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	Figure 1. The structure of the α1C subunit and its NH2 terminus. (A) Schematic presentation of the α1C subunit of the L-type Ca2+ channel, Cav1.2. Numbering is shown according to rabbit long-NT α1C-wt (Mikami et al., 1989). The principal NH2-terminal deletion mutants used in this work are indicated by short lines. (B) Comparison of protein sequences of NH2 termini of rabbit and human long-NT and short-NT isoforms. Dots stand for full sequence homology, dashes show gaps, and shaded areas show the conserved motif TxxYxP. Arrows indicate the location of the inserted methionine at the beginning of each NH2-terminal deletion mutant. The initial segments of long-NT and short-NT isoforms are encoded by exons 1a and exon 1, accordingly; the beginning of the transmembrane segment IS1 corresponds to the beginning of exon 3.
	Figure 2. Effects of NH2-terminal deletions and T10A/Y13F/P15A mutation on IBa in the absence of Cavβ (the α1Cα2δ subunit composition). (A) The standard procedure used to monitor IBa, and an averaged I-V curve. See explanations in the text. (B) Comparison of IBa in α1C-wt and α1CΔ5 (2.5 ng RNA/oocyte of each subunit). Panel a shows representative current traces recorded at +40 mV in oocytes of one batch (donor). A current trace obtained in an oocyte expressing the α1Δ46 mutant is shown for comparison. Panel b shows averaged I-V curves from α1C-wt and α1Δ5 groups (n = 9 oocytes, N = 2 batches). The normalized values of I40 are shown in panel c. (C) Panel a shows the summary of relative I40 in the various mutants. I40 in each oocyte was normalized to the average I40 of α1C-wt of the same batch, as explained in Materials and Methods. Panels b and c show normalized I-V and G-V curves, respectively, of the indicated channel constructs, averaged from oocytes of two representative batches (n = 8–14, N = 2). Amount of injected RNA was varied from 0.3 ng/oocyte in NT mutants to 5 ng/oocyte in α1-wt to reach comparable IBa amplitudes in order to minimize the fitting artifacts. Note that Boltzmann fits have been performed separately in each oocyte. For illustration purposes, in averaged I-V and G-V curves shown in C and in the following figures, the solid lines through averaged experimental points were drawn using Boltzmann equation with values of V1/2 and Ka from Table I. In this and the following figures, the numbers above bars indicate the number of cells tested, and asterisks indicate statistically significant differences, as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.
	Figure 3. NT mutations and deletions do not alter the surface expression of α1C. (A) A simplified presentation of the method used to measure the surface expression of α1C in giant membrane patches (see Materials and Methods for details). A devitellinized intact oocyte is placed for 10–20 min on coverslip with its animal (dark) hemisphere facing the coverslip (a). After attachment, the oocyte is swept away and patches of membrane (usually >100 μm in diameter) remain stuck to the coverslip (b). The cytosolic surface of the PM, facing the external solution, is thoroughly washed until the membrane appears transparent (c) and then stained with an antibody directed against a cytosolic part of the channel. (B) Examples of confocal images of α1C-wt, α1CΔ46, α1CΔ20, and α1CTYP in giant patches of oocytes of the same batch. (C) NT mutations do not alter the surface expression of α1C (a), despite the typical differences in IBa (measured at +20 mV) in oocytes of one of these batches (b). The amount of injected RNA was 5 ng/oocyte.
	Figure 4. Coexpression of β2b increases the surface expression of α1C-wt and of NT mutants. (A) Examples of confocal images of α1C-wt, α1CΔ46, α1CΔ20, and α1CTYP in giant PM patches, in absence and presence of β2b subunit. (B) Surface expression of the mutant proteins in α1α2δ subunit composition, without β2b, normalized to α1C-wt. This series of experiments included two oocyte batches and was separate from that shown in Fig. 3. Injected RNA was 5 ng/oocyte. (C) Summary of the effects of β2b on the surface expression of α1C mutants. For each construct, the surface expression in the presence of β2b was normalized to the average expression measured without β2b. (D) The effect of β2b on surface expression of α1C-HA; panel a shows representative confocal images of whole oocytes, either native (uninjected) or expressing α1C-HA + α2/δ (“α1C-HA”) or α1C-HA + α2/δ + β2b (“α1C-HA+β2b”). Injected RNA was 5 ng/oocyte. The dimensions of the image are 0.45 × 0.45 mm. The rightmost image exemplifies the method used to analyze the intensity of fluorescent labeling. A rectangle of a fixed width was superimposed on the image, and a profile of optical density (shown in b) along the axis approximately perpendicular to the PM (arrow) was obtained using the TINA software. (Optical density and distance were measured in arbitrary units inherent to the software). (b) Quantitative analysis of optical density profiles. The intensity was defined as the integral of the shaded area. The width of this area was defined as constant for all images taken in an experiment. The net intensity in each α1C-HA–expressing oocyte was calculated by subtracting the average intensity measured in uninjected oocytes of the same experiment, and then normalized to the average net intensity of [α1C-HA+α2/δ]-expressing oocytes of this experiment. The normalized values, shown in c, were summarized across all experiments (N = 3).
	Figure 5. Effects of mutations in NT, and of the β2b subunit, on voltage dependence of activation of IBa. (A) Representative net IBa of α1C-wt and α1CΔ46 constructs in the absence and presence of coexpressed β2b subunit, recorded at +40 mV. (B) I-V curves of channels in α1α2δ and α1α2δβ2b subunit compositions (n = 6–8, N = 1). Representative averaged I-V curves are shown for α1C-wt, α1CΔ5, α1CΔ6-20, and α1CΔ46. (C and D) Normalized averaged I-V curves (C) and G-V curves (D) from all experiments and all mutants tested, in absence (gray symbols) and presence (pink symbols) of the β2b subunit. The solid lines were drawn for illustration using the Boltzmann equation with the averaged parameters of V1/2 and Ka from Table I and Vrev from Table S1. Data are from 2–11 experiments, 14–56 cells.
	Figure 6. The NTI module regulates the effect of β2b subunit. (A) The increase in IBa caused by coexpression of β2b (relative to currents observed in the same α1C constructs without β2b) is much smaller in NT deletion mutants than in α1C-wt, at all voltages. Shown are results of two representative experiments (n = 10–18). (B) A general summary of the effect of coexpression of β2b. Shown is the fold increase in relative I40 in the various constructs; numbers of assayed cells are indicated above the bars. N = 2–7 experiments. (C) The increase in IBa caused by coexpression of β2b is potentiated in the α1CΔ5 mutant. N = 2.
	Figure 7. Design and surface expression of α1C-short, short/long NT chimeras, and α1CΔ21-46. (A) The design of initial NT segments of new constructs. Dashes show gaps. The homologous 15-aa sequence in long- and short-NT is highlighted in yellow and magenta, respectively. (B and C) Surface expression of the NT chimeric and deletion constructs in the absence and presence of β2b monitored in giant membrane patches (B) or in intact oocyte by imaging the external HA tag (C). The amount of injected RNA was 2.5 ng/oocyte in B and 5 ng in C. Examples of confocal images are shown in panel a, and the summary of the surface expression in the absence of β subunit (relative to α1C-wt) is shown in b. The effect of coexpression of β2b for each one of the constructs tested in these experiments is summarized in c. Numbers above bars indicate the number of patches (B) or oocytes (C) imaged.
	Figure 8. Chimeras of long- and short-NT help demarcate the NTI module. Two separate series of experiments, that used different doses of RNA, are shown in A–C. In Aa, B, and Ca, all RNAs were injected at 1 ng/oocyte; in Ab and Cb, at 2.5 ng/oocyte. (A) Comparison of normalized I40 in the absence of coexpressed β subunit. N = 2–3. (B) Effect of β2b on the voltage dependency of IBa in the same experiments as in Aa. Normalized averaged G-V curves are shown, in the absence (black symbols) and presence (red symbols) of the β2b subunit. (C) Summary of the effect of coexpression of β2b on I40. (D) The voltage dependency of the relative effects of β2b on IBa in selected NT mutants. The results were summarized from experiments shown in Figs. 6 B and 8. In each experiment, the increase in IBa caused by the coexpression of β2b was normalized to the average increase observed in α1C-wt group of oocytes at the same voltage, and the results were averaged across all experiments. The linear regression (dotted lines) is shown for illustrative purposes.

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