ß3 simply enhanced N- and R-type calcium channel currents even at higher concentrations, where the current enhancement for R-type channels was much less pronounced than for N-type channels. A similar, less pronounced enhancing effect of a ß subunit (ß2a) on R-type channels has been reported previously using oocytes (Olcese et al., 1996). The ß3-induced potentiation of N- and R-type channel currents is likely to be caused by enhanced channel trafficking to the plasma membrane and an increased channel open probability, which have been observed for L- and R-type channels (Jones et al., 1998; Yamaguchi et al., 1998; Gao et al., 1999; Gerster et al., 1999).
On the other hand, the mechanism of ß3-induced current inhibition observed at -80 mV appears to be due mainly to voltage-dependent channel inactivation and therefore it is unlikely that ß3 blocks channel trafficking as current inhibition was almost undetectable at -120 mV. In the absence of the 2 subunit, however, higher concentrations of ß3 also reduced N-type calcium channel currents at -120 mV. Although the cause of this inhibition is not clear, it cannot be attributed to a toxic effect of excess amounts of ß3 cRNA, since an inhibitory effect was not observed for N-, R- , and L-type channel currents in the presence of the 2 subunit. Steady-state inactivation of N-type channels revealed two components in the inactivation curves in the presence of ß3, namely HVI with the V1/2, inact of approximately -45 mV and LVI with a more hyperpolarized V1/2, inact of approximately -85 mV with 3-min HPs. Similarly, the V1/2, inact values for HVI and LVI of R-type channels were approximately -65 and -90 mV, respectively. Thus, it was determined that the effect of ß3 on inactivation is to transfer the channel state/composition from exhibiting HVI to LVI. The hyperpolarized V1/2, inact of LVI provides a plausible explanation for the voltage-dependent current suppression by ß3 at a HP of -60 or -80 mV. Prolonged HPs of 30 min induced a negative shift of inactivation curves of N-type channels with the V1/2, inact of -54.4 and -93.5 mV for HVI and LVI, respectively, suggesting more pronounced current inhibition with a high concentration of ß3 at a physiological range of resting membrane potential (-55 to -80 mV). In this study, ß3 subunit exhibited clear biphasic, bell-shaped effects on both peak current amplitude of I-V curves and Gmax of N-type channels at the HP of -80 mV (see Table I). The concentration dependence of ß3-induced increase in Gmax was found to be 2-fold more sensitive to the ß3 concentration than increase in %LVI. A similar but more pronounced (7-fold) difference in concentration dependence of increase in Gmax and %LVI has been demonstrated previously (Canti et al., 2001). The biphasic effect of ß3 on Gmax at the HP of -80 mV and the difference in concentration dependence strongly support two proposed models for interactions of 1 and ß subunits. The two site-model proposes that in addition to the primary "high" affinity AID that is involved in ß-induced channel trafficking (Pragnell et al., 1994; De Waard et al., 1995), other "low" affinity interaction sites exist for ß subunits at the COOH and NH2 terminus of R- and P/Q-type calcium channel 1 (Cav2.3 and 2.11) subunits (Qin et al., 1997; Tareilus et al., 1997; Walker et al., 1998, 1999). The alternate single-site model proposes that there is only one ß subunit binding site on the 1 subunit, namely AID. In this model, the affinity of AID to a ß subunit changes from "high" to "low" after 1 subunit incorporation into the plasma membrane. This results in the dissociation of the ß subunit from the 1 subunit and gives a chance of reassociation of ß at low affinity. Fitting our data to these two models, it is assumed that ß3 subunits at low concentrations bind to high affinity AID of Cav2.2 or 2.31, thereby enhancing channel expression and macroscopic currents. At higher concentrations, ß3 subunits interact with a second low-affinity site of the 1 subunits or alternatively, reassociate with AID, then promote the transfer from HVI to LVI and inhibit N- or R-type channel currents.
Together, we speculate that low concentrations of ß3 promote the expression of channels that produce HVI and high concentrations of ß3 transfer channel state/composition to LVI without affecting expression levels. When channels are expressed in the absence or presence of a low concentration of ß subunits, the participation of endogenous ß3 (ß3xo) in the channel composition or properties should be considered. However, based on the results obtained with exogenously applied ß3xo, we conclude rat ß3–induced shift of inactivation curves from HVI to LVI was independent on the presence of ß3xo.
In this study, we showed that the steady-state inactivation of N- and R-type calcium channels occurred in the "closed-state" and revealed extremely slow kinetics with at least two exponential components. It is surprising that the properties of inactivation were observed not only for channels coexpressed with a high concentration of ß3 but also for channels without ß3. Thus, although the subunit stoichiometry of N-type channels expressed in oocytes is unclear, higher concentrations of ß3 simply cause a negative shift of the inactivation keeping its slow and "closed-state" inactivation properties. Given that VDCC inactivation has been intensively studied in the "open-state", the physiological role and mechanism of VDCC "closed-state" inactivation are poorly understood. The "open-state" inactivation has been classified into three types, Ca2+-dependent and fast and slow voltage-dependent inactivation (for review see Hering et al., 2000). Time constants for the onset of fast and slow voltage-dependent inactivation of non-L-type (P/Q-, N-, and R-type) calcium channels range from 40 ms to 1 s (includes fast and slow components; e.g., Stephens et al., 2000; Stotz et al., 2000) and from 30 to 70 s (Sokolov et al., 2000), respectively. In this study, the time constants for fast and slow components of the N-type channel "closed-state" inactivation were 15 s and 10 min, respectively. To distinguish this process from the slow inactivation reported by Sokolov et al. (2000), we propose the term "ultra-slow" inactivation, analogous to "ultra-slow" inactivation of sodium channels (compare Todt et al., 1999).
Similar slow closed-state inactivation has been reported for native and recombinant N-type channels. In bullfrog sympathetic neurons, which predominantly express N-type calcium channels, channel inactivation was developed at HPs of -40, -50, and -60 mV from a HP of -80 mV over a time range of 10 min with 2 mM Ba2+ as the charge carrier (Jones and Marks, 1989). Another slowly developed inactivation was observed using 5 mM Ba2+ for N-type channels (Cav2.21/ß3/2 = 1:0.75:1) expressed in Xenopus oocytes (Degtiar et al., 2000). In this condition, the slow inactivation was negligible when cells were held at -80 mV, but became apparent at -70 and -60 mV over 3-min recordings. The amount of channel inactivation at -60 mV after 3 min is comparable with our results obtained from steady-state inactivation with 3-min HPs when channels were expressed with 2.5 ng ß3 (Cav2.21/ß3/2 = 1:1:1). Interestingly, syntaxin 1A promoted this slow inactivation, but did not show a clear effect on fast inactivation (Degtiar et al., 2000).
Patil et al. (1998) proposed a "preferential closed-state inactivation" model to account for pronounced channel inactivation during a train of action potential-like waveforms compared with a single square pulse for N-, P/Q-, and R- type calcium channels. N- and R-type but not L-type channels coexpressed with ß3 subunits exhibit the preferential closed-state inactivation (Patil et al., 1998) and ß3-induced current suppression (this study). Furthermore, the absence of the preferential closed-state inactivation for N-type channels coexpressed with ß2a subunits (Patil et al., 1998) is consistent with the lack of an inhibitory effect of ß2a on N-type channel currents (unpublished data). The inactivation process of N-type calcium channels occurred over several hundred milliseconds and the half time of the recovery from the inactivation at -100 mV was 300 ms (Patil et al., 1998). This is significantly faster than the "ultra-slow" process observed here. It remains to be determined if these two "closed-state" inactivation processes have a common underlying mechanism.
Possible Physiological and Pathophysiological Roles of ß3-induced Current Inhibition
It is interesting to consider what physiological role the ß3 subunit–induced suppression of N- and R-type calcium channel currents may play. N- and R-type channels are expressed predominantly in presynaptic nerve terminals of central and peripheral neurons and play a critical role in neurotransmitter release (for review see Waterman, 2000; Fisher and Bourque, 2001). Given that the ß3 subunit causes a biphasic effect on N- and R-type channel current amplitude, it appears that one role of ß3 is enhancement of the current amplitude to promote transmitter release at synapses. However, high levels of ß3 relative to 1 inactivate these calcium channels, thereby providing a negative feedback on the current amplitude. Interestingly, syntaxin A, which triggers vesicle fusion in nerve terminals, inactivates N- and R-type channels (Degtiar et al., 2000). It is also possible that ß3 more actively plays a role as a negative regulator for N- and R-type channels as the expression of 1 and ß subunits has been shown to be individually regulated during brain ontogeny (Jones et al., 1997; Vance et al., 1998).
The neuronal expression levels of VDCC subunits can change dramatically in diseases such as diabetes (Iwashima et al., 2001), temporal lobe epilepsy (Lie et al., 1999; Djamshidian et al., 2002) and neuropathic pain (Kim et al., 2001; Luo et al., 2001), indicating a potential pathophysiological role of the ß-dependent current modulation. Given that the ß3 subunit–induced current suppression is voltage dependent, the change in the resting membrane potential during hypoxia-induced nerve injury (Fujiwara et al., 1987; Shimizu et al., 1996; Hyllienmark and Brismar, 1999) may produce different outcomes depending on the levels of ß3 in particular neurons.
In conclusion, our results demonstrate a novel role of calcium channel ß3 subunits as a negative regulator of N- and R-type calcium channels approaching normal physiological conditions. We show that N- and R-type calcium channels display "ultra-slow" and "closed-state" voltage-dependent inactivation, and that ß3 causes a significant hyperpolarizing shift of the voltage-dependent inactivation.
|
ACKNOWLEDGMENTS |
|---|
This work was supported by grants from the National Health and Medical Research Council of Australia and Australian Research Council. T. Yasuda is a recipient of a UQIPRS scholarship.
Olaf S. Andersen served as editor.
Submitted: 28 October 2003
Accepted: 13 February 2004
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2/-subunit on ionic and gating current in transiently transfected HEK 293 cells. Am. J. Physiol. Heart Circ. Physiol. 270:H1521–H1528.1 subunit contains an endoplasmic reticulum retention signal antagonized by the ß subunit. Neuron. 25:177–190.[CrossRef][Medline]1B calcium channels. Biophys. J. 81:1439–1451.[Medline]1 subunit. Evidence for isoforms, brain distribution, and chromosomal localization. FEBS Lett. 338:1–5.[CrossRef][Medline]1A Ca2+ channel expressed in Xenopus oocytes. J. Physiol. 485:619–634.1-ß anchoring site in voltage-dependent Ca2+ channels. J. Biol. Chem. 270:12056–12064.1 subunits in temporal lobe epilepsy with Ammon's horn sclerosis. Neuroscience. 111:57–69.[CrossRef][Medline]-1 subunit of an 
-conotoxin-sensitive calcium channel. Proc. Natl. Acad. Sci. USA. 89:5058–5062.2 auxiliary subunit gene (CACNA2D2). J. Biol. Chem. 275:12237–12242.1C and ß subunits generate the necessary signal for membrane targeting of class C L-type calcium channels. J. Biol. Chem. 274:2137–2144.1C subunit are independent functions of the ß subunit. J. Physiol. 517:353–368.2 and 1 subunits. J. Biol. Chem. 272:18508–18512.1 subunit (1C-b) by the ß2a subunit: a peptide which inhibits binding of ß to the I-II linker of 1 induces functional uncoupling. Biochem. J. 348:657–665.[CrossRef][Medline]1E calcium channels. J. Gen. Physiol. 112:125–143.1B) calcium channel variants lacking the synaptic protein interaction site. J. Neurosci. 22:82–92.1 gene expression in rat dorsal root ganglia following peripheral nerve injury. Brain Res. Mol. Brain Res. 96:151–156.[Medline]2 subunit. J. Neurosci. 19:684–691.2 calcium channel subunit and its correlation with allodynia in spinal nerve-injured rats. J. Neurosci. 21:1868–1875.1E calcium channels. J. Physiol. 497:675–686.1-subunit. Nature. 368:67–70.[CrossRef][Medline]1E-type calcium channel by 2-subunit. Am. J. Physiol. Cell Physiol. 274:C1324–C1331. with a C-terminal Gß-binding domain of the Ca2+ channel 1 subunit is responsible for channel inhibition by G protein-coupled receptors. Proc. Natl. Acad. Sci. USA. 94:8866–8871.1A subunit. Eur. J. Neurosci. 14:987–997.[CrossRef][Medline]2/ and ß subunits in Xenopus oocytes: contribution of changes in channel gating and 1 protein level. J. Physiol. 489:55–62.1B Ca2+ channel amino terminus contributes determinants for ß subunit-mediated voltage-dependent inactivation properties. J. Physiol. 525:377–390.1B and ß subunits during rat brain ontogeny. J. Biol. Chem. 273:14495–14502.1 subunit and the effects of a cardiac ß subunit. Biochem. Biophys. Res. Commun. 196:1170–1176.[CrossRef][Medline]1A subunit. J. Biol. Chem. 273:2361–2367.1A subunit controls P/Q-type Ca2+ channel activation. J. Biol. Chem. 274:12383–12390.-conotoxin-sensitive human N-type calcium channel. Science. 257:389–395.1 subunit interaction domain. J. Biol. Chem. 270:18088–18093.1 L-type channels activate at relatively hyperpolarized membrane potentials and are incompletely inhibited by dihydropyridines. J. Neurosci. 21:5944–5951.1C subunit. J. Biol. Chem. 273:19348–19356.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Facebook 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  S.-N. Yang and P.-O. Berggren The Role of Voltage-Gated Calcium Channels in Pancreatic {beta}-Cell Physiology and Pathophysiology Endocr. Rev., October 1, 2006; 27(6): 621 - 676. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Vendel, M. D. Terry, A. R. Striegel, N. M. Iverson, V. Leuranguer, C. D. Rithner, B. A. Lyons, G. E. Pickard, S. A. Tobet, and W. A. Horne Alternative splicing of the voltage-gated Ca2+ channel beta4 subunit creates a uniquely folded N-terminal protein binding domain with cell-specific expression in the cerebellar cortex. J. Neurosci., March 8, 2006; 26(10): 2635 - 2644. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Mould, T. Yasuda, C. I. Schroeder, A. M. Beedle, C. J. Doering, G. W. Zamponi, D. J. Adams, and R. J. Lewis The {alpha}2{delta} Auxiliary Subunit Reduces Affinity of {omega}-Conotoxins for Recombinant N-type (Cav2.2) Calcium Channels J. Biol. Chem., August 13, 2004; 279(33): 34705 - 34714. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
