Mechanism of Auxiliary Subunit Modulation of Neuronal alpha 1E Calcium Channels

Lisa P. Jones, Shao-kui Wei, and David T. Yue

From the Program in Molecular and Cellular Systems Physiology, Departments of Biomedical Engineering and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Voltage-gated calcium channels are composed of a main pore-forming alpha 1 moiety, and one or more auxiliary subunits (beta , alpha 2delta ) 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 alpha 1E calcium channels in combination with a beta  (beta 1-beta 4) and/or the alpha 2delta 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 alpha 2delta with beta  subunits, of transfecting two different beta  subunits simultaneously, and of COOH-terminal truncation of alpha 1E to remove a second beta  binding site. The main results are as follows. (a) The alpha 2delta and beta  subunits modulate alpha 1E in fundamentally different ways. The sole effect of alpha 2delta is to increase current density by elevating channel density. By contrast, though beta  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 beta  isoforms produce nearly indistinguishable effects on activation. However, beta  subunits produced clear, isoform-specific effects on inactivation properties. (b) All the beta  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 beta  subunits. (d) The modulatory features found here for alpha 1E do not generalize uniformly to other alpha 1 channel types, as alpha 1C activation gating shows marked beta  isoform dependence that is absent for alpha 1E. Together, these results help to establish a more comprehensive picture of auxiliary-subunit regulation of alpha 1E calcium channels.

Key words: calcium channelsalpha 1Egating currentssubunit modulationheterologous expression
    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha 1 subunit, a cytoplasmic beta  subunit, and a disulfide-linked alpha 2delta subunit (for review, see Perez-Reyes and Schneider, 1994; De Waard et al., 1996). So far, seven different genes encoding alpha 1A,B,C,D,E,S,G subunits, and four different genes encoding beta 1,2,3,4 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 (beta , alpha 2delta ) can have striking effects on channel gating and/or channel expression, the specific effects observed vary across studies, even using the same alpha 1 subunit. At least some of the differences in subunit effects may reflect isoform-specific variations in the effects of distinct beta  subunit isoforms on alpha 1 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 alpha 1 isoform individually, and to undertake comprehensive studies with uniform experimental conditions.

Although most previous work has focused on alpha 1C, neuronal alpha 1E 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 alpha 1E has potential physiological relevance, as alpha 1E (presumed "R-type") channels have been implicated in neuronal functions including neurotransmitter release (Wu et al., 1998). Second, alpha 1E demonstrates an exceptional capacity for high-level recombinant expression, which permits well-resolved measurements of both ionic and gating currents, even when the alpha 1E 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, beta  subunits may affect alpha 1E expression in a uniquely different manner than observed with other pore-forming alpha 1 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 alpha 1 subunits, beta  subunits caused little change or even a decrease in overall alpha 1E 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, beta  subunits increased overall alpha 1E current density (Williams et al., 1994; Stephens et al., 1997). Here, however, no alpha 1E gating-current measurements have been made to permit assessment of underlying changes in channel density and open probability. Finally, alpha 1E is one of the channels in which a second beta  binding site has been explicitly identified (Tarelius et al., 1997; Walker et al., 1998). Characterization of mutant alpha 1E constructs lacking this site would allow determination of the functional importance of the secondary site.

Here, we therefore examine subunit modulation of alpha 1E channels coexpressed with various combinations of auxiliary subunits (beta 1-beta 4, alpha 2delta ) 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 alpha 1E 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 beta  binding site in alpha 1E? Through addressing these questions, this study helps to establish a more refined picture of auxiliary-subunit modulation of alpha 1E calcium channels.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha 1E (Soong et al., 1993), alpha 1C (Wei et al., 1991), beta 1b (Pragnell et al., 1991), beta 2a (Perez-Reyes et al., 1992), beta 3 (Castellano et al., 1993b), beta 4 (Castellano et al., 1993a), and alpha 2delta (Tomlinson et al., 1993) were subcloned into mammalian expression plasmids (pMT2; Genetics Institute, Cambridge, MA, for beta 4, pZEM229R; ZymoGenetics, Inc., Seattle, WA, for alpha 2delta , pGW1; British Biotechnologies, Cowley, Oxford, UK for all others). alpha 1EDelta was constructed by replacing the Bst 1107I (alpha 1E: nucleotide 4299, given start codon at nucleotide 1) and SalI (3' polylinker) region of alpha 1E 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 (alpha 1E or alpha 1C) and mixed with 10 µg of each desired auxiliary subunit (none, a beta  subunit, and/or the alpha 2delta subunit). If the amount of DNA totaled <30 µg, pBluescript was added to make up the difference. For certain experiments, both beta 2a and beta 3 were simultaneously transfected either in a 1:1 ratio (10 µg of each plasmid) or a 5:1 ratio (15 µg of beta 3, 3 µg of beta 2a). 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 beta 1b, 10 µg of alpha 2delta , 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 beta 2a 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 alpha 1 (A, B, C, D, E) or beta  (1b, 2e, 3a, 4) subunits, and only low levels of alpha 2delta (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 alpha 2delta with alpha 1E potentiated current by approximately threefold suggests that trace expression of endogenous alpha 2delta 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 MOmega , 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(infinity )-V relations shown in Fig. 11 for alpha 1C 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.


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Fig. 11.   (A) Representative whole-cell records obtained using the same voltage protocol and ionic conditions as in Fig. 2, but now from cells transfected with either alpha 1Cbeta 2a (cell 83_2, top) or alpha 1Cbeta 3 (cell 337_6, bottom). Traces are plotted for test pulse potentials of -30, -20, -10, 0, and 30 mV. Note the slow activation of alpha 1Cbeta 3 compared with alpha 1Cbeta 2a channels. (B) Average G-V relations for alpha 1Cbeta 2a (n = 10, diamond ) and alpha 1Cbeta 3 (n = 9, down-triangle). The solid lines are Boltzmann fits to the alpha 1Cbeta 3 (z = 1.55, V = -2.53 mV) and alpha 1Cbeta 2a (z = 2.7, V = -12.4 mV) data. (C) h(infinity )-V relations using the protocol in Fig. 7 for alpha 1Cbeta 3 (n = 2-3, down-triangle) and alpha 1Cbeta 2a (n = 2-3, diamond ), with 10 mM Ba2+ external, using a different internal solution (see MATERIALS AND METHODS). Solid lines are Boltzmann fits to alpha 1Cbeta 2a (z = 2.7, V = -30 mV), and alpha 1Cbeta 3 data (z = 2.3, V = -33 mV).


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Fig. 2.   Modulation of ionic current density by beta  subunits. (A) Ionic currents in response to the protocol (top) used to measure the voltage dependence of ionic activation. Currents were measured with 2 mM Ba2+ as charge carrier in response to voltage steps ranging from -50 to 50 mV in 10-mV increments for the indicated subunit combinations. (Cells 93_16 [alpha 1E], 174_3 [+alpha 2delta ], 296_4 [+beta 1b], 252_5 [+beta 2a], 306_21 [beta 3], and 296_25 [beta 4].) (B and C) Comparison of the effects of auxiliary subunits on expressed current density, assayed by normalizing either the maximal conductance (Gmax) derived from the peak tail current (B) or the peak current Imax (C) by the cell capacitance. Values are listed in Table I.


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Fig. 7.   Auxiliary subunits modulate inactivation. (A, top) Voltage protocol used to approximate the steady state inactivation (h(infinity )-V) relation. Voltage commands were given from a holding potential of -120 mV every 60 s. The test pulse potential was chosen to be the peak of the I-V relation, typically -5 mV. All h(infinity )-V measurements were made with 10 mM Ba2+ as a charge carrier unless otherwise indicated. (bottom) Representative whole-cell records for a cell transfected with alpha 1Ebeta 2a (cell 325_19) and a cell transfected with alpha 1Ebeta 3 (cell 327_11) for prepulse potentials of -100 to -30 mV (beta 2a) and -100 to -60 mV (beta 3), respectively. Only the first 200 ms of the prepulse were acquired and displayed. (B) Average h(infinity )-V relations derived by normalizing the peak test pulse current data from the protocol in A by the peak test pulse current in the absence of a prepulse and averaging across cells. All subunit combinations are plotted, with the identical legend as in Fig. 6, with the addition of alpha 1Ebeta 3alpha 2delta (×). Solid lines represent single-Boltzmann fits with values {subunit combinations [z, V1/2 (mV)]}: alpha 1E (2.4 , -66), +alpha 2delta (2.3, -62), +beta 1b (3.9, -74), +beta 2a (2.8, -46), +beta 3 (3.6, -81.3), +beta 4 (3.7, -75), +beta 2aalpha 2delta (3.2, -47), and +beta 3alpha 2delta (3.5, -74). Average fit values are summarized in Table III.

                              
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Table I
Comparison of Subunit Effects on Current Density (Gmax) and Charge Density (Qon, Qoff)


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Fig. 6.   Effects of auxiliary subunits on the kinetics and voltage dependence of activation. (A) Comparison of activation kinetics for channels containing different beta  subunits. Ionic currents recorded in 2 mM Ba2+ for alpha 1E in combination with either beta 1b, beta 2a, beta 3, or beta 4 (cells 296_4, 252_5, 306_21, and 296_25, respectively) were scaled and superimposed for test pulse potentials of -30, 10, and 30 mV to illustrate activation kinetics. (B) Effect of addition of a beta  or the alpha 2delta on activation kinetics. Ionic currents for alpha 1E (cell 171_3, solid line), alpha 1Ealpha 2delta (cell 174_3, dashed line), and alpha 1Ebeta 1b (cell 296_4, gray line) were scaled and superimposed for test pulse voltages of -30, -10, and 10 mV. (C) Averaged G-V relations for all the different subunit combinations (alpha 1E, black-down-triangle ; +alpha 2delta , bullet ; +beta 1b, square ; +beta 2a, diamond ; +beta 3, down-triangle; +beta 4, triangle ; +beta 2aalpha 2delta , open circle ). This key applies to D and E also. G-V curves were derived using the protocol in Fig. 2 by normalizing peak tail currents during repolarization to -50 mV by an estimate for the maximal tail current (MATERIALS AND METHODS). The continuous curves represent single-Boltzmann fits to the averaged alpha 1E and alpha 1Ebeta 2a data, with fit parameters (z = 2.3, V1/2-14.6 and z = 3.3, V1/2-20.8, respectively). Dashed curves represent dual-Boltzmann fits to the averaged alpha 1E and alpha 1Ebeta 2a data with parameters (Vlow-23.3, zlow = 4.3, Vhigh-10.5, zhigh = 2.1, with the fraction of the low threshold component equal to 0.29 for alpha 1E and 0.71 for alpha 1Ebeta 2a). (D) Averaged current voltage relations for the same cells as in C. I-V relations for individual cells were normalized by the peak current before averaging. (E) Voltage dependence of charge movement for all the different subunit combinations. Q-V relations were derived using the protocol in Fig. 4 A by integrating the ON transient and normalizing by an estimate for the maximum amount of mobile charge (Qon,max, see MATERIALS AND METHODS) before averaging across cells. Solid lines correspond to single-Boltzmann fits to alpha 1E data (z = 2.4, V = -23.7 mV) and to alpha 1Ebeta 2a data (z = 2.5, V = -29 mV). Values are summarized in Table II.

                              
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Table III
Comparison of Average h(bullet )-V Fit Parameters for alpha 1E and alpha 1EDelta


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Fig. 4.   Isolation of alpha 1E gating currents. (A) Gating currents in response to the protocol used for measuring the voltage dependence of charge movement (Q-V). Representative traces are illustrated for an alpha 1Ebeta 2a-transfected cell (249_13) during ionic current blockade with 2 mM Mg2+/0.2 mM La3+ for 15-ms test pulse depolarizations to -40, -20, 0, 20, and 40 mV from a holding potential of -110 mV. (B) Lack of gating current in a mock transfected cell (no alpha 1E), demonstrating the lack of appreciable amounts of endogenous voltage sensors. (C) Whole cell record in block solution from the cell in A in response to the indicated voltage protocol. The current trace is the difference of currents in response to positive and negative voltage jumps of opposite magnitudes, and illustrates that no nonlinear charge movement contaminates our leaks. (D) Charge movement occurs before activation of ionic currents. The voltage dependence of charge movement was derived by integrating the "ON" transient during depolarization (Qon-V, open circle ) and the "OFF" transient during repolarization, respectively (Qoff-V, diamond ), and normalizing by an estimate for the maximum amount of mobile charge. The ionic activation curve (G-V, bullet ) was derived from the voltage protocol in Fig. 2 A by normalizing peak tail currents (see MATERIALS AND METHODS). Solid lines represent the single-Boltzmann fits to the Qon-V (fit parameters: z = 2.7, V1/2 = -27.7 mV) and to the G-V (z = 3.1, V1/2-17 mV). (E) Plot of ionic current density (Gmax) versus the gating charge density (Qmax) for the cells used in calculating the Gmax/Qmax ratio for alpha 1Ebeta 2a (Table I). The plot illustrates the correlation between the magnitude of expressed current and mobile gating charge. Each data point represents one cell. Line is the identity relation.

                              
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Table II
Comparison of Average Boltzmann Fit Parameters for both the Voltage Dependence of Ionic Activation (G-V) and Charge Movement (Q-V)

For measurement of alpha 1E 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 alpha 1Ebeta 2a 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.


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Fig. 5.   Comparison of gating currents for alpha 1E in combination with different auxiliary subunits. (A) Representative gating currents were obtained using the same voltage protocol and block solutions as in Fig. 4. Traces are for cells transfected with the indicated subunit combinations in response to test pulse voltages of -40, -20, 0, 20 and 40 mV, respectively. Same cells as in Fig. 2. (B) Comparison of the average Qmax values for different subunit combinations. The maximum amount of mobile charge (Qmax) was calculated from the saturating values of Boltzmann fits to Qon-V. (C) Comparison of the average Gmax/Qmax values for different subunit combinations. The maximum conductance (Gmax) was determined from the saturating value of the Boltzmann fit to the tail-activation curves measured in 2 mM Ba2+. Average Gmax, Qmax, and Gmax/Qmax values are summarized in Table I.

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 (bullet ) and 2 mM MgCl2/0.2 mM LaCl3 (open circle ). The identity of the two curves, absent the expected surface-potential shift, provides additional strong support that La3+ does not perturb activation gating.


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Fig. 1.   La3+ control experiments. (A) La3+ does not change the voltage dependence of charge movement. Comparison of average Q-V relations in 2 mM Mg2+ (bullet , n = 6) and 2 mM Mg2+/0.2 mM La3+ (open circle , same cells). Solid lines represent Boltzmann fits to the data without (V1/2 = 32.8 mV, z = 2.4) and with (V1/2 = 28.5 mV, z = 2.4) La3+. (B) Estimate of surface charge shift between 2 mM Ba2+ and 2 mM Mg2+/0.2 mM La3+. (main plot) Comparison of averaged Qon-V relations (n = 9) measured in 2 mM Ba2+ (open circle ) and 2 mM Mg2+/0.2 mM La3+ (diamond ) to demonstrate the absence of an appreciable surface charge shift. Solid line is a dual-Boltzmann fit by eye to the 2 mM Mg2+/0.2 mM La3+data. (inset) Plot of averaged Qon-V (open circle ) and Qoff-V (bullet ) relations measured in 2 mM Ba2+ to illustrate the threshold of activation (~65 mV, dotted vertical line). For voltages past the threshold of activation, the presence of ionic current shifts the baseline used in the calculating Qon; as a result, Qon is overestimated and appears larger than Qoff for these voltages.

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, open circle ) and Qoff-V (inset, bullet ) 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+ (open circle ) and 2 mM MgCl2/0.2 LaCl3 (diamond ) 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(infinity )-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(infinity )-V relations were averaged across cells. For cells transfected with two beta  subunits, the h(infinity )-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(infinity )-V relations, we used a function of the form B(V) = flow{1 + exp[z low F(V - V1/2,low)/RT]}-1 + fhigh{1 + exp[z high 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.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Enhancement of Expressed Current Density by Auxiliary Subunits

Transfection of HEK 293 cells with the alpha 1E 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 beta  subunits. All beta  subunits were approximately equipotent in this regard, although the average beta 3 effect was slightly smaller (approximately sevenfold). Addition of alpha 2delta to alpha 1E produced a weaker increase in current (about threefold), and the combination of alpha 2delta and beta  subunits yielded no appreciable current enhancement over the coexpression of beta  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), IpeaknPo[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 alpha 1E Subunit

To determine the origin of the increased current density (nPo,max), we wished to measure the maximum amount of mobile gating charge (Qmaxnq, 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 alpha 1B subunit (Jones et al., 1997a), but not the alpha 1C subunit (Kamp et al., 1996). To determine the feasibility of La3+ blockade of calcium channels containing alpha 1E, 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, bullet ), and did not alter Qrev during repetitive stimulation in the presence of La3+ (open circle , 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 alpha 1E + beta 2a (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.


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Fig. 3.   Control experiments on alpha 1Ebeta 2a-transfected cells to assay the effect of La3+ block on channel gating. (A) Whole-cell currents elicited by 15-ms test depolarizations from a holding potential of -110 mV. For each test potential, the currents measured with either 2 mM Ba2+ (dashed line) or 2 mM Mg2+/0.2 mM La3+ (solid line) in the external solution are superimposed to demonstrate that ionic current blockade by La3+ does not alter the gating-charge movement. Cell 320_6. (B) Diary plot recorded from an alpha 1Ebeta 2a-transfected cell in response to 15-ms depolarizations to the reversal (~40 mV) every 15 s. Qrev was derived by integrating the "ON" gating current transient; Itail was measured as the peak tail current upon repolarization to -50 mV. Bar indicates the application of the 2 mM Mg2+/0.2 mM La3+ block solution. Cell 292_12.

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 alpha 1Ebeta 2a (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 alpha 1E alone was characteristically small, with a mean of 0.8 ± 0.1 fC/pF (n = 9). beta  Subunits induced the strongest enhancement of Qmax, ranging from fourfold for beta 3 to sevenfold for beta 4. Coexpression of alpha 2delta also produced clear augmentation of Qmax, though the effect was less potent than for beta  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 alpha 1E channels (increasing total amount of alpha 1E protein), or an increase in the fraction of functional alpha 1E protein in the membrane (with no increase in total amount of alpha 1E protein) by the alpha 2delta and beta  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 beta  subunits approximately doubled Gmax/Qmax, but alpha 2delta left the ratio unchanged. Table I reports further details of the calculations. The data in Fig. 5 suggest that beta  subunits enhance alpha 1E 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 alpha 2delta 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 beta  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 beta  subunits suggest that beta  subunits produce channels with similar activation kinetics. Fig. 6 B shows the identical analysis for channels expressed from alpha 1E alone (solid trace) or from alpha 1E + alpha 2delta (dashed trace). The records for alpha 1E + beta 1b (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 alpha 2delta subunit had little effect on the G-V (Fig. 6 C) or I-V (Fig. 6 D) relations. The lack of effect of alpha 2delta 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 alpha 1E. In contrast, single-Boltzmann function analysis (Fig. 6 C, solid curves, and Table II) clearly demonstrates that coexpression of beta  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 alpha 1E to 3.6 for alpha 1Ebeta 2a). As expected from the shift in the G-V relation, coexpression of beta 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 beta  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 beta  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 beta  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 beta  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 beta  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 beta  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, alpha 2delta had no effect on the Q-V relation. All the beta  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 beta  subunits act primarily to modulate a single locus of weakly voltage-dependent steps late in the activation pathway (see DISCUSSION).

Functional Stoichiometry of beta  Subunit Interaction

Previous reports in Xenopus oocytes indicate that alpha 1E channels containing different beta  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 beta  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 alpha 1Ebeta 2a and alpha 1Ebeta 3 channels illustrate the extremes of inactivation behavior observed with the different subunit combinations. beta 2a dramatically slowed inactivation, while beta 3 accelerated inactivation. beta 1b and beta 4 subunits also accelerated inactivation during the test pulse (not shown), though not as strongly as beta 3. To provide a robust indication of the differences in inactivation properties, we used such records to calculate steady state inactivation curves (h(infinity )V curves; Fig. 7 B). While addition of alpha 2delta did not affect the h(infinity )V relation, coexpression of beta  subunits induced striking modulation of steady state inactivation: beta 1b, beta 3, and beta 4 all left-shifted h(infinity )V curves by ~10, 15, and 10 mV, respectively; beta 2a imparted a right shift of ~15 mV. The profound distinction between the effects of beta 2a and the other beta  subunits has been reported in previous studies of alpha 1E (Olcese et al., 1994), and of other neuronal calcium channels, including alpha 1A (Stea et al., 1994) and alpha 1B (Patil et al., 1998). Table III summarizes the Boltzmann analysis of h(infinity )V data.

To investigate whether multiple beta  subunits can concomitantly specify the functional properties of a single calcium channel, we took advantage of the vast difference between the h(infinity )V relations for alpha 1Ebeta 3 and alpha 1Ebeta 2a channels. If there are multiple beta  subunit sites on alpha 1E that specify inactivation properties, then cotransfection of both beta 2a and beta 3 subunits should result in mixed-composition channels (e.g., alpha 1Ebeta 2abeta 3) whose inactivation behavior should be distinct from that of pure alpha 1Ebeta 2a- or alpha 1Ebeta 3-like channels. However, if there is only one functionally active beta  subunit site per channel, the aggregate h(infinity )V relation should possess only two components. Fig. 8, A and B, shows the results for one such experiment in which beta 3 and beta 2a 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(infinity )V data correspond closely to the steady state inactivation properties of pure alpha 1Ebeta 2a and alpha 1Ebeta 3 channels (Fig. 7 B and Tables III and IV). Cotransfection of beta 3 and beta 2a 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 alpha 1Ebeta 2a and alpha 1Ebeta 3 channels.


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Fig. 8.   Simultaneous cotransfection of two beta  subunits (beta 2a and beta 3) leads to two distinguishable channel populations. (A) Current traces recorded in 10 mM Ba2+ in response to the same h(infinity )-V protocol as in Fig. 7 (top), for a cell (330_18) transfected with alpha 1E in combination with equal amounts of beta 2a and beta 3. Traces displayed are for prepulse potentials ranging from -120 to -30 mV in 10--mV increments. (B) Plot of peak test-pulse currents (bullet ) for the cell in A, illustrating the bimodal nature of inactivation. The thick line corresponds to a dual-Boltzmann fit to the data with parameters (low: -397 pA, z-3.4, V1/2-81.2 mV; high: -164 pA, z-4.7, V1/2-39 mV), in good correspondence with the average steady state inactivation properties of alpha 1Ebeta 3 and alpha 1Ebeta 2a channels, respectively (Table III). The dotted and dashed lines illustrate the two components of the dual-Boltzmann fit. (C) Plot of average residuals (n = 26). The difference between the data and the fit value was normalized by the peak test-pulse current before averaging across cells. (D) Bar graph illustrating the dependence of the fraction of the low threshold Boltzmann component on the ratio of transfected beta 2a and beta 3. For the 1:1 ratio, n = 17, and for the 5:1 ratio, n = 9. (E) Comparison of average V1/2 derived from dual-Boltzmann fits to the h(infinity )-V for beta 2abeta 3-transfected cells (diagonal striped), with the average fit values derived from single-Boltzmann fits to h(infinity )-V data for beta 2a- or beta 3-transfected cells (gray). Fit values and cell numbers are summarized in Table III (for beta 2a or beta 3 alone) and Table IV (for beta 2abeta 3-transfected cells).

                              
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Table IV
Average Values for Dual-Boltzmann Fits to h(bullet )-V Data for Cells Transfected with both beta 2a and beta 3 Simultaneously

As a further test for the possible functional role of a second beta  subunit site (Tareilus et al., 1997), we examined how auxiliary subunits modulated the properties of a COOH-terminal truncation of the alpha 1E construct (alpha 1EDelta , amino acids 1-1871 of alpha 1E [1-2251]) that lacks the secondary binding site. Fig. 9 A displays ionic currents for channels composed of alpha 1EDelta + alpha 2delta or alpha 1E + beta 2a + alpha 2delta subunits. Coexpression of beta 2a with alpha 1EDelta 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 alpha 1E (Fig. 2 B). Similarly, modulation of activation by beta 2a is unchanged by the COOH-terminal deletion, as demonstrated in Fig. 9 B by the identical subunit modulation of G-V relations for alpha 1EDelta (circles) and wild-type alpha 1E (squares). Finally, we compared beta  subunit modulation of the steady state inactivation properties of alpha 1EDelta (Fig. 9, C, traces, and D, symbols; Table III) with data obtained with wild-type alpha 1E (Fig. 9 D, lines). The alpha 1E 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 beta  subunit coexpression (e.g., alpha 1EDelta + alpha 2delta in Fig. 9 D); this small shift likely reflects a difference in the intrinsic inactivation behavior of the alpha 1 backbone (Soldatov et al., 1997), rather than a change in the modulatory action of beta subunits. The close correspondence between h(infinity )-V relations for alpha 1E (lines) and alpha 1EDelta (symbols) in the Fig. 9 D overlay illustrates that beta  subunit modulation of inactivation is similar for the two constructs. Although the small difference between modulation of alpha 1EDelta and alpha 1E inactivation (most apparent for alpha 1EDelta beta 3alpha 2delta , Fig. 9 D, down-triangle) could reflect a minor contribution of a second beta subunit binding site, all the results in Figs. 8 and 9 support the view that a single beta  subunit binding site predominates in specifying inactivation properties. If present, the potential contribution of a second site appears to be small by comparison.


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Fig. 9.   Activation and inactivation properties of a COOH-terminal truncated version of alpha 1E (alpha 1EDelta , amino acids 1-1871) that lacks a putative beta  binding site. All measurements for both alpha 1E and alpha 1EDelta are in 10 mM Ba2+. (A) Traces illustrating activation of ionic currents for alpha 1EDelta alpha 2delta (cell 375_19) or alpha 1EDelta beta 2aalpha 2delta (cell 370_6) using the same voltage protocol as in Fig. 2. Data are shown for test pulse potentials of -30, -20, -10, 10, and 50 mV. (B) Comparison of G-V for alpha 1EDelta and alpha 1E indicates that modulation of alpha 1EDelta activation by beta  subunits is preserved. Symbols correspond to data for alpha 1EDelta and wild-type alpha 1E (alpha 1EDelta beta 2aalpha 2delta , open circle , n = 4; alpha 1EDelta alpha 2delta , bullet , n = 11; alpha 1Ealpha 2delta , black-square, n = 11; and alpha 1Ebeta 2a, square , n = 8). Lines are single-Boltzmann fits to the alpha 1Ealpha 2 and alpha 1Ebeta 2a data with fit parameters z = 2.4, V1/2-10.1; and z = 3.4, V1/2-18.2, respectively. The shift in V1/2 values relative to Fig. 6 C corresponds to a surface potential shift between 10 mM (used here) and 2 mM Ba2+ (Fig. 6). (C) Steady state inactivation. Traces after a 20-s prepulse (as in Fig. 7) to the indicated potentials are shown for alpha 1EDelta in combination with alpha 2delta (cell 376_9), beta 3 (cell 375_4), beta 2aalpha 2delta (cell 370_11), and beta 3alpha 2delta (cell 371_10). (D) Comparison of h(infinity )-V relations for alpha 1EDelta (symbols) and alpha 1E (same data as in Fig. 7, shown as lines connecting mean data values, without explicit reproduction of data points as symbols). Symbols correspond to the following constructs: alpha 1EDelta alpha 2delta , bullet ; alpha 1EDelta beta 2aalpha 2delta , open circle ; alpha 1EDelta beta 3, down-triangle; alpha 1EDelta beta 3alpha 2delta , ×. Data for alpha 1E is shifted by -7 mV to overlay the alpha 1EDelta data. Average fit values and cell numbers are summarized in Tables V (G-V) and III [h(infinity )-V] for both alpha 1E and alpha 1EDelta .

                              
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Table V
Comparison of Average Boltzmann Fit Values to G-V Relations for alpha 1E and alpha 1EDelta

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha 1 subunit under the same experimental conditions. Here, we have performed a systematic evaluation of auxiliary subunit regulation of expression and gating of alpha 1E calcium channels in HEK 293 cells. The experiments lead to three main conclusions. (a) The alpha 2delta and beta  auxiliary subunits differ fundamentally in the manner by which they induce an overall increase in current density. Coexpression of alpha 2delta with the pore-forming alpha 1E 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 beta  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 alpha 2delta had no appreciable effect on activation gating, beta  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 beta  isoforms produced nearly indistinguishable effects in regard to both current potentiation and activation gating. (c) Little functional evidence for a secondary beta  subunit binding site was found, fitting with earlier biochemical evidence for a 1:1 stoichiometry of alpha 1 and beta  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 alpha 1E and, where relevant, other calcium channels. For clarity, we will discuss alpha 2delta and beta  subunit effects sequentially, as independent parts. A kinetic mechanism is then developed to explain how beta  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 alpha 1 isoforms.

Modulation of alpha 1E by alpha 2delta

The alpha 2delta subunit produced an approximately threefold increase in alpha 1E current, which arose almost exclusively from elevated channel expression (Qmax). The alpha 2delta subunit had no other clear modulatory effects, except to slightly antagonize the effect of beta 3 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 alpha 1E alone. Similar effects on inactivation and expression were reported for doe1 (marine ray analog of alpha 1E) expressed in Xenopus oocytes (Ellinor et al., 1993). However, in contrast to our results, in studies of rat alpha 1E in COS-7 cells (Stephens et al., 1997) and human alpha 1E in Xenopus oocytes (Wakamori et al., 1994), coexpression of alpha 2delta 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 beta 3 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 alpha 2delta subunit on other alpha 1 subunits also varies widely, sometimes agreeing with our findings, other times not. For example, with regard to modulation of alpha 1C channel density, the alpha 2delta 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 alpha 1C, maximal dihydropyridine binding is not increased by alpha 2delta coexpression. Similarly, activation kinetics of alpha 1C 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.

beta Subunits Act Differently than the alpha 2delta Subunit

By contrast to the alpha 2delta subunit, coexpression of beta  subunits (beta 1-beta 4) 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 alpha 1E gating currents in Xenopus oocytes (Olcese et al., 1996), coexpression of beta 2a with alpha 1E 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 beta  subunit in modulating Po,max comes from a separate study that used fluctuation analysis to determine the effects of beta 2a and beta 1a on alpha 1E open probability (Noceti et al., 1996).

Fitting with the doubling of the Gmax/Qmax ratio, the beta  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 beta  subunits is also somewhat controversial. Although most studies of alpha 1E 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), beta 1b coexpression with the human alpha 1E in Xenopus oocytes was found to slow activation kinetics substantially. With respect to gating currents, the only other study of alpha 1E charge movement (Olcese et al., 1996) also found that the beta  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 beta ) shift in the Q-V reported here, Olcese et al. (1996) found that the beta  subunit produced no significant change in the Q-V. Again, these discrepancies may reflect differences between clones (human versus rat alpha 1E) or expression systems (Xenopus oocytes versus HEK 293 cells). Despite these minor differences, all the results indicate a role for beta  subunits in modulating activation gating.

In other calcium channels, the reported effects of the beta  subunits vary even more widely than for the alpha 2delta subunit. However, at least in some respects, beta  subunit modulation of other alpha 1 subunits appears similar to what we find for alpha 1E. For example, there are reported shifts in the voltage dependence of ionic activation for alpha 1A (Stea et al., 1994; De Waard and Campbell, 1995) and alpha 1C (Wei et al., 1991; Neely et al., 1993). Increased current density has also been observed for many of the alpha 1 subunits including alpha 1A (Mori et al., 1991), alpha 1B (Williams et al., 1992a), alpha 1C (Perez-Reyes et al., 1992), alpha 1D (Williams et al., 1992b), and alpha 1S (Ren and Hall, 1997). Furthermore, for alpha 1C, 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 beta  subunit modulation of ionic current activation is not associated with much shift in the Q-V, similar to what we find for alpha 1E. On the other hand, even these few gating current studies disagree in other regards. While coexpression of beta 1a (Kamp et al., 1996) or beta 3 (Josephson and Varadi, 1996) with alpha 1C increased both current density and Qmax in HEK 293 cells similar to our results for alpha 1E, beta 2a increased current without changing Qmax in Xenopus oocytes (Neely et al., 1993). Therefore, as with alpha 1E, the specific effects observed with beta  coexpression appear to depend on as yet unknown distinctions between expression systems, perhaps the endogenous expression of beta XO subunits in Xenopus oocytes (Tareilus et al., 1997).

Different beta  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 beta  subunits act by a similar mechanism to modulate activation and expression of alpha 1E, despite very different effects on inactivation. While no other study has compared gating currents of alpha 1E channels containing different beta  subunits, measurements of alpha 1E ionic-current G-V curves in Xenopus oocytes support this finding (Olcese et al., 1994). However, for other alpha 1 subunits, modulation of expression and activation may differ across beta  subunits. For example, there clearly is isoform specificity in the beta  subunit modulation of current potentiation in alpha 1A (Stea et al., 1994; De Waard et al., 1994) and alpha 1S (Ren and Hall, 1997). This fits with the binding affinity differences in vitro of various beta  subunits to alpha 1A (De Waard et al., 1995). However, binding of various beta  subunits to the I-II linker of alpha 1B 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 beta  Subunit May Predominate in Directing Baseline Channel Properties

Most previous studies have implicitly assumed that only one beta  subunit is involved in modulating channel properties, consistent with biochemical evidence for a 1:1 stoichiometry of alpha 1 and beta 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 beta  subunit binding site on the COOH terminus of alpha 1E, raising the possibility that two or more beta  subunits might collectively determine channel gating properties. Here, we found little evidence that two beta  subunits modulate the properties of the alpha 1E channel, either in regard to expression or gating.

Mechanism of beta  Subunit Modulation of alpha 1E Gating

To account for beta  subunit effects on G-V and Q-V curves, previous studies have proposed that the beta  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 alpha 1E 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.


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Fig. 10.   (A) Scheme used to model steady state activation. The equilibrium constant between two states is assumed to have the form Kialpha i(V)/beta i(V) = exp{[ziF(V - Vi)]/(RT)}, where alpha i(V) and beta i(V) are rate constants (millisecond-1), zi gives the valence of the gating charge moved in the transition, and Vi is related to the zero-potential free energy difference between the two states. Transitions through states C0-C3 correspond to Hodgkin-Huxley-like behavior of three identical gating particles, each characterized by the same equilibrium constant, K0. The movement of the three gating particles is followed by a weakly voltage-dependent step, which has a separate equilibrium constant, K1. Finally, there is a voltage-independent transition, with equilibrium constant K2 (z2 = 0). (B) Plot of the G-V (bullet ) and Q-V (open circle ) data from Fig. 6 for alpha 1E. The solid lines correspond to model fits with parameters: z0 = 1.32, V0-15.2 mV, z1 = 0.39, V1-104 mV, K2 = 0.63. (C) Plot of the G-V (bullet ) and Q-V (open circle ) data from Fig. 6 for alpha 1Ebeta 2a. Solid lines are the model fits produced by changing K2 for alpha 1E to 3.14, leaving all other equilibrium parameters the same. This change in K2 corresponds to a 1.96-fold increase in the maximum open probability [Po,maxK2/(K2 + 1)]. (D) Model fits for kinetics of activation. Pure ionic currents (thick gray line) were obtained for Vtest-30, -10, and 10 mV by subtracting gating currents measured in 2 mM Mg2+/0.2 mM La3+ from the currents measured in 2 mM Ba2+ (alpha 1Ebeta 1b, cell 296_4, same cell as in Fig. 6 A). The solid line is the model fit with kinetic parameters (f0 = 1.8, d0 = 0.4, f1 = 1.75, d1 = 0.5, f2 = 3) consistent with the steady state model parameters from B for alpha 1E alone. (E) The dashed line illustrates the kinetics that result when K2 is changed from 0.63 to 3.14, while otherwise maintaining the same model parameters as in D. This effect may be offset by setting f1 to 6 and f2 to 0.8 (solid line). Note that it is not necessary to modify parameters for the early transitions (f0, d0) to obtain a reasonable fit.

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) = alpha i(V)/ beta i(V), where alpha i(V) is the forward rate constant and beta i(V) is the backward rate constant (seconds-1). We chose alpha i(V) = fi exp{[di ziF(V - Vi)]/(RT)}, and beta i(V) = alpha i(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 alpha 1E 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 alpha 1Ebeta 1b 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 alpha 1E alone. Changing K2 to accord with the twofold increase in maximal open probability produced by beta  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 beta  subunit modulation of alpha 1E (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 alpha 1 Isoforms?

From the previous discussion, it is clear that generalization across alpha 1 subtypes is a difficult proposition. Nevertheless, we wondered whether some of the most robust properties of alpha 1E modulation by subunits would translate to a different alpha 1 subunit. In particular, there was striking adherence to two "rules" for alpha 1E modulation by subunits: (a) all beta  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 beta  subunits impart vastly different steady state inactivation curves. Do these features of subunit modulation hold true as general tenets for other alpha 1 subunits?

Fig. 11 tests this proposition for the alpha 1C calcium channel. The results indicate a complete reversal of the behavior found with alpha 1E. Now the kinetics of activation are clearly different for beta 3 and beta 2a subunits. In addition, the different beta  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 alpha 1E and alpha 1C subunits have interesting implications for the structure-function relations underlying alpha 1-beta modulation. The leading candidates for structural interaction between these two subunits are a small motif on the I-II linker of alpha 1 subunits known as the "alpha interaction domain" or AID (Pragnell et al., 1994), and another small motif in the middle of beta  subunits known as the "beta 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 alpha 1 subunits (De Waard et al., 1995). The key points with regard to our findings are that the BID is highly (~70%) homologous across beta  subunits, and the AID is also highly conserved across different alpha 1 subunits (Pragnell et al., 1994; De Waard et al., 1995). The profound differences in beta  isoform selectivity for entirely different gating properties, depending on the alpha 1 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 beta  subunit modulation of alpha 1 (Chien et al., 1996). Distinguishing between these two possibilities and identifying any secondary interaction sites will be important challenges for the future.

    FOOTNOTES

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: dyue{at}bme.jhu.edu

Original version received 12 March 1998 and accepted version received 15 June 1998.

We thank K.P. Campbell for the beta 1b clone, T.P. Snutch for the alpha 1E and alpha 2delta clones, E. Perez-Reyes for the alpha 1C, beta 2a, beta 3, and beta 4 clones, M. deLeon for construction of alpha 1EDelta , 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).

    Abbreviation used in this paper

I-V, current-voltage.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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