Facilitation by the beta2a subunit of pore openings in cardiac Ca2+ channels

Abstract

1. Single channel recordings were performed on the cardiac calcium channel (alpha1C) in order to study the effect of coexpression of the accessory beta2a subunit. On-cell patch clamp recordings were performed after expression of these channels in Xenopus oocytes. 2. The alpha1C subunit, when expressed alone, had similar single channel properties to native cardiac channels. Slow transitions between low and high open probability (Po) gating modes were found as well as fast gating transitions between the open and closed states. 3. Coexpression of the beta2a subunit caused changes in the fast gating during high Po mode. In this mode, open time distributions reveal at least three open states and the beta2a subunit favours the occupancy of the longest, 10-15 ms open state. No effect of the beta2a subunit was found when the channel was gating in the low Po mode. 4. Slow gating transitions were also affected by the beta2a subunit. The high Po mode was maintained for the duration of the depolarizing pulse in the presence of the beta2a subunit; while the alpha1C channel when expressed alone, frequently switched into and out of the high Po mode during the course of a sweep. 5. The beta2a subunit also affected mode switching that occurred between sweeps. Runs analysis revealed that the alpha1C subunit has a tendency toward non-random mode switching. The beta2a subunit increased this tendency. A chi2 analysis of contingency tables indicated that the beta2a subunit caused the alpha1C channel to gain 'intrinsic memory', meaning that the mode of a given sweep can be non-independent of the mode of the previous sweep. 6. We conclude that the beta2a subunit causes changes to the alpha1C channel in both its fast and slow gating behaviour. The beta2a subunit alters fast gating by facilitating movement of the channel into an existing open state. Additionally, the beta2a subunit decreases the slow switching between low and high Po modes.

Figure 1. Effect of the β_2a subunit on single α_1C channels

Coexpression of the β_2a subunit affects both fast and slow gating in α_1C. Single channel currents through the pore-forming subunit (α_1C) of the cardiac Ca²⁺ channel expressed with or without the β_2a subunit in oocytes in the presence of 10 μM Bay K 8644. Consecutive sweeps, pulses to +15 mV from a holding potential of -50 mV: α_1C channel expressed alone (A);α_1C+β_2a low P_o (B) and high P_o (C) traces. Corresponding ensemble averages (EA) and macroscopic patch currents (MP) from another oocyte are shown below the single channel records. D,P_o frequency histogram from seven patches expressing the α_1C subunit alone. E,P_o frequency histogram from eight patches co-expressing the α_1C+β_2a subunit. Bin width is 0.01. Vertical dashed line indicates P_o= 0.3.

Figure 2. Open dwell time histograms

Coexpression of the β_2a subunit increases the proportion of long openings. Fits of the open dwell time histograms to exponential distributions using the maximum likelihood method. α_1C (A) and α_1C+β_2a (B). The histograms are obtained from all data combined together, bin width is 0.07. In both panels, the continuous lines are the fits to the individual components and to the total histogram. Values of the time constants (τ_o1, τ_o2 and τ_o3, in ms) and of the respective relative amplitudes (A_o1, A_o2 and A_o3) are given for both α_1C and α_1C+β_2a. Recordings were performed in the presence of 10 μM Bay K 8644.

Figure 3. Open dwell time histograms for low and high P_o

The β_2a subunit favours the occurrence of long openings only in high P_o. Simultaneous fits of open dwell time histograms, with (α_1C+β_2a, C and G) and without (α_1C, A and E) the β_2a subunit, after separation of high P_o (E and G) and low P_o (A and C) sweeps. The histograms have been fitted in pairs, α_1C and α_1C+β_2a in each mode, forcing the time constants to the same values for the pair and allowing the respective amplitudes to change for each histogram, bin width is 0.07. Ensemble averages are shown to the right of corresponding histograms (B,D, F and H). Recordings were performed in the presence of 10 μM Bay K 8644.

Figure 4. Closed dwell time histograms

Changes in the overall closed time distribution in the presence of the β_2a subunit were not detected when using all mode sweeps. Closed time histograms for α_1C (A) and α_1C+β_2a (B), bin width is 0.07. As in Fig. 2, all events have been combined and fits to the individual components are shown together with the fits to the total histograms. Bay K 8644 (10 μM) is present.

Figure 5. Closed dwell time histograms for low P_o and high P_o

Coexpression of the β_2a subunit increases the proportion of the briefer closed times in high P_o. Closed time histograms separated into low P_o (A) and high P_o (B), bin width is 0.07. The continuous lines are the fits to the individual peaks in each histogram and to the total histograms. Time constants (in ms) and relative amplitudes of the individual components are listed for each histogram. Bay K 8644 (10 μM) is present in the solution.

Figure 6. The β_2a subunit caused an increase in the clustering of sweeps in the same mode

Clustering of sweeps of the same mode is more likely to occur in the presence of the β_2a subunit. Contingency tables from a pair of experiments comparing low P_o and high P_o gating in the α_1C(A) and the α_1C+β_2a(B) channel. In the presence of the β_2a subunit, the mode of a sweep (n) becomes non-independent of the mode of the previous sweep (n - 1). The (high P_o-high P_o) column is larger in B than in A, indicating clustering of consecutive high P_o sweeps in α_1C+β_2a channels, as opposed to frequent switches back and forth between the two modes in α_1C alone channels. The experiment analysed in A had a χ² value of 0.01, meaning that, in this case, the two characteristics that define the contingency table are not significantly related (P= 0.9185). The χ² value for the experiment in B was 8.51, meaning that the two characteristics that define the contingency table are significantly related (P= 0.0035). The relatively large (low P_o-low P_o) columns in both A and B are due to the fact that the majority of active traces gate in low P_o.

Figure 7. A multistate model for the effect of the β_2a subunit on α_1C single channel gating

A scheme depicting the open (O), closed (C) and inactivated (I) states of the channel and the effect of the β_2a subunit on the transitions between them.