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2a subunit of pore openings in cardiac Ca2+ channels
Received 3 July 1997; accepted after revision 16 October 1997.
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ABSTRACT |
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1C) in order to study the effect of coexpression of the accessory 2a subunit. On-cell patch clamp recordings were performed after expression of these channels in Xenopus oocytes.
1C 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.
2a 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 2a subunit favours the occupancy of the longest, 10-15 ms open state. No effect of the 2a subunit was found when the channel was gating in the low Po mode.
2a subunit. The high Po mode was maintained for the duration of the depolarizing pulse in the presence of the 2a subunit; while the 1C channel when expressed alone, frequently switched into and out of the high Po mode during the course of a sweep.
2a subunit also affected mode switching that occurred between sweeps. Runs analysis revealed that the 1C subunit has a tendency toward non-random mode switching. The 2a subunit increased this tendency. A 
2 analysis of contingency tables indicated that the 2a subunit caused the 1C channel to gain 'intrinsic memory', meaning that the mode of a given sweep can be non-independent of the mode of the previous sweep.
2a subunit causes changes to the 1C channel in both its fast and slow gating behaviour. The 2a subunit alters fast gating by facilitating movement of the channel into an existing open state. Additionally, the 2a subunit decreases the slow switching between low and high Po modes.
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INTRODUCTION |
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1 subunit that contains the voltage sensor and the ion conducting pore, as well as several accessory subunits , 
and 2/
(Perez-Reyes et al. 1989; Catterall, 1991; Hofmann, Biel & Flockerzi, 1994). The function of the accessory subunits has been extensively studied at the macroscopic current level. The 2a subunit increased the magnitude and the speed of activation of 1C macroscopic ionic currents (Lacerda et al. 1991; Wei, Perez-Reyes, Lacerda, Schuster, Brown & Birnbaumer, 1991; Singer, Biel, Lotan, Flockerzi, Hofmann & Dascal, 1991; Varadi, Lory, Schultz, Varadi & Schwartz, 1991; Williams et al. 1992; Lory, Varadi, Slish, Varadi & Schwartz, 1993; Olcese et al. 1994; Pérez-García, Kamp & Marban, 1995). This current potentiation occurred without significant changes in the movement of the voltage sensor, recorded as gating currents (Neely, Wei, Olcese, Birnbaumer & Stefani, 1993). These studies indicated that the 2a subunit facilitated pore opening by improving the coupling between the movement of the voltage sensor and the opening of the channel pore.
Voltage-gated ion channels exhibit fast gating transitions between open and closed states, as well as slow transitions between different modes that have distinct gating behaviour (Moczydlowski & Latorre, 1983; Bean, 1989). In native cardiac Ca2+ channels, individual sweeps have been grouped into three modes of gating: (a) null or silent traces with no channel activity; (b) mode 1, low probability of opening (low Po) traces with infrequent brief openings, and (c) mode 2, medium to high Po traces with openings clustered forming bursts of activity (Reuter, Stevens, Tsien & Yellen, 1982; Hess, Lansman & Tsien, 1984; Bean, 1990). Native cardiac Ca2+ channels can remain in a single mode during sequential sweeps; changes between these three modes of activity are referred to as slow gating transitions. The dihydropyridine agonist Bay K 8644 has been shown to enhance single channel calcium current by increasing the single channel open probability through an increase in the likelihood of mode 2 gating (Hess et al. 1984; reviewed by McDonald, Pelzer, Trautwein & Pelzer, 1994). In this study, we investigated whether the regulatory 2a subunit can modify the single channel gating properties of the cardiac 1C subunit expressed in Xenopus oocytes in the presence of 10 µM Bay K 8644. The 2a subunit affects fast gating transitions by favouring pore opening and facilitating the open state during high Po gating; slow gating transitions are affected by a stabilization of a given gating mode, without a change in the proportion of a given mode.
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METHODS |
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Molecular biology and oocyte preparation
The cRNAs were prepared from two plasmids bearing the a- splice variant of the type C or cardiac 1 (1C-a) formerly also CaCh2a, and the type 2a cardiac 2a subunit. The 1C cDNA was digested with Hind III as previously described (Wei et al. 1991) and 2a cDNA with Not I (Perez-Reyes et al. 1992). Linearized plasmids (0·5 µg) were transcribed at 37°C in a volume of 25 µl containing 40 mM Tris-HCl (pH 7·2), 6 mM MgCl2, 10 mM dithiothreitol, 4 mM spermidine, 0·4 mM each of adenosine triphosphate, guanosine triphosphate, cytosine triphosphate and uridine trisphosphate, 1 mM 7-methyl guanosonine (5') triphosphate (5') guanosine and 10 units of T7 RNA polymerase (Boehringer-Mannheim). The cRNA products were extracted with phenol-chloroform, recovered by precipitation with ethanol and suspended in double-distilled water to a final concentration of 0·2 µg µl-1 of each species, 50 nl were injected per oocyte.
Xenopus frogs were anaesthetized by immersion in water containing 0·15 % tricane methanesulphonate (Sigma) for at least 20 min until fully immobile. The ovaries were surgically removed under sterile conditions by abdominal incision. The animals were then killed by decapitation. Animal protocols were performed with the approval of the Institutional Animal Care Committee of the University of California, Los Angeles. Oocytes were maintained at 19·5°C in modified Barth's solution containing 90 mM NaCl, 2 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM Hepes, 50 µg ml-1 gentamicin, adjusted to pH 7·2 with NaOH.
Channel recording and data analysis
Recordings were performed 4-12 days following cRNA injection. Single channel activity was recorded with an EPC-7 patch clamp amplifier (List Electronic) from cell-attached patches of Xenopus oocyte membrane after removing the vitelline layer. Recording pipettes had tip diameters of 2-6 µm with a final resistance of 0·5-4 M
. Stray capacitance was reduced by coating the shank of the pipettes with a mixture of Parafilm and light mineral oil. The pipette solution contained 75 mM Ba(CH3SO3)2, 5 mM BaCl2, 10 µM (-)-Bay K 8644 and 10 mM Hepes, titrated to pH 7·0. The bath saline was 110 mM KCH3SO3 with 10 mM Hepes titrated to pH 7·0. We used a high (-)-Bay K 8644 concentration, about 200 times higher than its Kd, to minimize any potential effect of the 2a subunit on the binding of (-)-Bay K 8644 to the channel. The data presented were collected from one channel in a patch for any given experiment. Patch potentials were maintained at -50 mV and 200 ms pulses to 15 mV were delivered at 1 Hz. Analysis was performed on seven 1C and eight 1C+2a patches in which we succeeded in recording up to 1000 sweeps. Data were digitized at 50 or 100 µs (point)-1 after low-pass filtering at 2 kHz. Single channel records were corrected off-line for linear leak and capacity currents using customized software. Open and closed transitions were detected by the half amplitude threshold criterion using TRANSIT software (VanDongen, 1996). Open and closed time histograms were fitted to multiple exponential functions using the maximum likelihood method (Sigworth & Sine, 1987) and the adequacy of the fit with different numbers of distributions was statistically evaluated, and accepted when P < 0·01.
Separation of low and high Po sweeps
Categorization of sweeps was achieved by defining null sweeps as those containing no channel activity, low open probability (low Po) sweeps were defined as those that exhibited channel activity with an open probability of Po 
0·3, and high Po sweeps were defined as those that exhibited channel activity with a Po > 0·3.
Runs analysis and 2 contingency table analysis
Each sweep was categorized into null, low Po or high Po mode to perform runs and contingency analysis. Runs analysis evaluates whether the clustering of sweeps with the same mode (i.e. a run) represents a deviation from randomness. This deviation can occur in two ways: (a) the number of runs can be smaller than would randomly occur, in which case the distribution is 'contagious', or (b) the number of runs can be larger than would randomly occur, in which case the distribution is 'uniform'. In our case, the number of runs was always smaller than would randomly occur, thus we tested our data for 'contagiousness'. To do this, we performed one-tailed tests on the statistic Z (the standard random variable) in the case of three nominal categories. Z should a normal deviate for a large number of runs (n > 30, Brownlee, 1965; Zar, 1974), and we formulate the null hypothesis in these terms: 'the distribution of Z is non-contagious'. Values of Z are compared to tabulated critical values. The distribution is contagious when Z 
Zcritical and u µu, where u is the experimental number of runs and µu is the mean of the distribution, with standard deviation 
u. The distribution would be uniform if Z Zcritical and u µu.
Another approach to the clustering of sweeps with the same mode is the 2 analysis of contingency tables. Contingency tables (2 × 2) of elements were constructed. The null hypothesis in this case is that the row frequencies (mode during the second sweep) are independent of the column frequencies (mode during the first sweep). In other words, the null hypothesis states that the process has no intrinsic memory and the occurrence of a given mode sweep is independent of the mode of the channel during the previous sweep.
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RESULTS |
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Low and high Po gating behaviour is present in the 1C subunit
The single channel traces in Fig. 1 are representative of the fast and slow gating behaviour present in the 1C and 1C+2a channels. Channel openings are downward deflections. Ensemble averages (EA) of single channel experiments are shown below the single channel records. Macroscopic patch recordings (MP) from a different oocyte, using the same recording solutions, are superimposed on the ensemble averages for both the 1C and the 1C+2a channel. The close agreement in the time course of the macroscopic currents and the ensemble averages are evidence that the single channel recordings are representative samples. These channels have fast gating transitions between the open and closed states as well as bursts of openings that are closely spaced and that occur during the time interval of a sweep (200 ms). In addition to this fast gating behaviour, a slow gating process can be observed that switches the channel between low and high Po modes. In both 1C (Fig. 1A) and 1C+2a (Fig. 1B and C), we found null traces, traces with brief openings close to the pulse onset which became infrequent toward the end of the pulse (low Po) and traces with bursts of channel openings that can occur throughout the duration of the trace (high Po). Thus, as previously described for native Ca2+ channels (Hess et al. 1984), expressed Ca2+ channels can gate in low and high Po modes. Furthermore, these data indicate that the 1C subunit, when expressed alone, encodes these modes of gating. The examination of the traces in Fig. 1A, B and C suggests that coexpression of the subunit modified both fast and slow gating behaviours. The 2a subunit affected the fast gating properties by increasing burst duration and the proportion of long openings during a high Po burst. The 2a subunit affected the slow gating properties by stabilizing a given gating mode during consecutive sweeps.
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Figure 1. Effect of the
Coexpression of the
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Long openings during bursts of activity are favoured by coexpression of the subunit
We defined the three modes of gating for further analysis as follows: null sweeps were the sweeps with no channel activity; low Po sweeps were those with a Po 0·3, and high Po sweeps were those with a Po > 0·3. Examples of these modes can be seen in the single channel traces of Fig. 1, for the 1C channel, null (sweeps 1, 11 and 13), low Po (sweeps 2, 7, 8 and 9) and high Po (sweeps 3, 5, 10, 14 and 15). 1C+2a openings are shown in Fig. 1B (low Po), and Fig. 1C (high Po). Low Po sweeps are indistinguishable between 1C and 1C+2a channels. High Po openings are, however, quite distinct when comparing 1C and 1C+2a channels (e.g. compare sweep 10 in Fig. 1A with any sweep in Fig. 1C). High Po can be identified for both clones by clusters of openings separated by closings generating bursts of channel activity. The 1C+2a bursting pattern is different in that the openings during the burst, and the duration of the bursts, are longer (Fig. 1C), and consequently the closures during a high Po burst are shorter. These differences were quantitatively investigated by the construction and analysis of open and closed dwell time histograms.
Coexpression of the 2a subunit stabilizes bursting activity during high Po
Coexpression of the 2a subunit also caused changes in slow gating processes. In the absence of the 2a subunit, the channel frequently switched into, and out of, high Po during a sweep (Fig. 1A), whereas in the presence of the 2a subunit, a sweep that began in a particular mode tended to remain in that mode for the duration of the sweep (Fig. 1B and C). These gating properties are reflected in the Po frequency histograms compiled from all of the experiments for the 1C and the 1C+2a channels (Fig. 1D and E). In the presence of the 2a subunit, the histogram closely resembled that seen for native channels (Hess et al. 1984): there is a peak at very low Po, and a peak at very high Po, with relatively few sweeps exhibiting intermediate values of Po (between 0·3 and 0·6). This form of the histogram indicated that most of the sweeps were clearly separated as low or high Po. In the absence of the 2a subunit (Fig. 1D), the histogram had a similar peak at very low Po, but it did not have a distinct peak at higher Po, and it had a shallow distribution at intermediate Po values. This shallow Po distribution in conjunction with the visual examination of raw sweeps suggested that the intermediate Po sweeps, in the absence of the 2a subunit, were due to bursts of high Po gating that were not maintained for the entire duration of the sweep (see sweeps 5, 10, 14 and 15 in Fig. 1A).
Coexpression of the 2a subunit does not change the proportion between the different gating modes
To investigate whether the 2a subunit affected the proportion of the different gating modes, we compared the proportion of null, low Po and high Po sweeps for 1C versus 1C+2a. The gating modes were categorized as described above. The insets in Fig. 1D and E show that the 2a subunit caused no significant change in the overall proportion of null, low Po or high Po sweeps. In all of the experiments combined for 1C and 1C+2a, respectively, the fraction of null sweeps was 0·19 ± 0·05 and 0·24 ± 0·15, the fraction of low Po sweeps was 0·55 ± 0·07 and 0·62 ± 0·02, and, the fraction of high Po sweeps was 0·25 ± 0·06 and 0·13 ± 0·13.
Effect of 2a subunit coexpression on open dwell times
Changes to fast gating kinetics caused by
Figure 2. Open dwell time histograms
Coexpression of the
Table 1. Effect of coexpression of
To investigate whether low or high Po modes were preferentially altered by the
Figure 3. Open dwell time histograms for low and high Po
The
Figure 4. Closed dwell time histograms
Changes in the overall closed time distribution in the presence of the
Ca2+ channel inactivation follows only brief openings in low Po
Ensemble average currents are to the right of each corresponding open time histogram in Fig. 3. The average current was maintained during the pulse length in high Po sweeps, while in low Po sweeps the currents inactivated. This was seen at the single channel level as brief openings clustered in the first half of the sweep during low Po, while during high Po the openings occurred throughout the duration of the sweep. This finding suggests that the inactivated state is reached from the low Po mode of gating.
Effect of
The results of the analysis for the closed dwell times are shown in Figs 4 and 5. Figure 4 illustrates the closed dwell time histograms for
Figure 5. Closed dwell time histograms for low Po and high Po
Coexpression of the
Table 2. Effect of coexpression of
Effect of
We performed runs analysis (Plummer & Hess, 1991) to determine whether the
Figure 6. The
Clustering of sweeps of the same mode is more likely to occur in the presence of the
Taken together, the runs and contingency table analysis indicate that a non-random pattern of mode switching occurs between sweeps in the
A minimum model for the action of
The effect of the
Figure 7. A multistate model for the effect of the
A scheme depicting the open (O), closed (C) and inactivated (I) states of the channel and the effect of the
Single channel modal gating is a highly conserved feature of voltage gated calcium and other channels. Our results indicate that in the presence of Bay K 8644, the
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Acknowledgements
This work was supported by NIH grants AR38970 to E. S. and AR43411 to L. B. N. Q. is the recipient of the AHA Scientist Development Grant 9630053N.
Corresponding author
E. Stefani: UCLA School of Medicine, Department of Anesthesiology, BH-612 CHS, Box 951778, Los Angeles, CA 90095-1778, USA.
Email: 656}Email: {tracking}estefani{at}ucla.edu
Author's present address
J. Costantin: Department of Neurology, UCLA School of Medicine, UCLA, Los Angeles, CA 90024, USA.
Email: jcostant{at}ucla.edu
This article has been cited by other articles:
2a subunit coexpression were studied by measuring the distributions of open and closed dwell times. In all cases, open times could be fitted to the sum of three distributions with a fast 
near 1 ms, an intermediate near 2-4 ms and a slow near 10-15 ms. The histograms in Figs 2 and 3 correspond to all events combined for 1C alone (54 392 events from 7 patches, upper panel) and for 1C+2a (29 135 events from 8 patches, lower panel). Table 1 summarizes the means ± 1C and 1C+2a. The number in parentheses is the statistical significance for each value, comparing 1C and 1C+2a. The histograms and table show that the open times, with and without 2a, could be fitted by similar time constant values but with differing weights. The main action of the 2a subunit on the fast gating kinetics was to increase the fraction of the third open time distribution from 0·04 to 0·10 (Fig. 2, Ao3 values). The fact that the 2a subunit did not significantly change the time constant values of the open time distributions, suggests that coexpression of the 2a subunit did not create a new open state, but it favoured the frequency of occupancy of an existing long open state.
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2a subunit increases the proportion of long openings. Fits of the open dwell time histograms to exponential distributions using the maximum likelihood method. 1C (A) and 1C+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 (o1, o2 and o3, in ms) and of the respective relative amplitudes (Ao1, Ao2 and Ao3) are given for both 1C and 1C+2a. Recordings were performed in the presence of 10 µM Bay K 8644.
2a subunit on open times
1 (ms)A1 (pA) 2 (ms)A2 (pA) 3 (ms)A3 (pA) Mean open time (ms) 1CAll sweeps 0·76 ± 0·16 0·34 ± 0·15 2·55 ± 0·45 0·63 ± 0·26 8·82 ± 1·98 0·04 ± 0·07 2·02 ± 0·24 High Po 1·10 ± 0·18 0·44 ± 0·13 2·95 ± 0·23 0·61 ± 0·09 8·49 ± 0·89 0·06 ± 0·02 2·68 ± 0·30 Low Po 1·15 ± 0·23 0·77 ± 0·11 2·91 ± 0·18 0·40 ± 0·11 - - 1·66 ± 0·13 1C+2a All sweeps 0·56 ± 0·06 0·34 ± 0·04 2·26 ± 0·42 0·49 ± 0·10 14·46 ± 2·74 0·31 ± 0·06 5·55 ± 1·35 (0·31) (1·00) (0·62) (0·26) (0·05) (0·008) (0·04) High Po - - 2·53 ± 0·55 0·47 ± 0·09 13·34 ± 1·17 0·61 ± 0·10 10·99 ± 1·46 (0·37) (0·19) (0·01) (0·001) * (0·001) * Low Po 1·32 ± 0·16 0·91 ± 0·04 5·10 ± 0·72 0·09 ± 0·04 - - 2·34 ± 0·12 (0·74) (0·28) (0·04) (0·13) (0·35) 1C and 1C+2a, respectively. Numbers in parentheses are Student's t test-derived probabilities with two independent samples and two-tailed distribution. * Statistically significant.2a subunit, we constructed separate open dwell time histograms from sweeps containing only low (Fig. 3A and C) or high Po (Fig. 3E and G) sweeps. The sweeps were categorized into high and low Po separated at Po = 0·3 as described in Methods. These histograms were simultaneously fitted with three time constants in each mode, with and without the 2a subunit. The data show that the 2a subunit did not greatly modify the open times during low Po, while it increased the mean open time during high Po gating. This large increase in the mean open time in high Po results from a larger fraction of long openings (o3) which increased from 0·04 in 1C to 0·59 in 1C+ (Fig. 3E and G). The coexpression of the 2a subunit also increased the burst duration during high Po (Fig. 1). Bursts were identified as gating activity separated by closed time longer than 8·5 ms, which was derived from the closed dwell time histograms (Fig. 4). The mean burst duration increased from 35 ms in 1C alone to 44 ms in 1C+2a channels, while the average number of openings per burst did not significantly change (9·0 events in 1C and 7·4 events in 1C+2a). In summary, coexpression of the 2a subunit favoured pore opening during high Po by increasing the frequency of long openings during bursts of activity and increasing the mean duration of the bursts.
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2a subunit favours the occurrence of long openings only in high Po. Simultaneous fits of open dwell time histograms, with (1C+2a, C and G) and without (1C, A and E) the 2a subunit, after separation of high Po (E and G) and low Po (A and C) sweeps. The histograms have been fitted in pairs, 1C and 1C+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.
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2a subunit were not detected when using all mode sweeps. Closed time histograms for 1C (A) and 1C+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.
subunit coexpression on closed times
1C and 1C+2a channels for all traces, while in Fig. 5 low and high Po traces were separated. We could not detect any effect of the coexpression of the 2a subunit on the overall closed time distribution in the histograms containing all modes. To investigate possible actions of the 2a subunit further, we performed a separate analysis for low and high Po sweeps. In low Po, the data are indistinguishable, but in high Po coexpression of the 2a subunit significantly increased (see Table 2) the proportion of the briefer closed times, which resulted in a modest reduction of the mean closed times. The faster exit from the closed state is in agreement with the higher frequency of long openings with the 2a subunit. In summary, by increasing the rates from the closed to the open states the 2a subunit favours channel opening without changing the conformation of the open channels.
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2a subunit increases the proportion of the briefer closed times in high Po. Closed time histograms separated into low Po (A) and high Po (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.
2a subunit on closed times
1 (ms)A1 (pA) 2 (ms)A2 (pA)< 3 (ms)A3 (pA) Mean closed time (ms) 1C All sweeps 0·74 ± 0·12 0·66 ± 0·07 4·37 ± 0·51 0·24 ± 0·03 33·73 ± 6·70 0·10 ± 0·05 4·06 ± 0·78 High Po 0·64 ± 0·10 0·56 ± 0·04 2·74 ± 0·43 0·37 ± 0·07 12·50 ± 1·87 0·07 ± 0·07 2·14 ± 0·96 Low Po 0·79 ± 0·12 0·59 ± 0·07 6·77 ± 0·66 0·27 ± 0·02 41·30 ± 7·86 0·14 ± 0·06 7·17 ± 2·01 1C+2aAll sweeps 0·56 ± 0·03 0·54 ± 0·06 6·57 ± 0·71 0·28 ± 0·34 49·90 ± 7·80 0·18 ± 0·05 9·71 ± 1·82 (0·18) (0·19) (0·03) (0·32) (0·12) (0·30) (0·02) High Po 0·47 ± 0·05 0·77 ± 0·03 3·80 ± 0·80 0·20 ± 0·03 17·15 ± 3·94 0·05 ± 0·01 1·65 ± 0·24 (0·15) (0·001) * (0·20) (0·05) (0·23) (0·70) (0·67) Low Po 0·81 ± 0·13 0·43 ± 0·05 7·24 ± 0·68 0·35 ± 0·04 38·18 ± 5·16 0·22 ± 0·06 10·71 ± 1·95 (0·94) (0·06) (0·08) (0·05) (0·95) (0·26) (0·12) 1C and 1C+2a, respectively. Numbers in parentheses are Student's t test-derived probabilities with two independent samples and two-tailed distribution. * Statistically significant.2a subunit coexpression on mode switching between consecutive sweeps
2a subunit caused the channel to remain in a particular gating mode for a longer series of consecutive sweeps (i.e. a run). The three population runs analysis compared runs of sweeps that had been classified into modes as described above. The 1C+2a channels had consistently higher Z values (see Methods) between 3·11 and 13·6 (n = 8), which were all above the critical value of 1·64 (P 0·05). Thus, in the presence of the 2a subunit, the channels did not randomly switch between modes. In the 1C channels we obtained lower Z values ranging between 0·31 and 8·91 (n = 7). In two 1C experiments the Z values (0·31 and 0·42) were lower than the critical 1·64 Z value, indicating randomness. The remaining five 1C experiments showed a tendency to clustering. The overall higher Z values obtained for the runs analysis in the presence of the 2a subunit show a further deviation from random mode switching, which indicate that the 2a subunit further stabilizes a given mode of gating.
2 analysis of contingency tables (Plummer & Hess, 1991) was performed to determine whether a given gating mode during a sweep (n), was contingent on the mode of the previous sweep (n - 1). The 2 values obtained from the 1C+2a contingency tables were between 8·5 and 144·1 for the observed versus expected frequencies (n = 4). These values of 2 indicate that the 1C+2a channels show a non-random pattern of clustering of sweeps. Stated differently, the mode of a given (n) sweep is non-independent of the mode during the previous (n - 1) sweep when the 2a subunit is present. The 2 values obtained from the 1C channel had a large variability ranging between 0·002 and 101·9. In three of seven experiments, the 2 values indicated independence in mode switching during the time window between two consecutive sweeps. The remaining ones showed non-independence. An example of the tables obtained for two individual experiments is shown in Fig. 6. The 2a subunit increased the number of high Po sweeps followed by another high Po sweep (Fig. 6B).
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2a subunit caused an increase in the clustering of sweeps in the same mode2a subunit. Contingency tables from a pair of experiments comparing low Po and high Po gating in the 1C (A) and the 1C+2a (B) channel. In the presence of the 2a subunit, the mode of a sweep (n) becomes non-independent of the mode of the previous sweep (n - 1). The (high Po-high Po) column is larger in B than in A, indicating clustering of consecutive high Po sweeps in 1C+2a channels, as opposed to frequent switches back and forth between the two modes in 1C alone channels. The experiment analysed in A had a 2 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 2 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 Po-low Po) columns in both A and B are due to the fact that the majority of active traces gate in low Po.
1C+2a channels. The 1C channels showed some variability but a tendency of non-independence in consecutive sweeps. The 2a subunit significantly increased the tendency toward clustering as seen using both methods of analysis.
DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References
2a subunit
2a subunit on the fast and slow gating of the 1C channel can be represented by a minimal multistate model. The scheme (Fig. 7) summarizes the open, closed and inactivated states and the transitions between them. The two closed states C1n and C2n represent the final closed states for each mode before channel opening, the three dots represent the pathway to deeper closed states. The three open states O21, O22, and O23 represent the three open states regularly seen in high Po open time histograms. In the absence of the 2a subunit, high Po openings are predominantly transitions from C2n to O21 and O22 with less frequent transitions to O23. The main effect of the 2a subunit on fast C2n to O transitions in high Po is an increase in transitions from C2n to O23 and a decrease in transitions to O22. The facilitation by the 2a subunit of a long open state during high Po gating occurs through an increase in the forward rates to channel opening and not by a decrease in the backward rates away from channel opening, no new open time is created by the 2a subunit and thus the backward rates leading away from the three high Po open states are unchanged. The open state O11 represents the low Po open states lumped together into one, and I1 represents the inactivated state reached from the low Po open states. The model also describes the decrease in mode switching caused by the 2a subunit. The stabilization of high Po gating seen within individual sweeps is incorporated into the model by decreasing the vertical rates between high and low Po. The increase in clustering of sequential sweeps caused by the 2a subunit can also be represented as a decrease in the vertical rates between the different modes. There is not a change in the proportion of null, low Po and high Po sweeps, suggesting that the ratio of these vertical rates between channel states remains unchanged.
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2a subunit on 1C single channel gating2a subunit on the transitions between them.
2a subunit confers significant changes to both fast and slow gating processes without creating any new open state. The 2a subunit alters fast gating by facilitating movement of the channel into an existing open state. Additionally, the 2a subunit decreases the slow switching between modes both within the time course of a single sweep, and during the interval between sweeps, effectively creating a protein that is less willing to undergo slow conformational changes between gating modes.
REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References
1 and subunits of the skeletal muscle dihydropyridine-sensitive Ca2+ channel. Nature 352, 527-530.
subunit of the ratio of the ionic current to the charge movement in the cardiac calcium channel. Science 262, 575-578. subunit sets rates of channel inactivation independently of the subunit's effect on activation. Neuron 13, 1433-1438.
1 and subunits. Journal of General Physiology 105, 289-306. subunit of the L-type calcium channel. Journal of Biological Chemistry 267, 1792-1797.
1-subunit of the dihydropyridine receptor from skeletal muscle. Nature 340, 233-236.
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