Modulation of human neuronal alpha 1E-type calcium channel by alpha 2delta -subunit

Ning Qin1, Riccardo Olcese1, Enrico Stefani1,2, and Lutz Birnbaumer1,3

Departments of 1 Anesthesiology, 2 Physiology, and 3 Biological Chemistry and Molecular Biology Institute, School of Medicine, University of California, Los Angeles, California 90095

    ABSTRACT
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Abstract
Introduction
Methods
Results & Discussion
References

Calcium channels are composed of a pore-forming subunit, alpha 1, and at least two auxiliary subunits, beta - and alpha 2delta -subunits. It is well known that beta -subunits regulate most of the properties of the channel. The function of alpha 2delta -subunit is less understood. In this study, the effects of the calcium channel alpha 2delta -subunit on the neuronal alpha 1E voltage-gated calcium channel expressed in Xenopus oocytes was investigated without and with simultaneous coexpression of either the beta 1b- or the beta 2a-subunit. Most aspects of alpha 1E function were affected by alpha 2delta . Thus alpha 2delta caused a shift in the current-voltage and conductance-voltage curves toward more positive potentials and accelerated activation, deactivation, and the installation of the inactivation process. In addition, the efficiency with which charge movement is coupled to pore opening assessed by determining ratios of limiting conductance to limiting charge movement was decreased by alpha 2delta by factors that ranged from 1.6 (P < 0.01) for alpha 1E-channels to 3.0 (P < 0.005) for alpha 1Ebeta 1b-channels. These results indicate that alpha 2delta facilitates the expression and the maturation of alpha 1E-channels and converts these channels into molecules responding more rapidly to voltage.

calcium channel alpha 2delta -subunit; coupling efficiency; facilitation of expression

    INTRODUCTION
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Abstract
Introduction
Methods
Results & Discussion
References

CALCIUM CHANNELS ARE a molecularly diverse group of multisubunit proteins that are composed of a pore-forming and voltage-sensing alpha 1-subunit and at least two auxiliary or regulatory subunits: beta  and alpha 2delta (4). The molecular diversity of these channels is due to the existence of six distinct genes encoding alpha 1-subunits, one gene encoding alpha 2delta , and four beta -genes. Most of these subunits are differentially expressed in neurons, skeletal muscle, smooth muscle, and a variety of secretory cells. A further cause of molecular diversity in voltage-gated Ca2+ channels is that most of these subunits display variations in amino acid sequence because of alternative splicing (2).

The regulatory roles of beta -subunits have been extensively studied, and it is now accepted that beta -subunits facilitate channel assembly and recruitment to the cell surface (5, 27, 33), increase the coupling of voltage sensing to pore opening (19), and thereby facilitate activation by voltage (17, 20). For neuronal type alpha 1-subunits, e.g., alpha 1E, beta -subunits also modulate voltage-induced inactivation in a subunit- and splice variant-specific manner. For example, beta 1b promotes inactivation by accelerating its establishment and by shifting the voltage-inactivation curve to a more negative potential, whereas the beta 2a-subunit has the opposite effect (22).

Studies on the actions of alpha 2delta have been less extensive, and its roles are less clear. alpha 2delta is encoded in a single gene (8, 10) and is posttranslationally cleaved into alpha 2 plus delta , which however remain linked by disulfide bonds (8, 13). alpha 2delta is widely distributed, being present in all tissues expressing Ca2+ channels studied thus far, including skeletal, smooth, and cardiac muscles and neurons (6, 7, 10, 39, 41). Molecular cloning has identified splice variants in skeletal muscle (10), brain (14, 39), and aorta (3) that are 95% identical and of as yet unknown functional significance. Recently, experiments by Gurnett et al. (11) and Wiser et al. (40) showed that alpha 2delta , which has the potential of traversing the membrane three times (10, 14), is a single transmembrane protein anchored in the membrane by the COOH-terminally placed delta . As a consequence, all of alpha 2 and 50% of delta  are extracellular. alpha 2delta is highly glycosylated (8, 13), a feature that appears to be necessary for its regulatory function as seen in experiments in which removal of the carbohydrate by digestion with protein N-glycosidase F led to loss of channel activity (11). Coexpression of alpha 2delta has been shown to increase alpha 1S and alpha 1C dihydropyridine binding sites in L and COS cells (31, 34, 36) and to a lesser extent of alpha 1B omega -conotoxin binding sites in HEK 293 cells (38). In agreement with these findings, alpha 2delta increases ionic currents of alpha 1A and alpha 1C in Xenopus oocytes (12, 15, 16, 27) and to a lesser extent also of alpha 1B (3, 38). alpha 2delta may thus participate in the general process of multisubunit assembly and transport to the plasma membrane (27, 36). However, exogenous alpha 2delta does not further increase ionic current of alpha 1E when expressed in Xenopus oocytes or COS-7 cells (30, 35).

At the functional level, Ca2+ channels without alpha 2delta seem to differ from Ca2+ channels with alpha 2delta , pointing to a regulatory function in addition to a structural role. Thus alpha 2delta modulates both voltage-dependent activation and inactivation of alpha 1A, alpha 1C, and alpha 1E, with the exact effect varying somewhat depending on the heterologous expression systems (9, 28, 30, 35, 37).

In the present study, we further explored the effects of alpha 2delta on the functional properties of neuronal alpha 1E and their interplay with those of beta -subunits. In Xenopus oocytes, alpha 1E is largely independent of an exogenous beta  or alpha 2delta for its expression but is functionally regulated by exogenously supplied beta - and alpha 2delta -subunits (22, 24, 26, 33). Although there is constitutive expression of endogenous beta -subunits (33) and probably of an alpha 2delta (29) in Xenopus oocytes, they do not appear to significantly affect the kinetics of the expressed exogenous channel (29, 33). We report below that exogenous alpha 2delta regulates the coupling of voltage sensing (charge movement) to pore opening as well as the kinetics of activation and inactivation of these channels. Similar to exogenous beta -subunits, alpha 2delta does not affect alpha 1E expression, i.e., the average levels of measurable macroscopic currents. Except for rates of activation and deactivation in response to voltage changes, the effects of alpha 2delta oppose those of beta -subunits.

    METHODS
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Abstract
Introduction
Methods
Results & Discussion
References

Subcloning of alpha 2delta . Rabbit skeletal muscle alpha 2delta , a generous gift from the late Professor S. Numa, was subcloned from the mammalian expression vector pKNH into pAGA2 (25). pAGA2 is a transcription competent vector that is based on pGEM3 (Promega) and has an engineered 5'-untranslated leader sequence from an alfalfa mosaic virus RNA, an idealized Kozak consensus translation initiation sequence, and a 3'-poly(A) tail of 92 adenylyls intended to facilitate the translation and to improve the stability of the transcript in Xenopus oocytes (25). First, a 3.6-kb cDNA fragment encoding the complete alpha 2delta -subunit was purified from an agarose gel after digestion with EcoR I and subcloned into the EcoR I site of pBluescript (Stratagene) (pBS) to give pBS-alpha 2delta . Second, the NH2-terminal 300 bp of alpha 2delta were amplified with Pfu thermostable DNA polymerase (Stratagene) using the PCR, a sense primer starting with an Nco I site (TAA TCC ATG GCT GCG GGC CGC CCG), and an antisense primer (TGA AGG GTC GAC CTC TCG) with a Sal I site. After digestion with Nco I and Sal I, the PCR fragment was subcloned into Nco I/Sal I digested pAGA2 to create pAGA2-alpha 2delta (N). Finally, a 3.2-kb DNA fragment encoding the COOH-terminal part of alpha 2delta was excised from pBS-alpha 2delta by digestion with Bgl II and Sal I and subcloned into the Bgl II/Sal I of pAGA2-alpha 2delta (N) to give pAGA2-alpha 2delta .

In vitro synthesis of cRNA. The expression constructs of human alpha 1E (26) and rabbit beta 2a (23) were linearized with Hind III, that of rabbit beta 1b (12) with Not I, and that of alpha 2delta with Xho I. The cRNAs were synthesized in vitro with T7 RNA polymerase using reagents and protocols of the mMESSAGE mMACHINE transcription kit (Ambion, Austin, TX), with the exception that the LiCl precipitation step was repeated twice. The cRNAs were suspended in diethylpyrocarbonate-treated H2O at a final concentration of 1-2 mg/ml.

Oocyte preparation. Frogs were anesthetized by immersion in water containing 0.15-0.17% tricaine methanesulfonate for ~20 min or until full immobility, and the ovaries were removed under sterile conditions by surgical abdominal incision. The animals were then euthanized by decapitation. The animal protocols were performed with the approval of the Institutional Animal Care Committee of the University of California, Los Angeles. Before injection, oocytes were defolliculated by collagenase treatment (type I, 2 mg/ml for 40 min at room temperature; Sigma, St. Louis, MO). Oocytes were maintained at 19.5°C in Barth solution (5 mM HEPES, pH 7.0, 100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, and 1 mM MgCl2). Recordings were done 4-12 days after the RNA injection.

Recording techniques. The cut-open oocyte voltage-clamp technique (32) was used to record ionic and gating currents from oocytes expressing alpha 1C and alpha 1E Ca2+ channels, alone or in combination with the regulatory beta 2a. The external solution (recording chamber and guard compartments) had the following composition: 10 mM Ba2+, 96 mM Na+, and 10 mM HEPES, titrated to pH 7.0 with MES. The lower chamber in contact with the fraction of the oocyte permeabilized with 0.1% saponin contained 110 mM potassium glutamate and 10 mM HEPES titrated to pH 7.0 with NaOH. Because oocytes expressing Ca2+ channels showed large outward Cl- current even with Ba2+ as the charge carrier, all the oocytes before recording were injected with 100-150 nl of 50 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-Na4 titrated to pH 7.0 with MES, to prevent activation of Ca2+- and Ba2+-activated Cl- channels (18). To remove contaminating nonlinear charge movement related to the oocytes endogenous Na+-K+-ATPase, 0.1 mM ouabain was added to all external solutions. Leakage and linear capacity currents were compensated and subtracted on-line using p/-4 protocol from -90 mV holding potential (SHP). Charge movement was detected for depolarizations more positive than -70 mV and that did not change with SHPs of -120 or -90 mV. These results indicate that negative subtracting pulses from -90 mV SHP are adequate to subtract linear components.

Signals were filtered with an eight-pole Bessel filter to one-fifth of the sampling frequency. All the experiments were performed at room temperature (22-23°C).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Methods
Results & Discussion
References

Neither exogenous beta  nor alpha 2delta enhances appearance of macroscopic alpha 1E Ca2+ channel currents. Previous studies had shown that ionic currents produced in Xenopus oocytes injected with alpha 1C or alpha 1A cRNA alone are very small, that coexpression with either beta  or alpha 2delta significantly increased the inward currents of both types of channels, and that currents are further enhanced by combining all three subunits (9, 27). Unlike these alpha 1-subunits, expression of the human alpha 1E alone in Xenopus oocytes produces relatively large inward ionic currents (26). This was confirmed in the present studies in which we observed peak inward currents that averaged 1.5 ± 1 (SE) µA (n = 15 oocytes from 7 batches; test potential, 10 mV from -90 mV holding potential) in oocytes injected with alpha 1E-cRNA alone. This was not significantly affected by coexpression of exogenous beta 1b (2.0 ± 1.0 µA, n = 12), alpha 2delta (1.7 ± 0.7 µA, n = 7), or both beta 1b and alpha 2delta (2.4 ± 0.8 µA, n = 16). In contrast, both beta  and alpha 2delta affected the kinetics of alpha 1E activation, deactivation, and inactivation and, as a result, the current-voltage (I-V), the conductance-voltage (G-V), and the steady-state voltage-inactivation relationships. Figure 1 illustrates the effect of subunit coexpression on the I-V relationships of alpha 1E; beta 1b shifted the I-V curve to more negative potentials, and alpha 2delta shifted the I-V curve to more positive potentials, indicating that alpha 2delta regulates the response to voltage of this neuronal Ca2+ channel.


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Fig. 1.   Current-voltage relationships of alpha 1 alone (open circle ), alpha 1/alpha 2delta (bullet ), alpha 1/beta 1b (triangle ), and alpha 1/beta 1b/alpha 2delta (black-triangle) channels. cRNAs were injected at molar ratio of alpha 1E-beta 1b-alpha 2delta  = 1:5:2. Each data point is mean peak normalized current from 5 or 6 oocytes. Currents were measured with 10 mM Ba2+.

alpha 2delta modulates alpha 1E Ca2+ channel activation by voltage. Figure 2, A-F, shows that the rates of alpha 1E activation were markedly increased by alpha 2delta , both in the absence and in the presence of coexpressed beta -subunits. To compare the rates of channel activation, we fitted the activation phase of time courses with a first-order exponential function and calculated the time constants for the different alpha 1E-complexes (Fig. 2G). As obtained with test potentials to 0 mV, alpha 2delta in all subunit combinations decreased the activation time constant (tau act). The tau act of alpha 1E decreased from 3.5 ± 0.1 ms (n = 6) to 1.7 ± 0.3 ms (n = 7). Similarly, in the presence of beta 1b and beta 2a, two beta -subunits with opposing effects on inactivation (22), the coexpression of alpha 2delta decreased the tau act from 4.5 ± 0.9 ms (n = 7) (beta 1b) to 2.3 ± 0.2 ms (n = 7) (beta 1b + alpha 2delta ) and from 3.9 ± 0.15 ms (n = 5) (beta 2a) to 2.4 ± 0.6 ms (beta 2a + alpha 2d) (n = 9). The rate of a channel activation is the sum of the rates at which a channel changes from its closed to its open states (alpha ) and from open to closed states (beta ). In addition to measuring the effects of alpha 2delta - and beta -subunits on the rates of channel activation at positive test potentials, we also measured their effects on the rates of channel closure upon membrane repolarization to -50 mV, since at this potential rates of deactivation are dictated primarily by beta  (open to closed rate). A composite plot of the calculated transition rates (Fig. 2G) showed that alpha 2delta increases not only the rate at which the alpha 1E-channel activates but also the rate at which it deactivates and that the effect on the open to closed rate is larger than that on the closed to open rate. This last finding accounts for the shifts of the G-V and I-V curves to more positive potentials in the face of faster activation kinetics (Fig. 3).


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Fig. 2.   Modulation of alpha 1E channel activation by alpha 2delta . A-F: Ba2+ currents elicited in oocytes expressing alpha 1E in different combinations with beta  and alpha 2delta by 50-ms depolarization pulses to -30, 0, and +30 mV from a holding potential of -90 mV. Current signals were digitized at 10 kHz and filtered at 2 kHz. G: rates of activation and deactivation as a function of test potential. Rates of activation (1/tau  = 1/alpha  + beta ) were determined by fitting activation phase of current traces with a single exponential function [I = Imax + A · exp(-t/tau )] and rate of deactivation at -50 mV (1/tau ~1/beta ) was determined by fitting deactivation phase of tail current obtained by 25-ms step from -16 to +36 mV with 4-mV increment, followed by repolarization to -50 mV. Values obtained from individual fits were averaged. Data shown on figure represent means ± SE (n = 5 for alpha 1E, n = 7 for alpha 1E/alpha 2delta , n = 6 for alpha 1E/beta 1b, n = 6 for alpha 1E/beta 1b/alpha 2delta , n = 5 for alpha 1E/beta 2a, and n = 7 for alpha 1E/beta 2a/alpha 2delta ).


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Fig. 3.   Modulation of alpha 1E channel voltage-conductance relationship by alpha 2delta in absence of beta  (A) and presence of either beta 1b (B) or beta 2a (C). Currents were evoked by 25-ms depolarizing steps from -88 to +132 mV in 4-mV increments, followed by repolarization to -50 mV. Relative fraction of channels activated during 25-ms activating pulse was measured as peak tail currents during repolarization to -50 mV. Data from individual oocytes were fitted by Im/Imax = I1/Imax/{1 + exp[z1(V1/2-1 - Vm)F/RT]} + I2/Imax/{1 + exp[z2(V1/2-2 - Vm)F/RT]}, where Im is instantaneous tail current developed after activation at test pulse potential (Vm), which equals sum of I1 and I2; V1/2 is midpoint; z is effective valence; Imax is current at saturating voltage obtained from fit. Data points correspond to means and error bars are ±SE of averaged normalized currents. Continuous lines are sum of 2 Boltzmann distributions using averaged I1, I2, V1/2-1, V1/2-2, z1, and z2 parameters. Dotted lines are first Boltzmann distribution (first component) using averaged I1, V1/2-1, and z1 parameters.

Previously, we reported that the G-V curves of alpha 1E-channels obtained in Xenopus oocytes injected only with the alpha 1E-cRNA could not be fitted by a single Boltzmann distribution but that acceptable fits could be obtained using the sum of two Boltzmann distributions with about equal relative amplitudes having midpoints of activation (V1/2) at ~0 and 50 mV. In those studies, the primary effect of coexpressing an exogenous beta -subunit was to increase the relative amplitude of the component with V1/2 around 0 mV from ~50 to 75-80%, without significantly changing the voltage dependence and position along the voltage axis of the two individual components (22). The mechanism by which exogenous beta -subunits change the ratio of the two components of the G-V curve is not clear so far. To account for the two components in the G-V curve, one could assume that there are two populations of Ca2+ channels in oocytes injected with alpha 1E alone (alpha 1E alone and alpha 1E with endogenous beta -subunit); however, our previous results suggested that this is unlikely (33). In the present studies, we reproduced the effects of the beta -subunits on the G-V curves. Coexpression of alpha 2delta with alpha 1E in the presence and absence of exogenous beta -subunits resulted in G-V curves that were shifted to more positive potentials along the voltage axis. These shifts can be accounted for by a decrease in the proportion of the more negative component of the G-V curve and by a shift to more positive potentials of both midpoints of the two Boltzmann distributions. These effects are consistent with the results depicted in Fig. 1, showing that alpha 2delta shifts the peak of the I-V curve to more positive potentials by ~10 mV. As was the case for the activation kinetics, the effect of alpha 2delta on the G-V relationship was also seen when a beta -subunit was coexpressed. The effects of either beta 1b (Fig. 3B) or beta 2a (Fig. 3C) were partially opposed by alpha 2delta so that the first component of the G-V curve was decreased from 72 to 62% (in the presence of beta 1b) and 74 to 62% (in the presence of beta 2a), respectively (Table 1). Changes in the midactivation potentials occurred as well.

                              
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Table 1.   Effect of alpha 2delta - and beta -subunits on activation and inactivation of alpha 1E-calcium channel

alpha 2delta markedly accelerates the installation of inactivation of alpha 1E but affects steady-state inactivation only slightly. The second distinct regulatory effect of coinjection of alpha 2delta on alpha 1E noticed by us was that alpha 2delta greatly accelerates the rate of alpha 1E inactivation. Figure 4 shows the inactivation time courses at a test potential of 10 mV. These time courses could not be fitted with a single exponential decay function. We thus analyzed the data empirically by comparing times required to decrease channel activities to 50% of peak and found the exogenous alpha 2delta to accelerate inactivation regardless of coexpression of an exogenous beta -subunit (Fig. 4). The fastest rates of inactivation were obtained upon coexpression of alpha 1E and alpha 2delta with exogenous beta 1b: half time at 10 mV = 43 ± 15 ms (n = 7). When steady-state inactivation was determined as a function of inactivating test potentials, we found no significant effect of alpha 2delta (Fig. 5 and Table 1). This suggests that alpha 2delta not only increases the rate of inactivation but also increases the rate of recovery from inactivation.


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Fig. 4.   alpha 2delta -Subunit accelerates inactivation of alpha 1E. Inactivation time courses of alpha 1E channels in combination with different beta - and alpha 2delta -subunits were obtained by a 800-ms pulse to 10 mV from holding potential of -90 mV. Times to decrease 50% of initial peak current during pulse (t1/2) are listed in parentheses as follows: alpha 1 alone (150 ± 52 ms; A), alpha 1/alpha 2delta (116 ± 11 ms; A), alpha 1/beta 1b (107 ± 17 ms; B), alpha 1/beta 1b/alpha 2delta (43 ± 15 ms; B), alpha 1/beta 2a (401 ± 157 ms; C), and alpha 1/beta 2a/alpha 2delta (282 ± 72 ms; C).


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Fig. 5.   alpha 2delta -Subunit does not significantly modulate steady-state inactivation of alpha 1E-channel. alpha 1E-Channels were inactivated by 10-s conditioning pulses ranging from -120 to +27 mV with 7-mV increments, then deactivated for 4 ms at -90 mV and finally activated by a 400-ms test pulse to +20 mV. Time between each episode was 30 s. Sampling frequency was 25 Hz during conditioning pulse and 500 Hz during test pulse, and current signals were filtered at 200 Hz. Relative fraction of channels available for activation was measured as peak current during test pulses. Data from individual oocytes were fitted by Im = Imin + Imax/{1 · exp[z(V1/2 - Em)F/RT]}. Data points correspond to means and error bars to ±SE of averaged normalized currents. Continuous lines show Boltzmann distributions that fitted averaged data of alpha 1E.

alpha 2delta increases alpha 1E-gating currents without concomitant increases in ionic currents. The coupling between the movement of the voltage sensor (i.e., charge movement) and pore opening of a channel is the efficiency with which voltage sensing is transduced into an increase in channel open probability and is a parameter determined by both the identity of alpha 1 and the effects that regulatory subunits and other modifiers may have on the alpha 1-subunit. We reported previously that the alpha 1E-channel shows a tight coupling between voltage sensing and pore opening when compared with that of alpha 1C (20, 21). We also reported that beta -subunits significantly improve this coupling efficiency, possibly by reducing the number of nulls (20). We now tested the effect of alpha 2delta on the efficiency with which voltage sensing is coupled to pore opening.

To study the effect of alpha 2delta on coupling efficiency in alpha 1E, we determined both the limiting conductance (Gmax) and limiting charge movement (Qmax) as a function of voltage by using the same approach described previously (21), and then related both values as seen for alpha 1E-channels in Xenopus oocytes injected with alpha 1E alone or with combinations of beta - and alpha 2delta -subunits. Previously, we reported that the charge moved reaches a maximum around +40 mV for channels formed in oocytes injected with alpha 1E alone or a mixture of alpha 1E and beta 2a. To confirm that all of the alpha 1E charge was moved at +40 mV when coexpressed with alpha 2delta , we determined charge movement as a function of increasing test potentials (Q-V curves) for channels formed by injection of alpha 1E/beta 1b/alpha 2delta . The results showed that the Q-V curves of alpha 2delta -containing channels (Fig. 6A) are identical to those of alpha 1E- and alpha 1E/beta 2a-channels reported previously (21). Therefore, the Qmax values were computed by integrating the maximal gating current developing at the reversal potential (approximately +60 mV, experimentally determined for each oocyte). The Gmax was then obtained by dividing the peak tail current that developed at -50 mV after a 25-ms conditioning pulse to +132 mV by the driving force E, given by the following equation: E = Em - EBa = -50 mV - (+60 mV) = -110 mV, where Em is membrane potential and EBa is the apparent reversal potential at 10 mM external Ba2+. The Gmax/Qmax was then used as the measure of coupling efficiency, interpreting a higher Gmax/Qmax as an indication of a higher coupling efficiency between charge movement and pore opening. Figure 6 shows the Gmax/Qmax for alpha 1E-channel by injection of alpha 1E alone and in combination with regulatory subunits. In contrast to beta -subunits, which increased the Gmax/Qmax of alpha 1E by more than twofold, the alpha 2delta -subunit decreased the alpha 1E Gmax/Qmax to less than one-half, from 0.42 ± 0.03 S/µC (n = 5) to 0.25 ± 0.02 S/µC (n = 7). Likewise, alpha 2delta decreased the Gmax/Qmax of alpha 1Ebeta 2a from 0.91 ± 0.10 S/µC (n = 5) to 0.46 ± 0.03 S/µC (n = 8) and that of alpha 1Ebeta 1b from 1.07 ± 0.15 S/µC (n = 7) to 0.35 ± 0.03 S/µC (n = 7). We found that the reduced coupling efficiency correlated with an increase in the charge moved (Qmax). For example, the Qmax of alpha 1E/beta 1b and alpha 1E/beta 1b/alpha 2delta are 104 ± 30 pC (n = 6) and 438 ± 53 pC (n = 7), respectively. These data are consistent with published reports showing that alpha 2delta increases charge movement of alpha 1C or the amount of alpha 1C-subunit in the plasma membrane in either Xenopus oocytes or HEK 293 cells (1, 27). Interestingly, unlike what happens with alpha 1C channels, the increase in alpha 1E charge movement is not accompanied by a significant increase of ionic current. It will be interesting to determine whether alpha 2delta affects charge movement in other alpha 1-subunits.


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Fig. 6.   alpha 2delta reduces coupling efficiency between charge movement and pore opening. A: representative charge movement (Q)-voltage (V) relationship of alpha 1C/beta 1b/alpha 2delta -channel. Gating currents from oocytes coinjected with alpha 1E, beta 1b, and alpha 2delta were recorded in an extracellular solution containing 2 mM Co2+ and 200 mM La2+. Values of Q were obtained by integrating gating currents during pulse after baseline correction. Data represent means ± SE from 3 oocytes. B: representative records of gating currents obtained at reversal potential of approximately +60 mV. Records are part of data sets for G-V relationships shown in Fig. 3. Qmax values are time integrals of gating currents between 0 and 25 ms. C: limiting conductance (Gmax)/limiting charge movement (Qmax) of alpha 1E-channel in combination with different beta - and alpha 2delta -subunits. Gmax values were obtained by dividing peak tail currents in 10 mM Ba2+ at -50 mV after a voltage step to +132 mV, by Ba2+ driving force (Em - EBa) = -110 mV, where Em (-50 mV) is membrane potential and EBa (60 mV) is apparent (experimental) reversal potential for Ba2+. Data points correspond to means and error bars to ±SE of number of tests shown.

In summary, our observations indicate that alpha 2delta modulates most aspects of voltage-dependent responses of alpha 1E: its activation and deactivation kinetics and its inactivation. Thus, as seen in time course studies, alpha 2delta increases both the rate at which the channel opens in response to depolarization and that at which it closes in response to repolarization. Indeed, alpha 2delta converted alpha 1E into a channel that opens more rapidly to changes in membrane potential at all potentials tested, and this was independent of coexpression of an exogenous beta -subunit. As deduced from steady-state analysis, the alpha 1E-channel under control of alpha 2delta requires higher potentials for 50% activation (Fig. 3). It should be noted that the rate of deactivation measured at -50 mV was increased more than the rate of activation at +50 mV (Fig. 2G). This accounts for the finding that G-V curves were shifted along the voltage axis to more negative potentials in the face of facilitated activation (Fig. 3). We saw no evidence for a strong interaction between the effects of beta -subunits on activation and those of the alpha 2delta , i.e., channel kinetics were regulated by alpha 2delta regardless of the presence of a beta -subunit and beta -subunits displayed their characteristic effects on both activation and inactivation in the absence and in the presence of alpha 2delta , with the exception that even though beta -regulated channels responded to alpha 2delta with faster inactivation kinetics, at steady state the effects of alpha 2delta were almost undetectable in alpha 1Ebeta 1b-channels.

The regulatory effect of alpha 2delta is dependent on the type of alpha 1-subunit. For some of the alpha 1-subunits, it increases the ionic current size (alpha 1S, alpha 1C, alpha 1D, alpha 1A, and alpha 1B) (3, 12, 15, 16, 27, 38) or drug binding sites (alpha 1S, alpha 1C, alpha 1A, and alpha 1B) (31, 34, 36), and for other alpha 1-subunits, it changes the rate of activation and inactivation in the presence of beta  (alpha 1A and alpha 1C) or absence of beta -subunit (alpha 1E and alpha 1C) (28, 37). Based on these results, we conclude that the function of alpha 2delta -subunit is not limited to playing a role in determining the subcellular distribution of Ca2+ channels or to facilitating assembly and targeting of Ca2+ channels to the cell surface (27) but that it also functions as a regulatory subunit modulating voltage-dependent activation and inactivation and the coupling efficiency between charge movement and pore opening. The most notable general effect of alpha 2delta on alpha 1E is to increase the speed at which the channel opens and closes in response to voltage. We conclude that, like beta -subunits, alpha 2delta contributes to the fine-tuning of Ca2+ channel activity.

    ACKNOWLEDGEMENTS

This study was supported in part by a National American Heart Association Scientist Development Grant (to N. Qin), by a Greater Los Angeles American Heart Association grant-in-aid (to R. Olcese), and by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants AR-38970 (to E. Stefani) and AR-43411 (to L. Birnbaumer).

    FOOTNOTES

Address for reprint requests: L. Birnbaumer, Dept. of Anesthesiology, UCLA School of Medicine, BH-612, CHS, Box 951778, Los Angeles, CA 90095-1778.

Received 20 October 1997; accepted in final form 22 January 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results & Discussion
References

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AJP Cell Physiol 274(5):C1324-C1331
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