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BMC Neurosci
2002 Nov 06;3:17. doi: 10.1186/1471-2202-3-17.
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Evidence for non-independent gating of P2X2 receptors expressed in Xenopus oocytes.
Ding S
,
Sachs F
.
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BACKGROUND: P2X2 receptor is an ATP-activated ion channel which is widely expressed in the nervous system, and mediates synaptic transmission.
RESULTS: We recorded currents of P2X2 receptors expressed in Xenopus oocytes from outside-out patches and have found that currents recorded from patches containing a single or multiple P2X2 channels differ in a manner suggesting positive cooperativity. First, the currents from multichannel patches exhibit simultaneous transitions more frequently than predicted from the activity of independent channels. Second, the mean open lifetime at the current level of a single channel in a multichannel burst is about six times longer than the open time of currents from single channel patches, a trend opposite to what is expected of independent channels. These results indicate that the channels have positive cooperativity and that the longer opening is due to a slower closing rate. Third, from kinetic analysis the likelihood of the cooperative model is significantly larger than that of the independent model. Fourth, the open channel noise of currents from patches containing multiple channels is less than half that from a single channel, which is consistent with the channel properties being different when they are active in groups.
CONCLUSION: Taken together, our results suggest that P2X2 receptors are non-independent, but interact with positive cooperativity.
Figure 1. Typical current from a multiple-channel patch. (A) The outside-out patch current activated by 10 μM ATP recorded at -120 mV from a Xenopus oocyte. The data were sampled at 10 kHz and filtered at 5 kHz. There are at least three channels active in this patch as indicated by the open level of current. There are a surprising number of what appear to be nearly simultaneous openings and closings, some of which are marked by arrows. These occurrences are the indication of channel interactions. The convention in this paper is that inward currents are downward. (B) The all-points amplitude histogram of currents from panel A (0.15 pA/bin). The distribution was fit by a sum of four Gaussians (lines), with means of 0, 4.1, 7.9 and 11.9 pA, representing the closed, 1st, 2nd, and 3rd open levels. The open channel noise of each level was ~1.2 pA. The probabilities, P(k)exp, that all channels are closed, one, two, and three channels are open from histogram are 0.210, 0.487, 0.270, 0.027, respectively in this patch. (C) Comparison of P(k)exp with P(k)binomial from the binomial distribution (Eq. 5). The ratio of P(k)exp/P(k)binomial increases with increasing k.
Figure 2. Dose-response curves of currents from multiple- and single channel patches. (A) Multiple channel currents activated by different ATP concentrations at -120 mV. The data were sampled at 10 kHz and filtered at 5 kHz. The bar indicates the application of ATP. (B) The dose-response curve of multiple channel currents (Circles, n = 3) and single channel currents (Diamonds, n = 2) recorded from outside-out patches. The multiple channel currents were normalized to the peak value of currents activated by 100 μM ATP and rescaled to the same po,max as the single channel current. po of single channel currents (Diamonds) were calculated from the all-points histogram (see Figure 4). The solid line is a fit of the Hill equation of multiple channel currents with an EC50 of 9.8 ± 0.8 μM and a Hill coefficient of 1.91 ± 0.29. The dash-dot line is the fit of the Hill equation of single channel currents with an EC50 of 11.2 ± 1.0 μM, a Hill coefficient of 2.3 ± 0.44, and po,max of 0.61.
Figure 3. Mean open channel lifetimes of currents from multiple channel currents and single channel currents. The currents were recorded from Xenopus oocytes at -120 mV in divalent free perfusion solutions and were sampled at 20 kHz and filtered at 5 kHz. (A) Single channel current (left) shown with the idealized current above and the histogram below. The mean amplitude and open channel noise are 4.1 pA and 2.4 pA. (B) Currents from a patch containing two channels (left) with the idealized currents shown above and the histograms below. The mean amplitude and open channel noise are 4.8 pA and 1.5 pA. (C) Mean open lifetimes of currents from patches containing one (n = 6), and two (n = 4) and three (n = 5) opening levels. (D) The relative open channel noise of single channel currents and of the 1st and 2nd opening levels of multiple channel currents. The amplitude difference between 1st and 2nd level of multiple channel currents is insignificant. However, the noise of multiple channels (either 1st or 2nd level) and single channel currents is significantly different (p > 0.05, t-test).
Figure 4. Simulated currents containing one, two, three channels, and mean times of each open level. (A) Simulated currents containing one (top), two (middle) and three (bottom) independent channels. We used a two state model (Scheme 1) with α = 250 s-1, and β = 400 s-1 to simulate the currents. The simulated currents were sampled at 100 kHz and filtered with a digital filter of 10 kHz. The mean closed and unitary current amplitude set to 0 and 1 pA respectively. We set the standard deviation for closed and open level of 0.1 and 0.3 pA to mimic the large open channel noise of P2X2 receptors. Oi represents the nth open level for multiple channel currents. (B) The mean times of each open level from 5 s of simulated data.
Figure 5. Open channel noise is independent of ATP concentration. (A) Single channel currents recorded at -120 mV from a Xenopus oocyte activated by different concentrations of ATP as indicated in the figure. The data were filtered at 10 kHz and sampled at 40 kHz. The perfusion solution contained no divalent ions. All of the current traces in this figure are from the same patch. This patch contained one channel because there is no overlap of current in all the range of ATP concentrations. (B) The all-points amplitude histograms of the currents from panel A (0.05 pA/bin), with the distributions fit to the sum of two Gaussians. The dot, dash dot, and solid lines are the fits of open peak, closed peak, and the summation of two Gaussians. The fitting parameters are listed in Table 1.
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