Modified Sympathetic Nerve System Activity with Overexpression of the Voltage-dependent Calcium Channel β3 Subunit*

N-type voltage-dependent calcium channels (VDCCs) play determining roles in calcium entry at sympathetic nerve terminals and trigger the release of the neurotransmitter norepinephrine. The accessory β3 subunit of these channels preferentially forms N-type channels with a pore-forming CaV2.2 subunit. To examine its role in sympathetic nerve regulation, we established a β3-overexpressing transgenic (β3-Tg) mouse line. In these mice, we analyzed cardiovascular functions such as electrocardiography, blood pressure, echocardiography, and isovolumic contraction of the left ventricle with a Langendorff apparatus. Furthermore, we compared the cardiac function with that of β3-null and CaV2.2 (α1B)-null mice. The β3-Tg mice showed increased expression of the β3 subunit, resulting in increased amounts of CaV2.2 in supracervical ganglion (SCG) neurons. The β3-Tg mice had increased heart rate and enhanced sensitivity to N-type channel-specific blockers in electrocardiography, blood pressure, and echocardiography. In contrast, cardiac atria of the β3-Tg mice revealed normal contractility to isoproterenol. Furthermore, their cardiac myocytes showed normal calcium channel currents, indicating unchanged calcium influx through VDCCs. Langendorff heart perfusion analysis revealed enhanced sensitivity to electric field stimulation in the β3-Tg mice, whereas β3-null and Cav2.2-null showed decreased responsiveness. The plasma epinephrine and norepinephrine levels in the β3-Tg mice were significantly increased in the basal state, indicating enhanced sympathetic tone. Electrophysiological analysis in SCG neurons of β3-Tg mice revealed increased calcium channel currents, especially N- and L-type currents. These results identify a determining role for the β3 subunit in the N-type channel population in SCG and a major role in sympathetic nerve regulation.

Voltage-dependent Ca 2ϩ channels (VDCCs) 2 are present in excitable tissues. VDCCs mediate the influx of Ca 2ϩ in response to membrane depolarization and regulate many fundamental functions, including muscle contraction, neurotransmitter release, and gene transcription (1). The VDCC ␣1 subunit families are classified into three main groups (CaV1.1-4, CaV2.1-3, and CaV3.1-3) based on their physiological properties and sequence similarities (2). VDCCs are heteromultimeric proteins, composed of four subunits (␣1, ␣2/␦, ␤, and ␥). The ␣1 subunit serves as the channel pore, voltage sensor, and the binding site for various channel modulators (1,3). The ␤ subunit, which is hydrophilic and has no transmembrane domain, is the most important auxiliary subunit in the VDCC complex, with four different genes having been identified. The ␤ subunits have distant homology with the Src homology region 3 and guanylate kinase module, which interact with the ␣1 subunit (4).
Several important roles of the ␤ subunit in channel properties, such as multifold increases in peak calcium current density and acceleration of inactivation kinetics, have been demonstrated using heterologous cDNA co-expression systems (5,6). The presence of the ␤ subunit and its interaction with the ␣1 subunit are rate-limiting steps in VDCC functioning in a variety of isolated cell systems (1,(5)(6)(7). This interaction between the ␤ and ␣1 subunits is dependent on the ␤ interaction domain and the ␣1 subunit interaction domain (AID), but channel modulation due to the ␤ subunit is independent of interaction with the AID (8).
Because of the technical progress in electrophysiology and molecular biology in recent decades, a large amount of knowledge about VDCCs has been accumulated, although discrepant results between native tissues and co-expression systems still exist. Furthermore, heterologous expression systems may be inadequate to analyze the trafficking mechanism of the channel subunit assembly. The physiological importance of accessory subunits such as the ␤ subunit is likely difficult to extrapolate in such systems. To identify the exact role of the auxiliary ␤ subunits, a recent approach using a transgenic animal model and a combination of molecular biological and physiological analyses has proven effective, although such experiments are painstaking (9). The sympathetic nervous system is an essential regulator in the "fight or flight" response, the key behavioral response to stress and danger. This system has a strong influence on the circulatory system. Its activity is increased in hypertension and heart failure and decreases the favorability of a prognosis. Neuronal (N)-type voltage-dependent Ca 2ϩ channels (N-VDCCs) play major roles in the release of norepinephrine at sympathetic nerve terminals (10,11). A strain of N-VDCC-deficient mice exhibited impaired positive inotropic contractions (11). Furthermore, they retained positive inotropic responses that were not dependent on -conotoxin GVIA, a specific N-VDCC inhibitor, but were primarily dependent on compensatorily upregulated R-type VDCCs (12). The physiologically important components of N-VDCCs are the CaV2.2 (␣1B) and ␤3 subunits (13). Gene-targeting experiments resulting in ␣1Band ␤3-deficient mice have shown that N-VDCCs play a major role in sympathetic nerve regulation (11,14). Through in vivo analysis of ␤3-deficient mice, we reported significantly modified sympathetic nervous transmission due to ␤3 gene ablation (14). However, mere ablation of an accessory subunit may not enable the determination of its physiological role. Thus, ␤3 overexpressing transgenic mice were also developed in an attempt to confirm the role of the ␤3 subunit in sympathetic nerve function in vivo.
In this study, we analyzed and clarified the physiological role of the ␤3 subunit by establishing ␤3-overexpressing transgenic mice and comparing them with other transgenic mice (␤3-deficient, CaV2.2-deficient) with regard to sympathetic nervous system function.

EXPERIMENTAL PROCEDURES
Generation of ␤3 Transgenic Mice-The ␤3 transgenic mice was generated as described previously with CAG promoter (15). The ␤3-deficient (␤3 Ϫ/Ϫ ) and the CaV2.2-deificient mice were generated by gene-targeting methods (11,16). All experiments were conducted in accordance with the Guidelines for the Use of Laboratory Animals of the Akita University School of Medicine.
ECG Telemetry-ECG telemetry was performed as described previously (14). During the recording period (60 min), animals were at liberty in their home cage. Analog ECG signals were transferred to a receiver device and digitized by a converter system (Mini-Digi-1A; Axon Instruments, Foster City, CA). The effect of -conotoxin GVIA (30 g/kg) was observed 30 min after its intraperitoneal injection.
Blood Pressure Measurement-Mice were anesthetized with urethane (1.5 g/kg, intraperitoneally). After the surgery, the preparation was allowed to stabilize for at least 30 min before the start of the experiment. The mean arterial blood pressure was obtained using arterial catheters surgically inserted into the right carotid artery (11). Following surgery, mean arterial blood pressure was measured with a microtip catheter pressure transducer connected to a carrier amplifier (AP-601G; Nihon Koden, Tokyo, Japan). The left carotid artery was ligated for 30 s to observe the baroreflex responses.
Langendorff Perfusion Experiments-The isolated heart was mounted on a Langendorff apparatus and perfused at a constant hydrostatic pressure of 75 mm Hg with an oxygenated Tyrode's solution (37°C) containing 5.5 M atropine (12). The heart was paced electrically at 400 beats/min. The electrical field stimulation (EFS) was applied in conjunction with the pacing stimulation (delay, 4 ms; duration, 1 ms for 5 s).
Isolation of SCG Neurons and Whole-cell Recordings-Electrophysiological measurements (whole-cell mode) were performed on the superficial cervical ganglion (11). Currents were recorded with an Axopatch 200B patch clamp amplifier (Axon Instruments) (16) Statistical Analysis-The data are presented as the mean Ϯ S.E. Differences were evaluated using unpaired Student's t tests and were deemed statistically significant at p Ͻ 0.05.

RESULTS
Overexpression of the ␤3 Subunit Increases the Ca 2ϩ Channel Population-The overexpression construct was generated by ligating the full-length ␤3 subunit coding sequence into the pCXN2 plasmid (gift from Dr. Miyazaki, Osaka University School of Medicine), under the CAG promoter, followed by intra-ribosomal entry site and enhanced green fluorescent protein (EGFP) sequences, and completed with a rabbit ␤-globin poly(A) signal (Fig. 1A, panel i). The plasmid insert from the promoter to the poly(A) sequence was prepared and injected into the pronucleus (15).
Transgenic offspring were genotyped by Southern blot analysis, confirming three transgenic lines (Tg-a, Tg-b, and Tg-c) with germ line transmission (Fig. 1A, panel ii, lanes 1-3, Tg-a, Tg-b, and Tg-c, respectively). The transgenic mice had no overt phenotype based on observations. By comparison with known amounts of plasmid construct, the Tg-a line was estimated to carry four copies of the transgene (Fig. 1A, panel ii, lane 1), Tg-b, two copies (lane 2), and Tg-c, eight copies (Fig. 1A, panel ii, lane 3). Because of its high copy number, the Tg-c mouse line was used for further analysis.
To investigate the influence of overexpressed ␤3 on the Ca 2ϩ channel population, we analyzed the expression of the ␣1 and ␤ subunits in the SCG of the ␤3-Tg mice and wild-type (WT) control mice by RT-PCR (Fig. 1B) and immunoblotting (Fig.  1C). Using the CAG promoter, the ␤3 subunit was significantly overexpressed in SCG (418 Ϯ 14.4% versus control, n ϭ 4; Fig.  1B). The mRNA expression levels of the ␤1, ␤2, and ␤4 subunits in the SCG of ␤3-Tg-mice were not significantly different (115 Ϯ 7, 103 Ϯ 4, and 100 Ϯ 3%, respectively, versus control, n ϭ 4 for each gene; Fig. 1B, RT-PCR). Transcription of the P/Q-type channel-forming CaV2.1 subunit was not changed (114 Ϯ 5% versus control, n ϭ 4), nor was the CaV2.2 signal changed (109 Ϯ 5% versus control, n ϭ 4). The ␤3-Tg mouse did not differ in expression of the R-type channel-forming CaV2.3 subunit (95 Ϯ 6% versus control, n ϭ 4). The level of the dihydropyridine-sensitive L-type channel-forming CaV1.2 significantly increased (234 Ϯ 24% versus control, n ϭ 4). In contrast, the expression of another L-type channel, the CaV1.3 subunit, was unchanged (119 Ϯ 10.9% versus control, n ϭ 4). The expression of ␤-actin confirmed that comparable amounts of RNA had been loaded in each reaction (Fig. 1B). The expression of intra-ribosomal entry site-EGFP was confirmed by RT-PCR, indicating overexpression of the transgene. We also conducted RT-PCR analysis of calcium channels in the heart; no significant difference in CaV1.2 expression was observed, whereas ␤3 gene expression was significantly increased (data not shown).
Western Blot Analysis-To further investigate the influence of ␤3 overexpression on Ca 2ϩ channel populations at the protein level, we analyzed the ␣1 and ␤ subunits in the SCG of ␤3-Tg and WT control mice by immunoblotting (Fig. 1C). The protein levels of the ␤1, ␤2, and ␤4 subunits in the SCG were not significantly different (97 Ϯ 4, 104 Ϯ 6, and 92 Ϯ 4%, respectively, versus control, n ϭ 4 for each gene), as in the RT-PCR analysis. Using the CAG promoter, the ␤3 subunit was overexpressed in the SCG (181 Ϯ 20.0% versus control, n ϭ 4; Fig. 1C).
By contrast, the level of CaV1.2 protein was elevated significantly (268 Ϯ 26.8% versus control, n ϭ 4; Fig. 1C), and this corresponds to its increased transcription in the RT-PCR analysis (Fig. 1B). The AID domain of CaV1.2 is known to bind all ␤ subunits (20). Therefore, the level of CaV1.2 protein in SCG should be affected by increased ␤3. Nevertheless, the increased CaV1.2 protein level is probably related to increased transcription of CaV1.2 and the interaction between CaV1.2 and ␤3.
Protein levels of other ␣1-forming subunits (CaV1.3, Cav2.1, and CaV2.3) were unchanged (data not shown). Both anti-GAPDH antibody binding (Fig. 1C, GAPDH) and Ponceau-S staining (data not shown) confirmed comparable loading of proteins in the lanes of the gel. Western blot analysis confirmed the expression of EGFP, indicating overexpression of the transgene (Fig. 1C, GFP); the EGFP protein level was low compared with the high EGFP mRNA level (Fig. 1B, EGFP). In the heart, the protein level of L-type channel forming CaV1.2 was apparently same in Tg mice (data not shown).
Increased Heart Rate on ECG-ECG of the mice overexpressing the ␤3 gene (␤3-Tg mice) revealed a regular pattern indicative of physiological pacemaking and excitation propagation ( Fig. 2A, panel i). The heart rate calculated over 1 h was increased significantly in the ␤3-Tg mice ( Fig. 2A, panel ii, red bar) and tended to be slower in the ␤3-null mice ( Fig. 2A, panel ii, blue bar). The administration of -conotoxin GVIA (30 g/kg) significantly decreased the heart rate in all three groups ( Fig. 2A, panel iii), with the ␤3-Tg mice showing an enhanced response to the toxin (asterisk).
Enhanced Baroreflex Response in ␤3-Tg Mice-As we observed significant heart rate changes in ␤3-Tg mice, we next examined blood pressure. Fig. 2B, panel i, shows representative blood pressure traces in WT (upper trace) and ␤3-Tg (lower trace) mice. We found no significant difference in the mean arterial blood pressure (mAP) in the basal state between the WT (85.7 Ϯ 1.6 mm Hg, n ϭ 19) and ␤3-Tg mice (85.4 Ϯ 2.2 mm Hg, n ϭ 8).
To assess sympathetic nerve function in the ␤3-Tg mice, we examined the carotid baroreflex function. The baroreflex function is dependent on carotid baroreceptors in the carotid sinus, which detect changes in aortic blood pressure and regulate blood pressure by activating sympathetic or parasympathetic nerves with impulses from the nucleus of the tractus solitarius in the brain stem. Fig. 2B, panel ii, summarizes the change in mAP at the end of bilateral carotid artery occlusion (30 s). In the WT mice, the mAP was increased by bilateral carotid artery occlusion (12.7 Ϯ 3.6 mm Hg, n ϭ 7), and the ␤3-Tg mice showed a significantly enhanced response (19.7 Ϯ 1.6 mm Hg, n ϭ 8).
Our results demonstrated that the ␤3-Tg mice showed an increased baroreflex response, which was predominantly mediated by N-type VDCCs, given that -conotoxin GVIA abolished this increase. Furthermore, the enhanced baroreflex response suggests that the ␤3-Tg mice have enhanced sympa-thetic nerve tone. These results were confirmed by echocardiography, which revealed ␤3-dependent enhancement in the ejection fraction (supplemental Fig. 5). The ␤3-Tg mice showed a significantly increased ejection fraction, whereas the ␤3-null mice showed a reduced ejection fraction (supplemental Fig. 5B) compared with WT mice. The N-VDCC blocker -conotoxin GVIA caused a significantly decreased ejection fraction in WT mice (supplemental Fig. 5C, white bar). The ␤3-Tg mice showed an enhanced response to -conotoxin GVIA (supplemental Fig. 5C, asterisk), confirming up-regulated sympathetic activity in the ␤3-Tg mice. On the other hand, the contractile inotropic responses of the right atrium to isoproterenol, a ␤-adrenergic stimulant, showed no significant difference among WT, ␤3-null, and ␤3-Tg mice, suggesting that post-adrenergic receptor cascades were not influenced (supplemental Fig. 6).
Langendorff Perfusion Results-The transgenic mouse showed increased heart rate and sensitivity to -conotoxin GVIA on ECG. Blood pressure measurement also revealed enhanced responses to carotid artery occlusion or -conotoxin GVIA. Furthermore, echocardiography revealed increased ejection fraction and enhanced responsiveness to -conotoxin GVIA. These results clearly suggest that sympathetic nerve activity in the ␤3-Tg mice is increased. On the other hand, contractile analysis results showed conserved contractility to ␤-adrenergic stimulants, indicating unchanged sensitivity to catecholamines, which probably means that post-adrenergic receptor cascades were not influenced. To further analyze the relationship between cardiac contraction and sympathetic nerve activity, we used a Langendorff apparatus to measure heart perfusion in response to EFS, where 100 nM isoproterenol was used to provoke a maximum response, which was designated as 1.0. Compared with the response in WT mice (Fig. 3, A,  upper panel and E, open circles), the response to EFS was significantly increased in the ␤3-Tg mice (Fig. 3, B, upper panel, and E, red squares) and was decreased in the ␤3-deficient mice (Fig.  3, C, upper panel, and E, blue triangles). These ␤3-dependent responses suggested that the modified sympathetic nerve terminals released norepinephrine. To determine the involvement of N-type VDCCs, we also evaluated CaV2.2-deficient mice in a Langendorff perfusion experiment; the CaV2.2-deficient mice showed the smallest response to EFS (Fig. 3, D, upper panel, and  E, green diamond).
To further examine the contribution of N-type VDCCs, we assessed the sensitivity of the response to -conotoxin GVIA in each mouse line (Fig. 3, A-D, lower panels). Perfusion with 1 M -conotoxin GVIA significantly reduced the response to EFS in WT, ␤3-Tg, and ␤3-null mice (Fig. 3, A-C), but it only marginally affected the response in CaV2.2-deficient mice (Fig. 3D). The ␤3-Tg mice were most sensitive to the toxin. Although less sensitive than the ␤3-Tg mice, the ␤3-deficient mice were more sensitive to the toxin than the CaV2.2-deficient mice, which were only marginally sensitive. Taken together, the results of the Langendorff experiments, which are summarized in Fig. 3E, indicate that the sensitivity to -conotoxin GVIA apparently depends on the N-type VDCC population. Furthermore, these results suggest that the neurotransmitter release at sympathetic nerve terminals was triggered primarily by N-type VDCCs. Increased Plasma Catecholamines in the ␤3-Tg Mice-To analyze whether the sympathetic tone is enhanced in the ␤3-Tg mice, we next evaluated plasma catecholamines. In vivo, it might happen that the enhanced release of catecholamines is down-regulated, and the animals do not have an enhanced sympathetic tone. Indeed, the ␤3-Tg mice showed increased plasma epinephrine and norepinephrine levels at the basal status (epinephrine, 2.40 Ϯ 0.51 ng/ml, n ϭ 7 (wild) and 5.84 Ϯ 1.26 ng/ml, n ϭ 6 (␤3-Tg mice), respectively; norepinephrine, 3.51 Ϯ 0.74 ng/ml, n ϭ 7 (wild) and 7.93 Ϯ 0.69 ng/ml, n ϭ 6 (␤3-Tg mice), respectively).
Modified Calcium Channels in SCG Neurons-The increased sensitivity of the ␤3-Tg mice to -conotoxin in the ECG, blood pressure, and Langendorff perfusion experiments was likely attributable to enhanced sympathetic nerve tone in these mice, given that other physiological results indicated an increased heart rate, enhanced responses to N-type calcium channel blockers, and increased ejection fraction in ␤3-Tg mice. Thus, we next examined SCG neurons, which contain sympathetic neurons. Rectangular pulses (50-ms duration) of various potentials were applied in the presence of 10 mM Ba 2ϩ , as a charge carrier. Fig. 4A shows examples of voltage-dependent Ca 2ϩ channel currents in WT and ␤3-Tg mice. At positive test potentials, inward Ba 2ϩ currents were observed in both groups (Fig. 4A). The mice overexpressing the ␤3 gene showed significantly increased peak current amplitude in the neurons of the SCG (75.3 Ϯ 3.6 pA/pF, n ϭ 15, and 87.4 Ϯ 3.6 pA/pF, n ϭ 12, in WT and ␤3-Tg mice, respectively; Fig. 4B, asterisks).
We examined the effects of the sequential application of VDCC blockers on the peak current evoked by voltage steps to Ϫ10 mV, delivered at 10-s intervals (Fig. 4C, upper  panel). The electrophysiological recordings revealed the co-existence of various types of channels,  The SCG neurons of mice overexpressing the ␤3 gene had significantly more N-type and L-type channel current densities than those of WT mice (N-type, 36.8 Ϯ 2.7* pA/pF versus 29.3 Ϯ 2.1 pA/pF, respectively, n ϭ 8 for both; L-type, 22.5 Ϯ 1.6* pA/pF versus 17.3 Ϯ 0.9 pA/pF, respectively, n ϭ 8 for both). There was no significant difference in the number of P/Q-type or R-type channel currents between the SCG neurons of ␤3-Tg and WT mice (P/Q-type, 11.0 Ϯ 0.8 pA/pF versus 12.6 Ϯ 1.5 pA/pF, respectively, n ϭ 8 for both; R-type, 16.0 Ϯ 1.6 pA/pF versus 15.0 Ϯ 1.0 pA/pF, respectively, n ϭ 8 for both). These results indicate that the total calcium channel current amplitude was greater in the SCG neurons of the ␤3-Tg mice because of increased numbers of -conotoxin-sensitive N-type and dihydropyridine-sensitive L-type channels. In contrast, the calcium channel currents in cardiac myocytes did not differ significantly among the WT, ␤3-null, and ␤3-Tg mice (supplemental Fig. 7).
To investigate whether the N-type channel population is dependent on the amount of ␤3 subunit, we examined the influence of ␤3 expression on the N-type channel population in cell lines. Specifically, we examined the influence of ␤ subunit expression on the channel population of the N-type channel pore-forming CaV2.2 subunit in HEK293T cells (supplemental Fig. 8). The amount of the CaV2.2 gene product was clearly dependent on the amount of co-transfected ␤3 gene plasmid, indicating a major role of the ␤3 subunit in channel localization in the membrane.

DISCUSSION
We constructed a transgenic mouse in which the voltage-dependent calcium channel ␤3 subunit was overexpressed. In this study, we focused on the sympathetic phenotype of the transgenic mouse line. The ␤3-overexpressing mice had increased expression of the ␤3 subunit, increased numbers of CaV2.2 (N-type) and CaV1.2 (L-type) calcium channels in the supracervical ganglion (SCG) neurons, and increased heart rate. Furthermore, they showed enhanced sensitivity to the N-type calcium channel-specific blocker -conotoxin GVIA with regard to ECG, blood pressure, and echocardiography. However, their atria showed normal contraction in response to isoproterenol, and their cardiac myocytes showed normal calcium channel currents, indicating unchanged calcium influx through the VDCC in cardiac myocytes. Langendorff heart perfusion experiments revealed an enhanced response to electric field stimulation in the ␤3-Tg mice, supporting the idea of increased sympathetic tone. Electrophysiological analysis of the SCG neurons in ␤3-Tg mice revealed increased calcium channel currents because of increased N-type and L-type currents. Furthermore, the plasma catecholamine levels were increased significantly in ␤3-Tg mice. Taken together, our present findings show increased sympathetic nerve activity in the ␤3-Tg mice, due primarily to increased channel populations in the SCG.
In our previous study with ␤3-deficient mice (16), the CaV2.2 channel population was significantly decreased in the dorsal root ganglion. In contrast, the ␤3-Tg mice showed increased CaV2.2 and CaV1.2 in the SCG (Fig. 1C). The increase in the CaV2.2 channel population was apparently the result of a posttranslational mechanism, because no significant change in mRNA expression was observed by RT-PCR, whereas Western analysis revealed a significantly increased CaV2.2 protein level. The significant effect of ␤3 subunit overexpression on the CaV2.2 channel population in HEK293T cells (supplemental Fig. 8) was likely because of an interaction between CaV2.2 and ␤3. By contrast, CaV1.2 showed increased mRNA levels using RT-PCR, suggesting that ␤3 induced the transcription of the CaV1.2 gene in the SCG. The regulatory mechanisms for ␣1 and ␤ subunit expression are thought to be independent, because the expression of the ␤ subunit protein was shown to be unchanged in CaV2.2 (␣1B)-deficient mice (12). Therefore, the increased CaV1.2 expression may have been an indirect effect of ␤3 overexpression. The molecular mechanisms underlying the stimulatory effect of the ␤ subunit on CaV1.2 gene expression remain unclear, but it is known that ␤3, with CaV1.2, forms most L-type calcium channels in smooth muscle cells, and the same ␤3, with CaV2.2, forms most N-type calcium channels in the brain. Recent studies have revealed various roles of ␤ subunits unrelated to physiological channel function, including regulatory effects of ␤3 on inositol 1,4,5-trisphosphate production (21) and the interaction between ␤4 and nuclear proteins, which results in the regulation of gene transcription (22). Furthermore, the ␤1 subunit physically docks with the ryanodine receptor (23). This interaction between the ␤1 subunit and the ryanodine receptor is key for the generation of calcium signals coupled to membrane depolarization, which strengthens excitation-contraction coupling, suggesting that each ␤ subunit might have specific or preferential roles. Moreover, recent crystal structure analysis of the core segment of the calcium channel ␤ subunit supports the idea that this subunit can serve as a multifunctional protein (4,24,25). Additionally, Ras-related small G-proteins such as Gem, Rad, and Rem bind to the ␤ subunit and inhibit VDCC activity (26). Thus, many intracellular proteins are likely to influence channel functions through ␤ subunits.
The ␤ subunits function as chaperone-like molecules in the trafficking of ␣1 subunits to the plasma membrane (27). Lacerda et al. (28) reported an enhanced effect of ␤ subunits on channel populations, and Chien et al. (27) described the effects of the ␤2 subunit on Cav1.2 (L-type) channel formation. The effects of the ␤ subunit on N-type channel populations have been reported in null-mutant mice (16), whereas in vivo overexpression experiments, such as a transgenic approach, have failed. Combined with our previous study (16), the evidence clearly indicates that ␤3 has a strong influence on N-type channel populations. Furthermore, the protein level of CaV2.2 in the microsomal fraction was apparently dependent on the co-expressed ␤3 subunit (supplemental Fig. 8), whereas other ␤ subunits showed lesser effects. Given that ␤3 has the highest affinity for CaV2.2, the effect of ␤3 may be related to this high affinity. Nevertheless, the ␤3 subunit had a preferential effect on N-type channel populations both in vivo (Fig. 1C) and in vitro (supplemental Fig. 8). The interaction between the ␣1 and ␤ subunits is thought to help target the channel pore to the membrane, resulting in increased channel populations and increased channel amplitudes. This effect of the ␤3 subunit on the N-type channel population probably caused the enhanced sympathetic nerve tone in the ␤3-Tg mice.
In conclusion, our findings indicate that ␤3-Tg mice have increased N-type channel populations in the SCG, resulting in enhanced sympathetic tone. This finding implies the physiological importance of the ␤3 subunit in a sympathetic regulatory system, which suggests that the ␤3 subunit may be a pharmacological target.