Single channel properties of human alpha3 AChRs: impact of beta2, beta4 and alpha5 subunits

Abstract

1. We performed single channel analysis on human alpha3 acetylcholine receptors (AChRs) in Xenopus oocytes and native AChRs from the human neuroblastoma cell line IMR-32. alpha3 AChRs exhibit channel properties that reflect subunit composition. 2. alpha3beta2 AChR open times were 0.71 +/- 0.14 and 3.5 +/- 0.4 ms with a predominant conductance of 26 pS. alpha3beta4 AChRs had open times of 1.4 +/- 0.2 and 6.5 +/- 0.8 ms and a predominant conductance of 31 pS. Burst times were 0.82 +/- 0.12 and 5.3 +/- 0.7 ms for alpha3beta2 and 1.7 +/- 0.1 and 16 +/- 1 ms for alpha3beta4. Desensitization was faster for AChRs with the beta2 subunit than for those with the beta4 subunit. 3. One open time for alpha3alpha5beta2 AChRs (5.5 +/- 0.3 ms) was different from those of alpha3beta2 AChRs. For alpha3alpha5beta4 AChRs, an additional conductance, open time and burst time (36 pS, 22 +/- 3 ms and 43 +/- 4 ms, respectively) were different from those for alpha3beta4 AChRs. 4. alpha3 AChRs were inhibited by hexamethonium or mecamylamine. The rate constants for block of alpha3beta4 by hexamethonium and of alpha3beta2 by mecamylamine were 1.2 x 107 and 4.6 x 107 M-1 s-1, respectively. 5. AChRs from IMR-32 cells had a predominant conductance of 32 pS and open times of 1.5 +/- 0.3 and 9.6 +/- 1.2 ms. These properties were most similar to those of alpha3beta4 AChRs expressed in oocytes. Antibodies revealed that 5 +/- 2 % of IMR-32 alpha3 AChRs contained alpha5 subunits and 6 +/- 2 % contained beta2 subunits. IMR-32 alpha3 AChRs are primarily alpha3beta4 AChRs.

Figure 5. Closed time distributions for α3 AChRs expressed in oocytes

Examples of closed time distributions were fitted with exponential functions as described in Methods. For these patches, four components could adequately describe the data. The time constants determined from the fits are given for each patch. These values were used to set the critical times for burst definitions. For α3β2 and α3α5β2 AChRs, the fastest time constant was defined as the intraburst closed time, while the three longer time constants were set as between burst closed times. For α3β4 and α3α5β4 AChRs, the two shortest time constants were set as intraburst closed times, while the two longest time constants were set as between burst closed times. Channels were activated by 1 μM ACh for α3β2 and α3α5β2, while 5 μM ACh was used for α3β4 and α3α5β4 AChRs. The holding potential was -80 mV.

Figure 1. Heterologous α3 AChR single channel currents

Each panel depicts representative single channel recordings for outside-out patches from oocytes that were injected with the indicated combination of human AChR subunit transcripts. ACh concentrations were 1, 5, 10 and 5 μM for α3β2, α3α5β2, α3β4 and α3α5β4, respectively. All channels were recorded at a holding potential of -80 mV. The data were filtered at 2 kHz for display and are not necessarily contiguous.

Figure 2. Representative open level amplitude histograms for α3 AChRs expressed in oocytes

For each subunit composition, open level amplitude histograms were constructed from events list files created to include all event amplitudes that could be discerned for a particular patch (either one or two conductance levels). The fits to Gaussian functions with the appropriate number of components are also shown. For the α3α5β4 AChRs, distributions for two patches are superimposed. One of the patches has two components and the other only one, while together they represent the three conductances observed for this subunit combination. The mean values represent the mean from several patches including the example shown. The holding potential was -80 mV.

Figure 3. Representative open time distributions for α3 AChRs expressed in oocytes

For each subunit combination, log-binned histograms were constructed from events list files for the predominant conductance for that channel. As shown, the histograms were fitted with exponential functions of the appropriate number of components using the method of maximum likelihood. Time constants reflect the mean values observed for all patches including components that may not have been present in the representative example. Percentages reflect the mean areas from the fits that correspond to each exponential component when present. The holding potential was -80 mV.

Figure 4. Representative burst durations for α3 AChRs expressed in oocytes

For each subunit combination, log-binned histograms were constructed from events list files for the predominant channel conductance. The burst criterion times were evaluated as described in Methods. The histograms were fitted with exponential functions of the appropriate number of components using the method of maximum likelihood. The time constants reflect the mean of values observed for all patches for that AChR. The percentages reflect the mean areas from the fits that correspond to each exponential component when present. The holding potential was -80 mV.

Figure 6. Segments of recording demonstrating the longer bursting channels of α3α5β4 AChRs

This recording was made at -80 mV and has been scaled so that it can be compared directly to the recordings presented in Fig. 1. Note the dramatic increase in duration of channel openings and bursts compared with α3β4 AChRs without the α5 subunit.

Figure 7. Open time versus time elapsed during recording of α3β4 AChRs and α3α5β4 AChRs in oocytes

These plots demonstrate the persistence of channels of all kinetic types throughout the duration of the recordings. Note the difference in ordinate magnitudes for the two channel types and the much longer duration channel openings for α3α5β4 versusα3β4 AChRs.

Figure 8. Channel blockade of oocyte-expressed α3 AChR single channel currents by hexamethonium or mecamylamine

A, all α3 subunit combinations were sensitive to inhibition by hexamethonium or mecamylamine. α3β2 and α3α5β2 AChRs were potently inhibited by mecamylamine as evidenced by the large reduction in opening frequency and shorter duration openings. Hexamethonium blocked α3β4 and α3α5β4 AChRs by reducing the channel open duration but did not reduce the channel open frequency. All traces were recorded at -80 mV. The block by both agents was reversible after 1 min washout. B, the relationship for reciprocal open time versus concentration of the blocking agent reveals their forward block rate constants. The relationship for hexamethonium block of α3β4 AChRs revealed a blocking rate of 1.2 × 10⁷ M⁻¹ s⁻¹. For mecamylamine block of α3β2 AChRs, the rate constant was 4.6 × 10⁷ M⁻¹ s⁻¹.

Figure 9. Native human α3 AChR single channel currents recorded from IMR-32 neuroblastoma cells

Representative examples are shown for single channel activity recorded from outside-out patches from IMR-32 cells for the distributions of amplitude, open duration and burst duration. Time constants and percentages were determined as described for figures depicting heterologously expressed AChRs. All data were recorded at -80 mV.

Figure 10. Native human α3 AChR single channel currents recorded from IMR-32 neuroblastoma cells resemble oocyte-expressed α3β4 AChRs under high sodium conditions

High sodium conditions had higher external sodium than previous experiments and no internal fluoride (see Methods). Representative examples are shown for channel activity recorded from outside-out patches along with the distributions of channel amplitudes, open durations and burst durations. Time constants and percentages were determined as described in earlier figures and reflect the means ±s.e.m. from three to five patches. All data were recorded at -60 mV. The arrow marks a clear transition to a subconductance state during the α3β4 AChR opening.

Figure 11. Fraction of IMR-32 α3 AChRs that contain β2 or α5 subunits

A, solid-phase RIAs were used to demonstrate the fraction of total IMR-32 α3 AChRs that contain the β2 subunit. Microwells coated with mAb 210 (to α3) or mAb 295 (to β2) were used to adsorb AChRs from Triton X-100 extracts from IMR-32 cells. The adsorbed AChRs were labelled with ³H-epibatidine for quantification. Typically, 5-12 fmol of AChR (0.15-0.20 fmol (mg wet weight cells)⁻¹) were bound by mAb 210, while mAb 295 bound 6 ± 2 % of this amount. B, immunoprecipitation of IMR-32 AChRs was used to demonstrate the fraction of total IMR-32 AChRs that contain the α5 subunit. Triton X-100 extracts were incubated with excess mAb 210 or α5 antiserum, labelled with ³H-epibatidine, and then precipitated with excess sheep anti-rat IgG as secondary antibody. The α5 antiserum precipitated 5 ± 2 % as much AChR as mAb 210.