Orientation of the calcium channel beta relative to the alpha(1)2.2 subunit is critical for its regulation of channel activity

Abstract

Background: The Ca(v)beta subunits of high voltage-activated Ca(2+) channels control the trafficking and biophysical properties of the alpha(1) subunit. The Ca(v)beta-alpha(1) interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that betas regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the alpha-interaction domain (AID) be a rigid structure.

Methodology/principal findings: The present study tests this hypothesis by altering the flexibility and orientation of this region in alpha(1)2.2, then testing for Ca(v)beta regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished beta2a and beta3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of beta2a to produce non-inactivating currents. Orientation of Ca(v)beta with respect to alpha(1)2.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Ca(v)beta subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca(v)beta subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms beta-sheet. The orientation of beta with respect to alpha was confirmed by the bimolecular fluorescence complementation assay.

Conclusions/significance: These results show that the orientation of the Ca(v)beta subunit relative to the alpha(1)2.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca(v)beta to regulate channel activity.

Figure 1. Altering the structure of the linker between AID and IS6.

Amino acid sequence of the amino-terminal portion of the I–II loop of α₁2.2, and location of the following mutations: poly-glycine (PG6), poly-alanine (PA6), and deletions (Bdel1, Bdel2, Bdel3). Dashes represent the deleted amino acids. To highlight the sequence conservation across the Ca_v2 family, the residues that are not conserved are underlined. Predicted secondary structure (SOPMA algorithm; [43]) is represented by h – for helix, c – for random coil.

Figure 2. Ca_vβ subunit regulation of α₁2.2.

(A) Average peak currents normalized to cell capacitance. Smooth curves represent fits to the average data using a Boltzmann-Ohm equation. The symbols defined in (A) apply to all panels. (B) Activation represented by the normalized conductance (G/G_max). (C) Normalized current traces obtained during 350 ms step depolarization to +20 mV from a holding potential of −90 mV. WT+β3 currents are represented by a thick grey line. (D) The residual current at 350 ms of depolarizing pulse was divided by the maximum inward current and plotted against the test potential. (E) Representative current traces obtained during a test pulse to +40 mV after 15 s prepulses to varying potentials from a holding potential of −90 mV. Traces recorded after prepulses to −60 and −20 mV are highlighted to emphasize the β3 induced shift in steady-state inactivation. Scale bar represents 20 ms and 200 pA. (F) Effects of β2a and β3 on steady-state inactivation. The mean normalized amplitude of the currents is expressed as a function of membrane potential and fit with a Boltzmann equation (smooth curves). Data represent mean±SEM, in which the number of cells used to calculate the average is reported in Table 1.

Figure 3. Introduction of the poly-glycine substitution in the α₁2.2 subunit disrupts (PG6), while poly-alanine substitution (PA6) preserves Ca_vβ regulation.

Panels A–D show data obtained with PG6, while panels E–H show data obtained with PA6. (A, E) Peak current-voltage relationships normalized to cell capacitance for the respective α₁ mutant expressed alone or with β2a or β3. (B, F) Activation represented by the normalized conductance (G/G_max). The residual current after either 25 ms (C) or 350 ms (G) of depolarization divided by the maximum inward current, and plotted against test potential. Representative traces normalized to the peak inward current are shown in the inset. (D, H) Comparison of the β2a and β3 effects on steady-state inactivation estimated using 15 s prepulses to varying potentials. Dotted lines represent steady-state inactivation measured for WT channels in the presence of β3 (α₁2.2+α₂δ1+β3).

Figure 4. Deletions in the linker between AID and IS6 (Bdels) affect β regulation.

Panels A–D show data obtained with Bdel1, panels E–H show data obtained with Bdel2, and panels I–L show data obtained with Bdel3. (A, E, I) Peak current-voltage relationships normalized to cell capacitance for Bdels expressed with β2a or β3. In the absence of a β, only Bdel1 produced detectable currents. (B, F,J) Normalized current traces recorded during depolarizing steps to +20 mV from a holding potential of −90 mV. Residual current after either 350 ms (C) or 25 ms (G, K) of depolarization divided by the maximum inward current, and plotted against test potential. (D, H, L) Comparison of the β2a and β3 effects on steady-state inactivation for Bdels estimated using 15 s prepulses to varying potentials. Dotted lines represent β3 effect on steady-state inactivation for WT channel.

Figure 5. Estimating P_o of wild-type and Bdel1 channels.

(Aa) Exemplar gating current at reversal potential (∼65 mV) for WT channels expressed with β2a. (Ab) Ionic current trace from the same cell recorded during a depolarizing step to +30 mV from a holding potential of −90 mV. Exemplar gating (Ba) and ionic (Bb) currents for the Bdel1 deletion mutant (also with β2a). Scale bars represent the same units as in panel A. (C) Plot of G_max versus Q_max where each symbol represents an individual cell. Solid line represents the fit to the data with linear regression. The slope, G/Q, is proportional to maximal channel open probability.

Figure 6. Probing the α₁-β orientation with bimolecular fluorescence complementation.

(A) Images of representative cells transfected with the RFP, mCherry, α₂δ1, and either WT α₁2.2, Bdel1, or Bdel2. Transfected cells were identified by their red fluorescence, imaged in the red channel (10 ms), then in the cyan channel (1500 ms). Images were collected with the 40× objective and the spinning disk out (confocal off). Images taken at 100× confirmed that channel proteins were excluded from the nucleus. The images were converted from 24 to 8 bit, and their intensity range was set to 1–260. Inset bar represents 5 µm, and applies to all panels. (B–D) Data used to calculate the specificity of bimolecular fluorescent complementation. Individual cells were imaged in the cyan channel (BiFC signal) and in the red channel (free RFP marker), and the ratio of these signals was calculated. The ratios were then binned using Excel, and plotted using Prism. (B) Results obtained with WT α₁2.2 tagged with the CFP 1–158 fragment and β3-core tagged with the CFP 159–238 fragment. The median ratio was 0.23. (C) Results obtained with tagged Bdel1, The median ratio was 0.77. (D) Results obtained with tagged Bdel2, The median ratio was 0.32. (E) Specificity of the BiFC signal was determined by dividing the median ratio of the cyan to red fluorescence for the Bdels by the WT median ratio. Results were obtained from the following number of cells: WT, 107; Bdel1, 112; and Bdel2, 111. Asterisks represent statistical significance at the P<0.001 (***) or P<0.5 (*) level.

Figure 7. Model showing possible orientations of β with respect to α₁ assuming a β sheet structure at the site of deletion.

(A) Model showing the orientation of β-subunit in wild-type, and (B) after deletion of 1 amino acid. The β3 core structure was modeled from PDB code 1VYT . The CFP, Cirulean, was modeled from PDB code 2QYT . The fragments of CFP were generated using PyMOLWin (Delano Scientific), where CFP-N corresponds to residues 1–158, and CFP-C corresponds to residues 159–238. The approximate size of the α₁2.2 domains and linkers were estimated using the method of Helton and Horne, where the volume occupied by each segment is calculated from the number of amino acids in each segment . The β3 subunit was scaled using the same method.