Voltage-gated calcium channels are composed of a main pore-forming α1 moiety, and one or more auxiliary subunits (β, α2δ) that modulate channel properties. Because modulatory properties may vary greatly with different channels, expression systems, and protocols, it is advantageous to study subunit regulation with a uniform experimental strategy. Here, in HEK 293 cells, we examine the expression and activation gating of α1E calcium channels in combination with a β (β1–β4) and/or the α2δ subunit, exploiting both ionic- and gating-current measurements. Furthermore, to explore whether more than one auxiliary subunit can concomitantly specify gating properties, we investigate the effects of cotransfecting α2δ with β subunits, of transfecting two different β subunits simultaneously, and of COOH-terminal truncation of α1E to remove a second β binding site. The main results are as follows. (a) The α2δ and β subunits modulate α1E in fundamentally different ways. The sole effect of α2δ is to increase current density by elevating channel density. By contrast, though β subunits also increase functional channel number, they also enhance maximum open probability (Gmax/Qmax) and hyperpolarize the voltage dependence of ionic-current activation and gating-charge movement, all without discernible effect on activation kinetics. Different β isoforms produce nearly indistinguishable effects on activation. However, β subunits produced clear, isoform-specific effects on inactivation properties. (b) All the β subunit effects can be explained by a gating model in which subunits act only on weakly voltage-dependent steps near the open state. (c) We find no clear evidence for simultaneous modulation by two different β subunits. (d) The modulatory features found here for α1E do not generalize uniformly to other α1 channel types, as α1C activation gating shows marked β isoform dependence that is absent for α1E. Together, these results help to establish a more comprehensive picture of auxiliary-subunit regulation of α1E calcium channels.

Voltage-gated calcium channels are molecular transducers that trigger cellular processes ranging from muscle contraction to neurotransmission. Modulation of these channels thereby constitutes a key potential mechanism for functional adaptation and plasticity. At least three different subunits are believed to comprise native calcium channels: a main, pore-forming α1 subunit, a cytoplasmic β subunit, and a disulfide-linked α2δ subunit (for review, see Perez-Reyes and Schneider, 1994; De Waard et al., 1996). So far, seven different genes encoding α1A,B,C,D,E,S,G subunits, and four different genes encoding β1,2,3,4 subunits have been identified, along with multiple splice variants. Given this heteroligomeric structure, regulation of channel properties by variations in subunit composition have been widely studied as a potential mechanism for tuning channel gating properties to support a given physiologic role.

Despite the potential importance of modulation by subunit combination, fundamental uncertainties remain about the effects of auxiliary subunits (for review see Perez-Reyes and Schneider, 1994; De Waard et al., 1996; Walker and De Waard, 1998). While coexpression studies have demonstrated that the addition of auxiliary subunits (β, α2δ) can have striking effects on channel gating and/or channel expression, the specific effects observed vary across studies, even using the same α1 subunit. At least some of the differences in subunit effects may reflect isoform-specific variations in the effects of distinct β subunit isoforms on α1 gating. Further variability may arise from the use of diverse expression systems, electrophysiological methods, and experimental solutions. These points underscore the need to explore subunit modulation of each α1 isoform individually, and to undertake comprehensive studies with uniform experimental conditions.

Although most previous work has focused on α1C, neuronal α1E channels (Soong et al., 1993) have recently emerged as important channels with which to attempt such comprehensive investigation for several reasons. First, subunit modulation of α1E has potential physiological relevance, as α1E (presumed “R-type”) channels have been implicated in neuronal functions including neurotransmitter release (Wu et al., 1998). Second, α1E demonstrates an exceptional capacity for high-level recombinant expression, which permits well-resolved measurements of both ionic and gating currents, even when the α1E subunit is expressed alone (which generally lowers overall expression of current). This capability enables examination of changes in both peak open probability and channel density (Olcese et al., 1996), two critical measures for resolving how auxiliary subunits affect the overall level of calcium current. Third, β subunits may affect α1E expression in a uniquely different manner than observed with other pore-forming α1 subunits, providing a potentially useful clue as to the underlying mechanism of subunit modulation. Olcese et al. (1994, 1996) provide the most biophysically detailed results in this regard, using Xenopus oocytes. In contrast to other α1 subunits, β subunits caused little change or even a decrease in overall α1E current density. This outcome resulted from decreased channel density, as assessed by maximal gating charge, countered by increased channel opening. By contrast, in mammalian expression systems, β subunits increased overall α1E current density (Williams et al., 1994; Stephens et al., 1997). Here, however, no α1E gating-current measurements have been made to permit assessment of underlying changes in channel density and open probability. Finally, α1E is one of the channels in which a second β binding site has been explicitly identified (Tarelius et al., 1997; Walker et al., 1998). Characterization of mutant α1E constructs lacking this site would allow determination of the functional importance of the secondary site.

Here, we therefore examine subunit modulation of α1E channels coexpressed with various combinations of auxiliary subunits (β1–β4, α2δ) in mammalian HEK 293 cells. The same recombinant expression system, along with a consistent set of experimental solutions and protocols, is used throughout to facilitate direct comparison of channels with differing molecular composition. Measurements of both ionic and gating currents permits in-depth analysis of subunit modulatory effects. We focus on three key questions. (a) To what degree does modulation of α1E current density reflect modulation of channel gating and/or number of functional channels? (b) How do different auxiliary subunits compare with regard to modulation of activation gating? (c) What is the functional impact of the secondary β binding site in α1E? Through addressing these questions, this study helps to establish a more refined picture of auxiliary-subunit modulation of α1E calcium channels.

Expression of N-Type Channels

HEK 293 cells, obtained from Dr. Jeremy Nathans (Johns Hopkins University; Gorman et al., 1990), were grown at 37°C in Dulbecco's modified Eagles medium (GIBCO BRL, Grand Island, NY), 10% fetal calf serum (GIBCO BRL), 1% L-glutamine (Sigma Chemical Co., St. Louis, MO), 1% penicillin-streptomycin (P0906; Sigma Chemical Co.), in 5% CO2. Low-passage number cells were used (<P20). cDNAs encoding channel subunits α1E (Soong et al., 1993), α1C (Wei et al., 1991), β1b (Pragnell et al., 1991), β2a (Perez-Reyes et al., 1992), β3 (Castellano et al., 1993b), β4 (Castellano et al., 1993a), and α2δ (Tomlinson et al., 1993) were subcloned into mammalian expression plasmids (pMT2; Genetics Institute, Cambridge, MA, for β4, pZEM229R; ZymoGenetics, Inc., Seattle, WA, for α2δ, pGW1; British Biotechnologies, Cowley, Oxford, UK for all others). α1EΔ was constructed by replacing the Bst 1107I (α1E: nucleotide 4299, given start codon at nucleotide 1) and SalI (3′ polylinker) region of α1E in pGW1 with a shorter polymerase chain reaction fragment, including a premature stop codon after the codon for amino acid 1871. The portion of the channel derived from PCR was verified in its entirety with the use of the fluorescent dideoxy terminator method of thermocycle sequencing on an automated DNA sequencer (Applied Biosystems Division 373a; Perkin-Elmer Cetus Instruments, Emeryville, CA). HEK 293 cells were transiently transfected using a standard, calcium-phosphate precipitation procedure (Brody et al., 1997) with a total of 30 μg of DNA per 10-cm plate. 10 μg of a plasmid containing a pore forming subunit was included (α1E or α1C) and mixed with 10 μg of each desired auxiliary subunit (none, a β subunit, and/or the α2δ subunit). If the amount of DNA totaled <30 μg, pBluescript was added to make up the difference. For certain experiments, both β2a and β3 were simultaneously transfected either in a 1:1 ratio (10 μg of each plasmid) or a 5:1 ratio (15 μg of β3, 3 μg of β2a). More than 20% of cells transfected with a pore forming subunit exhibited detectable high-threshold calcium currents.

“Mock-transfected” cells were transfected with 10 μg of β1b, 10 μg of α2δ, and 10 μg of pBluescript. In our usual ionic current recording conditions (detailed below), we observed no high threshold, voltage-gated, calcium-channel currents in such cells (n = 32 cells, over two independent rounds of transfection), or in cells transfected with the β2a subunit alone (n > 40 cells; Patil et al., 1998). In mock-transfected cells, we occasionally (∼10% of cells) observed endogenous, low threshold calcium channel currents of small amplitude (peak ionic current ∼20 pA in 10 mM Ba2+), as reported previously by Sun et al. (1994). Although endogenous currents of such small amplitude would contribute negligibly to our results, cells with low threshold activity were nevertheless rejected. At the biochemical level, Western blots performed on total membrane protein (30 μg/lane) from untransfected cells revealed no known high threshold α1 (A, B, C, D, E) or β (1b, 2e, 3a, 4) subunits, and only low levels of α2δ (personal communication, Mark Williams, SIBIA Neurosciences Inc., La Jolla, CA). Blots were probed individually with appropriate antibodies, and the lack of subunit proteins was gauged from the absence of bands that were clearly present using cells transfected with corresponding recombinant subunits. The result that coexpression of α2δ with α1E potentiated current by approximately threefold suggests that trace expression of endogenous α2δ did not significantly influence our results.

Electrophysiology

Whole-cell recordings were obtained at room temperature 48–72 h after transfection using an Axopatch 200A (Axon Instruments, Foster City, CA) and standard patch-clamp techniques. Cell capacitance ranged from 10–40 pF. Series resistance was typically <5 MΩ, and compensated 70–85%, resulting in a typical settling time of ∼80 μs. Voltage pulses were delivered every 15–20 s from a holding potential of −110 mV, except for prepulse inactivation protocols, where voltage pulses were given every minute from a holding potential of −120 mV to allow recovery from inactivation. Data were typically acquired at 50 kHz and filtered at 10 kHz (−3 dB, four-pole Bessel). Displayed traces have generally been additionally processed with a gaussian digital filter at 2 kHz. Leak and capacity currents were subtracted by a P/8 protocol (ionic currents) or P/−8 protocol (gating currents) from the −110-mV holding potential, unless otherwise noted (Armstrong and Bezanilla, 1974). To allow better resolution of small currents, we often subtracted a smooth curve fitted to the leak currents. In some cases, the first 200 μs after a voltage step contains a large leak subtraction artifact, which was zeroed when present before digital filtering.

The base external solution contained (mM) 155 N-methyl-d-glucamine (NMG) aspartate, 10 HEPES, 10 4-aminopyridine, 0.1 EGTA, pH 7.4 with NMG, 280–300 mOsm with no added charge carriers. The internal solution contained (mM) 150 NMG-methanesulfonate (MeSO3), 1 MgCl2, 4 MgATP, 10 HEPES, 10 EGTA, pH 7.3, with NMG, typically 280–290 mOsm. The h(∞)-V relations shown in Fig. 11 for α1C were obtained with an internal solution in which the 150 mM NMG-MeSO3 was replaced by 150 mM Cesium-MeSO3. For measurement of ionic currents, either 2 or 10 mM BaCl2 was added to the external solution. For typical gating current measurements, 0.2 mM LaCl2/2 mM MgCl2 was added. External solution flowed continuously at a rate of 1–2 ml/ min during recording. The bath solution was grounded by a 0.5 M KCl agar bridge attached to a Ag-AgCl wire. Measurements were started after >5 min of dialysis with the internal solution. In all cases, the junction potential between external and internal solutions was ∼5 mV (Neher, 1992). To determine the true applied potential, this value should be added to the voltages in the figures and text.

For measurement of α1E activation curves, 2 mM BaCl2 was the charge carrier throughout. Test depolarizations were 30 ms long and ranged from −70 to +70 mV (see Fig. 2 A, top) with repolarization to −50 mV to allow good resolution of tail currents. For each cell, plots of peak tail current at −50 mV (Itail) vs. test pulse voltage (Vtest) were normalized by an estimate of maximal peak tail current (Itail,max). Itail,max was taken as the saturating value of Boltzmann fits to the Itail-Vtest data. The resulting normalized relations are equivalent to normalized Po-V relations, and are referred to as G-V curves. G-V curves were then averaged across cells. Such G-V curves were indistinguishable from G-V relations obtained using 15-ms test depolarizations (data not shown). We did not correct tail currents for the contribution of the “OFF” gating current. To assay the magnitude of the error that such OFF gating currents might produce, we corrected G-V relations for six cells transfected with α1Eβ2a by subtracting the OFF gating currents measured during repolarization to −50 mV. We found that the average single-Boltzmann fit parameters for the corrected and uncorrected G-V curves were statistically indistinguishable (P < 0.05, Student's t test, uncorrected: z = 3.51 ± 0.4, V1/2 = −20.1 ± 3.2 mV; corrected: z = 3.57 ± 0.5, V1/2 = 19.7 ± 3.4 mV), although Itail,max was reduced by ∼5% (−2,638 ± 568 pA [uncorrected] vs. 2,516 ± 542 pA [corrected]). Gmax was calculated according to Gmax = Itail,max/(V − Vrev), where Vrev was +40 mV in 2 mM BaCl2. Therefore, the small error in Itail,max will lead to a slight overestimate of the Gmax/Qmax ratio, which may vary slightly for the different subunit combinations.

For gating currents, ionic currents were blocked by the external solution containing 0.2 mM LaCl3 (Bean and Rios, 1989). The effective free La3+ concentration was 0.1 mM due to the presence of 0.1 mM EGTA in all external solutions. The voltage protocol was the same as for ionic currents, except that the test pulse duration was decreased to 15 ms, and repolarization to −110 mV (see Fig. 5 A, top). Total charge moved during test depolarization (Qon) was obtained by integrating over the entire depolarizing epoch, taking as the zero baseline the average current over the last 3 ms of the test pulse. Total charge moved during repolarization (Qoff) was calculated similarly. For each cell, Qon-V and Qoff-V curves were normalized by an estimate of maximal mobile charge (Qmax), taken as the saturating value of the Boltzmann fit (detailed below) to the Qon-V or Qoff-V curves, as indicated in the text. Such normalized Qon-V and Qoff-V curves were averaged across cells.

To ensure that La3+ does not alter activation gating, we obtained Q-V relations both in the presence and absence of La3+ blockade. Fig. 1 A shows the results of the analysis, in which we compared Qoff-V curves acquired in 2 mM MgCl2 (•) and 2 mM MgCl2/0.2 mM LaCl3 (○). The identity of the two curves, absent the expected surface-potential shift, provides additional strong support that La3+ does not perturb activation gating.

To determine explicitly the surface-charge shift between solutions used for ionic and gating currents, we exploited the property that isolated gating currents can actually be measured in the solution for ionic current, so long as the voltage range is negative to the threshold (∼−65 mV, Fig. 1,B, inset) for ionic-current activation. We could then calculate the surface-charge shift by direct comparison of the rising “foot” of Q-V curves obtained in ionic and gating current solutions. Fig. 1,B shows the results of this approach. Before averaging across cells, Q-V data for a single cell was normalized by the value of Qon at −65 mV in 2 mM Ba2+. In the ionic-current solution containing 2 mM Ba2+, the Qon-V (Fig. 1,B, inset, ○) and Qoff-V (inset, •) curves matched at potentials negative to −65 mV, indicating that gating currents were isolated below this potential. The main graph in Fig. 1 B demonstrates that, over this range of voltages, Qon-V relations obtained in 2 mM Ba2+ (○) and 2 mM MgCl2/0.2 LaCl3 (⋄) are essentially indistinguishable, indicating that there is little if any surface-charge shift between solutions. To quantitate the value of the shift, for each cell the voltage shift required to fit the same dual-Boltzmann to both sets of Qon-V data was taken to be the surface potential difference. Averaging this value across cells gave a value of 3 ± 1 mV (n = 9). These results excluded the need for surface-charge correction between ionic and gating current measurements.

Steady state inactivation curves were approximated using a protocol in which a 20-s prepulse was followed by a step to peak of the current–voltage (I-V)1 curve (typically −5 mV) to measure the fraction of inactivated current. In some cases, a 10-ms normalizing prepulse at the test pulse potential was included before the 20-s prepulse to assay for the presence of cumulative inactivation or rundown. Steady state inactivation (h(∞)-V) curves were derived by normalizing test pulse currents by either the current during the normalizing test pulse, or by the value of the test pulse with no prepulse. All steady state inactivation curves were measured with 10 mM Ba2+ as charge carrier. Voltage commands were given every minute from a holding potential of −120 mV. Typically, prepulse voltages ranged from −120 to −20 mV in 10-mV increments. Normalized h(∞)-V relations were averaged across cells. For cells transfected with two β subunits, the h(∞)-V relation was fit with a dual-Boltzmann function to obtain parameters for the low and high threshold components, in addition to the relative contribution of each component.

Boltzmann fits to either G-V or Q-V relations were performed with functions of the form B(V) = Bmax/{1 + exp[−zF(V − V1/2)/ RT]}, where Bmax is the saturating value, z is the effective charge, and V1/2 is the midpoint of activation. Qon-V data above +40 mV were sometimes unreliable and were therefore excluded.

For dual-Boltzmann fits to h(∞)-V relations, we used a function of the form B(V) = flow{1 + exp[zlow F(V − V1/2,low)/RT]}−1 + fhigh{1 + exp[zhigh F(V − V1/2,high)/RT]}−1, where V1/2,low and V1/2,high are midpoints of activation, zlow and zhigh are the effective valences, and flow and fhigh are amplitudes of low and high threshold components. Fits were obtained using nonlinear, least-squares minimization. All reported values are mean ± SEM.

Enhancement of Expressed Current Density by Auxiliary Subunits

Transfection of HEK 293 cells with the α1E subunit alone, or in combination with various auxiliary subunits, led to the expression of well-resolved inward barium currents carried by recombinant calcium channels (Fig. 2,A). The relative magnitudes of the various sets of traces illustrate that addition of auxiliary subunits caused striking increases in the level of expressed current. To quantify the relative increase in current density, we calculated the maximum tail current upon repolarization to 50 mV [Gmax = nPo,max g(−50 mV)h], where g is the unitary conductance, h is the fraction of noninactivated channels at the end of the test pulse, n is the number of channels, and Po,max is the maximum open probability. Fig. 2,B compares the average values of Gmax for all different subunit combinations examined. The largest effect was the ∼12-fold enhancement of expressed current with the coexpression of β subunits. All β subunits were approximately equipotent in this regard, although the average β3 effect was slightly smaller (approximately sevenfold). Addition of α2δ to α1E produced a weaker increase in current (about threefold), and the combination of α2δ and β subunits yielded no appreciable current enhancement over the coexpression of β subunits alone. Since modulation of Gmax values may reflect not only changes in nPo,max, but also differences in the number of noninactivated channels (h) with different subunits, we examined another measure of current density (Fig. 2 C), Ipeak = nPo[Vpeak] i[Vpeak], where Po[Vpeak] and i[Vpeak] are the open probability and unitary current at the voltage (Vpeak) yielding the maximum test-pulse current. This measure (Ipeak), which is less sensitive to test pulse inactivation, gave similar results. Therefore, we are confident that Gmax can henceforth be used as a quantitative indicator of relative changes in current (nPo,max).

Isolation of Gating Currents from Channels Containing the α1E Subunit

To determine the origin of the increased current density (nPo,max), we wished to measure the maximum amount of mobile gating charge (Qmax = nq, q is the charge per channel), which provides a convenient assay for the relative number of functional channels (n). Measuring Qmax involves good resolution of the currents arising from gating charge movement (gating currents; Armstrong and Bezanilla 1977; Sigworth, 1994), which in turn requires a blocker that eliminates ionic currents without significantly perturbing channel gating behavior. Previous work indicates that the highly potent block by La3+ can be used to isolate gating currents of calcium channels containing the α1B subunit (Jones et al., 1997a), but not the α1C subunit (Kamp et al., 1996). To determine the feasibility of La3+ blockade of calcium channels containing α1E, we examined gating currents with either 2 mM Ba2+ or 2 mM Mg2+/0.2 mM La3+ added to the bath solution (Fig. 3,A). Although 2 mM Mg2+/0.2 mM La3+ (solid traces) completely blocked ionic currents, the early outward transients that are dominated by gating current were unchanged, arguing strongly that La3+ does not alter the voltage sensor movement that underlies activation gating. Furthermore, the block of ionic current was completely reversible (Fig. 3,B, •), and did not alter Qrev during repetitive stimulation in the presence of La3+ (○, obtained by integrating outward transients at the reversal potential). The lack of change of Qrev argues that La3+ does not promote channel inactivation, which would be apparent as a reduction in Qrev (Jones et al., 1997b) known as gating-charge immobilization (Armstrong and Bezanilla, 1977; Bezanilla et al., 1991). Similar results to those for α1E + β2a (Fig. 3, A and B) were obtained with the other subunit combinations (data not shown). Further experiments (see materials and methods) demonstrated that La3+ did not affect the voltage dependence of charge movement, and that the surface charge shift between solutions used for ionic and gating current measurements was ∼3 mV.

With assurance that La3+ does not detectably alter gating or surface-charge properties, we turned to analysis of extensive sets of currents recorded during La3+ block for α1Eβ2a (Fig. 4,A). These traces represent genuine calcium-channel gating currents for several reasons. First, no such currents are observed in mock-transfected cells (Fig. 4,B). Second, no nonlinear charge movement is present in the range of our leak pulses (Fig. 4,C). Third, the measured charge movement is not affected by the choice of the leak subtraction protocols (data not shown). Finally, these “nonlinear displacement” currents have the standard properties typically associated with gating currents (Fig. 4,D): time integrals of outward (Qon) and inward (Qoff) displacement currents saturated with increasing test depolarization; Qoff ∼ Qon in the absence of inactivation (Fig. 4,D); charge movement (Qon-V or Qoff-V curves) occurs before, and then parallels, ionic-current activation (G-V curve); and finally, the maximal amount of gating-charge (Qmax) is linearly correlated with maximal current density (Gmax) (Fig. 4 E).

Mechanism of Current Potentiation by Auxiliary Subunits

With the ability to isolate gating currents, we could now compare auxiliary subunits with regard to their mechanism for current potentiation. Fig. 5,A displays representative gating-current records for most of the different subunit combinations. These traces illustrate that all auxiliary subunits boost the maximum amount of gating charge (Qmax), which is taken as the saturating value of the Boltzmann fit to the Qon-V relation. Fig. 5,B compares the average values of Qmax for all different subunit combinations examined. Qmax for channels expressed from α1E alone was characteristically small, with a mean of 0.8 ± 0.1 fC/pF (n = 9). β Subunits induced the strongest enhancement of Qmax, ranging from fourfold for β3 to sevenfold for β4. Coexpression of α2δ also produced clear augmentation of Qmax, though the effect was less potent than for β subunits. Table I summarizes the complete details of the analysis. Given that gating-charge per channel (q) does not appear to be affected by auxiliary subunits (Noceti et al., 1996), the rise in Qmax likely reflects an increase in the number of functional channels (n). Hence, our results indicate that the enhancement of current density by auxiliary subunits arises, at least in part, from an increase in the number of functional channels. Such an increase in the number of functional channels may reflect either improved processing and trafficking of α1E channels (increasing total amount of α1E protein), or an increase in the fraction of functional α1E protein in the membrane (with no increase in total amount of α1E protein) by the α2δ and β subunits.

To determine whether an increase in the maximal open probability (Po,max) also contributes to higher ionic-current densities, we calculated the ratio Gmax/ Qmax, which is directly proportional to Po,max, so long as auxiliary subunits do not alter permeation properties of the channel (as in Fig. 6,D and Noceti et al., 1996). Fig. 5,C shows that all β subunits approximately doubled Gmax/Qmax, but α2δ left the ratio unchanged. Table I reports further details of the calculations. The data in Fig. 5 suggest that β subunits enhance α1E current density by jointly increasing the number of functional channels (as reported by Qmax) and the maximal open probability (as reflected by Gmax/Qmax). The enhancement of current by the α2δ subunit appears to be fundamentally different: there may be a pure increase in the number of functional channels, without change in Po,max.

Subunit Modulation of Activation Gating

A second goal of this study was to compare auxiliary subunit effects on the kinetics and voltage dependence of channel activation. To qualitatively compare activation kinetics for channels with different β subunits, we normalized the rising phases of exemplar ionic-current records (Fig. 2,A) evoked by voltage steps to −30, 10, and +10 mV (Fig. 6,A). The identical trajectories of traces from all four β subunits suggest that β subunits produce channels with similar activation kinetics. Fig. 6 B shows the identical analysis for channels expressed from α1E alone (solid trace) or from α1E + α2δ (dashed trace). The records for α1E + β1b (gray traces) are reproduced for comparison. Here again, the close correspondence between traces suggests that auxiliary subunits do not significantly modulate activation kinetics.

To examine whether auxiliary subunits affect the steady state voltage dependence of activation (G-V), we tested for subunit-dependent changes in G-V curves derived from peak tail currents (see materials and methods) (Fig. 6,C). Coexpression of the α2δ subunit had little effect on the G-V (Fig. 6,C) or I-V (Fig. 6,D) relations. The lack of effect of α2δ on the kinetics and voltage dependence of activation, as well as on Gmax/ Qmax (Fig. 5,C), suggests that this subunit is functionally uncoupled from any aspect of activation in α1E. In contrast, single-Boltzmann function analysis (Fig. 6,C, solid curves, and Table II) clearly demonstrates that coexpression of β subunits produces an ∼7-mV hyperpolarizing shift and a modest increase in the steepness of G-V relations (e.g., the Boltzmann valence (z) increases from 2.4 for α1E to 3.6 for α1Eβ2a). As expected from the shift in the G-V relation, coexpression of β subunits shifted the peak of the I-V relation leftward (Fig. 6 D) without altering the reversal potential.

The results in Fig. 6, C and D, are compatible with earlier work on β subunit effects in Xenopus oocytes (Olcese et al., 1994), in which G-V relations were fitted with dual-Boltzmann functions. In agreement with the earlier report, application of dual-Boltzmann analysis to our G-V data (Fig. 6,C, dashed curves) suggests that the apparent hyperpolarization and steepening of activation by β subunits could arise from an increase in the proportion of the low threshold Boltzmann component from ∼30 to ∼70%, without change in valence or midpoint parameters of individual Boltzmann functions. More in-depth interpretation of the data, like that introduced by dual-Boltzmann analysis, is deferred to the discussion, where explicit fits of a multistate kinetic model will be employed. For simplicity, in the remainder of the results, we retain single-Boltzmann analysis for first-order characterization of experimentally resolvable changes in activation. Regardless of the particular analytical functions used to describe the data, the results thus far (Figs. 5 and 6, A–D) clearly indicate that β subunits increase ionic current by simultaneously modulating the G-V relation and doubling the Gmax/ Qmax ratio, in agreement with the findings of Olcese et al. (1994, 1996).

To explore the mechanistic basis of the β subunit effects on activation, we investigated how auxiliary subunits influenced Q-V curves derived from gating currents (Fig. 6,E). The rising phase of Q-V curves is very sensitive to modulation of the early events in the activation pathway, and the interrelation of Q-V and G-V curves lends insight into steps that couple voltage sensor movement to channel openings (Jones et al., 1997a). Fig. 6,E illustrates that all β subunits produced essentially identical effects on the Q-V relation: a small hyperpolarizing shift in the midpoint (∼5 mV) with little change in the steepness (Boltzmann valence [z] ranges from 2.5 to 2.8, Table II). The effects of β subunits on Q-V curves are smaller than on G-V curves, thereby narrowing the gap between Q-V and G-V relations along the voltage axis. As expected from previous null results, α2δ had no effect on the Q-V relation. All the β subunit effects on gating (Figs. 5,C and 6, C–E), particularly the contraction between Q-V and G-V curves, fit nicely with the idea that all β subunits act primarily to modulate a single locus of weakly voltage-dependent steps late in the activation pathway (see discussion).

Functional Stoichiometry of β Subunit Interaction

Previous reports in Xenopus oocytes indicate that α1E channels containing different β subunits have strikingly different inactivation characteristics, despite very similar activation gating (Olcese et al., 1994). Here, we sought to confirm this effect in HEK 293 cells so that we could exploit this property to test whether multiple β subunits can simultaneously define the functional behavior of a calcium channel. To assay inactivation properties, we used a 20-s prepulse followed by a test pulse to the peak of I-V relations (Fig. 7,A). Typical currents for α1Eβ2a and α1Eβ3 channels illustrate the extremes of inactivation behavior observed with the different subunit combinations. β2a dramatically slowed inactivation, while β3 accelerated inactivation. β1b and β4 subunits also accelerated inactivation during the test pulse (not shown), though not as strongly as β3. To provide a robust indication of the differences in inactivation properties, we used such records to calculate steady state inactivation curves (h(∞)V curves; Fig. 7,B). While addition of α2δ did not affect the h(∞)V relation, coexpression of β subunits induced striking modulation of steady state inactivation: β1b, β3, and β4 all left-shifted h(∞)V curves by ∼10, 15, and 10 mV, respectively; β2a imparted a right shift of ∼15 mV. The profound distinction between the effects of β2a and the other β subunits has been reported in previous studies of α1E (Olcese et al., 1994), and of other neuronal calcium channels, including α1A (Stea et al., 1994) and α1B (Patil et al., 1998). Table III summarizes the Boltzmann analysis of h(∞)V data.

To investigate whether multiple β subunits can concomitantly specify the functional properties of a single calcium channel, we took advantage of the vast difference between the h(∞)V relations for α1Eβ3 and α1Eβ2a channels. If there are multiple β subunit sites on α1E that specify inactivation properties, then cotransfection of both β2a and β3 subunits should result in mixed-composition channels (e.g., α1Eβ2aβ3) whose inactivation behavior should be distinct from that of pure α1Eβ2a- or α1Eβ3-like channels. However, if there is only one functionally active β subunit site per channel, the aggregate h(∞)V relation should possess only two components. Fig. 8, A and B, shows the results for one such experiment in which β3 and β2a were cotransfected in a 1:1 weight ratio. This example demonstrates that inactivation is clearly biphasic, with a low threshold, readily inactivating component, as well as a high threshold, inactivation-resistant component. Only two Boltzmanns are required to produce an excellent fit of the data since the average residual for the dual-Boltzmann fit is close to zero (Fig. 8,C). Furthermore, the average fit parameters to the h(∞)V data correspond closely to the steady state inactivation properties of pure α1Eβ2a and α1Eβ3 channels (Fig. 7,B and Tables III and IV). Cotransfection of β3 and β2a in a 5:1 weight ratio merely decreased the relative amplitude of the low threshold component (Fig. 8,D), while preserving the intrinsic properties of the two components (Table IV). The only apparent deviation from parameters obtained with pure-composition channels is a small 7–9-mV increase in the V1/2 for the high threshold component (compare Tables III and IV). Although this increase could reflect a minor contribution of mixed-composition channels, the overall results are consistent with the functional dominance of pure α1Eβ2a and α1Eβ3 channels.

As a further test for the possible functional role of a second β subunit site (Tareilus et al., 1997), we examined how auxiliary subunits modulated the properties of a COOH-terminal truncation of the α1E construct (α1EΔ, amino acids 1–1871 of α1E [1–2251]) that lacks the secondary binding site. Fig. 9,A displays ionic currents for channels composed of α1EΔ+ α2δ or α1E + β2a + α2δ subunits. Coexpression of β2a with α1EΔ increased Gmax from −306 ± 115 pS/pF (n = 11) to −1,708 ± 251 pS/pF (n = 4), a 5.6-fold increase similar to the 4.1-fold increase in Gmax seen for wild-type α1E (Fig. 2,B). Similarly, modulation of activation by β2a is unchanged by the COOH-terminal deletion, as demonstrated in Fig. 9,B by the identical subunit modulation of G-V relations for α1EΔ (circles) and wild-type α1E (squares). Finally, we compared β subunit modulation of the steady state inactivation properties of α1EΔ (Fig. 9, C, traces, and D, symbols; Table III) with data obtained with wild-type α1E (Fig. 9,D, lines). The α1E data have been shifted uniformly by −7 mV in the Fig. 9,D overlay to account for a difference in inactivation that is present even without β subunit coexpression (e.g., α1EΔ + α2δ in Fig. 9,D); this small shift likely reflects a difference in the intrinsic inactivation behavior of the α1 backbone (Soldatov et al., 1997), rather than a change in the modulatory action of β subunits. The close correspondence between h(∞)-V relations for α1E (lines) and α1EΔ (symbols) in the Fig. 9,D overlay illustrates that β subunit modulation of inactivation is similar for the two constructs. Although the small difference between modulation of α1EΔ and α1E inactivation (most apparent for α1EΔβ3α2δ, Fig. 9,D, ▿) could reflect a minor contribution of a second β subunit binding site, all the results in Figs. 8 and 9 support the view that a single β subunit binding site predominates in specifying inactivation properties. If present, the potential contribution of a second site appears to be small by comparison.

Although auxiliary subunits clearly have a role in defining channel properties, specific modulatory effects vary widely across studies, underscoring the need to examine comprehensively the modulation of each α1 subunit under the same experimental conditions. Here, we have performed a systematic evaluation of auxiliary subunit regulation of expression and gating of α1E calcium channels in HEK 293 cells. The experiments lead to three main conclusions. (a) The α2δ and β auxiliary subunits differ fundamentally in the manner by which they induce an overall increase in current density. Coexpression of α2δ with the pore-forming α1E moiety produced a clear-cut enhancement of current, arising purely from an increase in the number of functional channels (n), without significantly affecting channel gating behavior. By contrast, coexpression of β subunits induced stronger potentiation of current by joint elevation of channel number (n) and maximal open probability (Po,max), suggesting effects on both channel assembly and gating. (b) While α2δ had no appreciable effect on activation gating, β subunits produced significant hyperpolarizing shifts in the voltage dependence of ionic-current activation and gating-charge movement, all without discernible change in activation kinetics. Importantly, different β isoforms produced nearly indistinguishable effects in regard to both current potentiation and activation gating. (c) Little functional evidence for a secondary β subunit binding site was found, fitting with earlier biochemical evidence for a 1:1 stoichiometry of α1 and β subunits for skeletal (De Waard and Campbell, 1995) and neuronal N-type (Witcher et al., 1993) channels. Together, these findings represent an important contribution to clarifying both the mechanism and structural determinants of auxiliary-subunit modulation of calcium channels.

In the sections to follow, we will first relate each of the conclusions to previous studies of α1E and, where relevant, other calcium channels. For clarity, we will discuss α2δ and β subunit effects sequentially, as independent parts. A kinetic mechanism is then developed to explain how β subunits can produce all the observed changes in gating, simply by alteration of the equilibrium between a single, weakly voltage-dependent transition near the open state. Finally, we consider the generality of our conclusions to other α1 isoforms.

Modulation of α1E by α2δ

The α2δ subunit produced an approximately threefold increase in α1E current, which arose almost exclusively from elevated channel expression (Qmax). The α2δ subunit had no other clear modulatory effects, except to slightly antagonize the effect of β3 on inactivation (Fig. 7,B). All measures of activation gating, including the maximal open probability (Gmax/Qmax), the voltage dependence of charge movement (Q-V), and the voltage dependence of ionic activation (G-V) were similar to α1E alone. Similar effects on inactivation and expression were reported for doe1 (marine ray analog of α1E) expressed in Xenopus oocytes (Ellinor et al., 1993). However, in contrast to our results, in studies of rat α1E in COS-7 cells (Stephens et al., 1997) and human α1E in Xenopus oocytes (Wakamori et al., 1994), coexpression of α2δ was found to produce a depolarizing shift in the G-V without modifying expressed current levels; however, these studies agree with our findings concerning the slight antagonism of β3 effects on inactivation (Fig. 7 B).

The applicability of our results to other channel types is unclear. However, it is interesting to note that the reported effects of the α2δ subunit on other α1 subunits also varies widely, sometimes agreeing with our findings, other times not. For example, with regard to modulation of α1C channel density, the α2δ subunit was found to increase ligand binding sites (Welling et al., 1993), protein levels (Shistik et al., 1995), and gating currents (Bangalore et al., 1996). By contrast, in other studies (Wei et al., 1995; Gurnett et al., 1997) of α1C, maximal dihydropyridine binding is not increased by α2δ coexpression. Similarly, activation kinetics of α1C accelerate in some studies (Singer et al., 1991; Bangalore et al., 1996), but not others (Mikami et al., 1989; Welling et al., 1993). The sources of this variability have yet to be determined.

β Subunits Act Differently than the α2δ Subunit

By contrast to the α2δ subunit, coexpression of β subunits (β1–β4) enhanced current density (Gmax) by increasing not only the number of functional channels (Qmax) but also the maximum open probability (Gmax/ Qmax). Similar effects on current density have been reported in COS 7 (Stephens et al., 1997) and HEK 293 cells (Williams et al., 1994), but not in Xenopus oocytes (Soong et al., 1993; Olcese et al., 1994, 1996). Furthermore, in a study of α1E gating currents in Xenopus oocytes (Olcese et al., 1996), coexpression of β2a with α1E actually decreased the number of functional channels (Qmax), although surprisingly they found a twofold increase of Gmax/Qmax that is qualitatively similar to our result. Additional support for the role of the β subunit in modulating Po,max comes from a separate study that used fluctuation analysis to determine the effects of β2a and β1a on α1E open probability (Noceti et al., 1996).

Fitting with the doubling of the Gmax/Qmax ratio, the β subunit also induced hyperpolarizing shifts of both G-V and the Q-V relations and slightly reduced the gap between the two, all while producing little effect on activation kinetics. Here, the action of β subunits is also somewhat controversial. Although most studies of α1E report effects on G-V relations and activation kinetics that are similar to ours (Witcher et al., 1993; Olcese et al., 1994; Stephens et al., 1997), in one case (Wakamori et al., 1994), β1b coexpression with the human α1E in Xenopus oocytes was found to slow activation kinetics substantially. With respect to gating currents, the only other study of α1E charge movement (Olcese et al., 1996) also found that the β subunit reduced the gap between the G-V and Q-V. However, in contrast to the small but statistically significant (P < 0.01, Student's t test, comparing cells with and without a β) shift in the Q-V reported here, Olcese et al. (1996) found that the β subunit produced no significant change in the Q-V. Again, these discrepancies may reflect differences between clones (human versus rat α1E) or expression systems (Xenopus oocytes versus HEK 293 cells). Despite these minor differences, all the results indicate a role for β subunits in modulating activation gating.

In other calcium channels, the reported effects of the β subunits vary even more widely than for the α2δ subunit. However, at least in some respects, β subunit modulation of other α1 subunits appears similar to what we find for α1E. For example, there are reported shifts in the voltage dependence of ionic activation for α1A (Stea et al., 1994; De Waard and Campbell, 1995) and α1C (Wei et al., 1991; Neely et al., 1993). Increased current density has also been observed for many of the α1 subunits including α1A (Mori et al., 1991), α1B (Williams et al., 1992a), α1C (Perez-Reyes et al., 1992), α1D (Williams et al., 1992b), and α1S (Ren and Hall, 1997). Furthermore, for α1C, studies of gating currents in both HEK 293 cells (Kamp et al., 1996; Josephson and Varadi, 1996) and Xenopus oocytes (Neely et al., 1993) find that β subunit modulation of ionic current activation is not associated with much shift in the Q-V, similar to what we find for α1E. On the other hand, even these few gating current studies disagree in other regards. While coexpression of β1a (Kamp et al., 1996) or β3 (Josephson and Varadi, 1996) with α1C increased both current density and Qmax in HEK 293 cells similar to our results for α1E, β2a increased current without changing Qmax in Xenopus oocytes (Neely et al., 1993). Therefore, as with α1E, the specific effects observed with β coexpression appear to depend on as yet unknown distinctions between expression systems, perhaps the endogenous expression of βXO subunits in Xenopus oocytes (Tareilus et al., 1997).

Different β Isoforms Have Similar Effects on Activation and Expression, but Not on Inactivation

There was little isoform dependence to the modulation of all the above measures of activation gating, suggesting that different β subunits act by a similar mechanism to modulate activation and expression of α1E, despite very different effects on inactivation. While no other study has compared gating currents of α1E channels containing different β subunits, measurements of α1E ionic-current G-V curves in Xenopus oocytes support this finding (Olcese et al., 1994). However, for other α1 subunits, modulation of expression and activation may differ across β subunits. For example, there clearly is isoform specificity in the β subunit modulation of current potentiation in α1A (Stea et al., 1994; De Waard et al., 1994) and α1S (Ren and Hall, 1997). This fits with the binding affinity differences in vitro of various β subunits to α1A (De Waard et al., 1995). However, binding of various β subunits to the I-II linker of α1B occurs with the same affinity (Scott et al., 1996). Whether differences in in vitro binding affinities translates into discernible gradations of functional effects remains to be established.

One β Subunit May Predominate in Directing Baseline Channel Properties

Most previous studies have implicitly assumed that only one β subunit is involved in modulating channel properties, consistent with biochemical evidence for a 1:1 stoichiometry of α1 and β subunits for skeletal (De Waard et al., 1996) and N-type (Witcher et al., 1993) channels. However, a recent report by Tareilus et al. (1997) identified a second β subunit binding site on the COOH terminus of α1E, raising the possibility that two or more β subunits might collectively determine channel gating properties. Here, we found little evidence that two β subunits modulate the properties of the α1E channel, either in regard to expression or gating.

Mechanism of β Subunit Modulation of α1E Gating

To account for β subunit effects on G-V and Q-V curves, previous studies have proposed that the β subunit acts mainly on the weakly voltage-dependent steps that “couple” channel opening to voltage sensor movement (Neely et al., 1993; Olcese et al., 1996). Here, we demonstrate that this mechanism may explain not only the modulation of G-V and Q-V curves, but also the doubling of maximum open probability. Fig. 10 depicts a channel gating model that closely resembles those previously used in the study of potassium channel gating (Zagotta and Aldrich, 1990; Schoppa et al., 1992). There are three independent, voltage-dependent transitions between the closed states (C0, C1, C2, and C3), each associated with an appropriately scaled equilibrium constant K0. These transitions are followed by a weakly voltage-dependent transition (C2–C3) with equilibrium constant K1 and a final voltage-independent step with equilibrium constant K2. K0 and K1 are voltage dependent according to a Boltzmann distribution, Ki = exp{[ziF(V − Vi)]/(RT)}. To obtain baseline model parameters (z0, z1, V0, V1, K2), we fit α1E alone Q-V and G-V data (Fig. 10,B). Then, to simulate the observed twofold change in the maximum open probability, we modified only K2, the equilibrium constant for the last voltage- independent transition leading to channel opening. This simple change reproduced well both the shift in the G-V relationship and the shift in the Q-V relationship (Fig. 10 C). In fact, such simulations indicate that modifying the coupling of charge movement to channel opening (K2) usually also perturbs the Q-V relation and, therefore, charge movement. Yet in several studies (Kamp et al., 1996; Josephson and Varadi, 1996; Neely et al., 1993; Olcese et al., 1996), shifts in ionic activation have been seen with little or no modification of charge movement. In these studies, it may be that the shift in charge movement is too small to be well resolved.

To determine the effect of modifying K2 on the time course of activation, we modeled the kinetics of activation. The choice of rate constants is constrained by the equilibrium constants, according to Ki(V) = αi(V)/ βi(V), where αi(V) is the forward rate constant and βi(V) is the backward rate constant (seconds−1). We chose αi(V) = fi exp{[di ziF(V − Vi)]/(RT)}, and βi(V) = αi(V)/Ki(V), giving us free parameters f0, d0, f1, d1, and f2, which we could vary to fit the activation kinetics. Fig. 10,D shows a model fit (thin solid line) consistent with α1E alone steady state model parameters (z0, z1, V0, V1, K2; Fig. 10,B). Representative ionic current data (Fig. 10,B, thick gray line) are derived by subtracting the gating currents (Fig. 5,A) from the α1Eβ1b whole cell records in Fig. 6,A. Because of the subunit invariance of activation kinetics, these ionic currents also represent the expected time course of α1E alone. Changing K2 to accord with the twofold increase in maximal open probability produced by β subunits can modify the activation kinetics (Fig. 10 E, dashed line), but this may be compensated for by altering only f1 and f2. This amounts to subtle changes in the absolute rate constants of the last two transitions, but only an alteration of the equilibrium constant of the last transition. Although no change in parameters corresponding to the more voltage-dependent steps (z0 and f0) is necessary, we found that we could not well reproduce the invariance of activation kinetics by just modifying f2, corresponding to the last voltage-independent step. Therefore, this simulation argues that all the effects of β subunit modulation of α1E (increased open probability, hyperpolarization of G-V and Q-V curves, and invariant activation kinetics) can be attributed to actions on one or a few weakly voltage-dependent steps before opening.

Generalizability of Results to Other α1 Isoforms?

From the previous discussion, it is clear that generalization across α1 subtypes is a difficult proposition. Nevertheless, we wondered whether some of the most robust properties of α1E modulation by subunits would translate to a different α1 subunit. In particular, there was striking adherence to two “rules” for α1E modulation by subunits: (a) all β subunits produce no change in the kinetics of activation, but induce identical but relatively small hyperpolarizing shifts of the G-V curve, and (b) distinct β subunits impart vastly different steady state inactivation curves. Do these features of subunit modulation hold true as general tenets for other α1 subunits?

Fig. 11 tests this proposition for the α1C calcium channel. The results indicate a complete reversal of the behavior found with α1E. Now the kinetics of activation are clearly different for β3 and β2a subunits. In addition, the different β isoforms led to large differences in G-V curves. On the other hand, steady state inactivation curves show only small isoform-dependent distinctions.

The diametrically opposite behaviors exhibited by α1E and α1C subunits have interesting implications for the structure–function relations underlying α1-β modulation. The leading candidates for structural interaction between these two subunits are a small motif on the I-II linker of α1 subunits known as the “alpha interaction domain” or AID (Pragnell et al., 1994), and another small motif in the middle of β subunits known as the “β interaction domain” or BID (De Waard et al., 1995). AID and BID peptides bind with high affinity (tens of nanomolar), and the BID region has documented importance for modulation of α1 subunits (De Waard et al., 1995). The key points with regard to our findings are that the BID is highly (∼70%) homologous across β subunits, and the AID is also highly conserved across different α1 subunits (Pragnell et al., 1994; De Waard et al., 1995). The profound differences in β isoform selectivity for entirely different gating properties, depending on the α1 subtype, suggest that either the AID–BID interaction is exquisitely sensitive to small sequence variations in the AID (Scott et al., 1996), or there are other features that contribute to β subunit modulation of α1 (Chien et al., 1996). Distinguishing between these two possibilities and identifying any secondary interaction sites will be important challenges for the future.

We thank K.P. Campbell for the β1b clone, T.P. Snutch for the α1E and α2δ clones, E. Perez-Reyes for the α1C, β2a, β3, and β4 clones, M. deLeon for construction of α1EΔ, J.G. Mulle for technical assistance, and David Brody and Carla DeMaria for discussion and comments.

This work was supported by the National Institutes of Health (NIH) to D.T. Yue, the National Science Foundation Presidential Faculty Fellowship (D.T. Yue), a Maryland American Heart Association Postdoctoral Fellowship (S.K. Wei), and an NIH Medical Scientist Training Program Award (L.P. Jones).

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Author notes

Address correspondence to David T. Yue, Program in Molecular and Cellular Systems Physiology, Departments of Biomedical Engineering and Neuroscience, Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, MD 21205. Fax: 410-955-0549; E-mail: [email protected]