Five different types of functional BK channels are observed following injection of a 1:1 mixture of mutant and WT cRNA into Xenopus oocytes. (A) Histograms of Vh values of single-channels from Fig. 4. WT channels expressed after injection of only WT cRNA are indicated by black bars, and G375R homotetrameric mutant channels expressed after injection of only G375R cRNA are indicated by blue bars. The 33 assembled channels expressed after a 1:1 injection of mutant and WT cRNA are indicated by red bars. The one assembled channel in the cluster of black bars is consistent with a channel comprised of four WT subunits. The four assembled channels within the blue bars are consistent with channels comprised of four mutant subunits. The 28 assembled channels with Vh values falling between those of the blue bars and black bars are consistent with hybrid (heterotetrameric) channels assembled from mixed subunits. The 28 hybrid channels appear to cluster into three groups with mean ± SEM Vh values of 1.4 ± 4.3 mV for the 7 channels in the left hybrid group, 59.6 ± 2.6 mV for the 11 channels in the middle hybrid group, and 96.4 ± 2.2 mV for the 10 channels in the right hybrid group. All Vh comparisons of the three hybrid groups were significantly different, P < 0.0001, two-tailed t test. The single assembled channel that fell within the WT (black) group had a Vh of 151.3 mV, which was significantly different from the 96.4 mV hybrid group, P < 0.0001, one sample two-tailed t test. The group of four hybrid channels that fell within the homotetrameric (blue) group had a Vh of −75.0 ± 22.0 mV which was significantly different from the 1.4 mV hybrid group, P = 0.0019, two-tailed t test. Hypothesized subunit compositions of the three apparent clusters of hybrid channels are depicted by schematics, where black circles indicate WT subunits and blue circles indicate G375R mutant subunits. Subunit structures for the homotetrameric mutant and WT channels are also indicated. (B) The experimentally observed percentages for each of the five types of 33 assembled channels are listed for comparison to the theoretical percentages from Fig. 1. (C)Vh for the five types of assembled channels is approximated by a linear incremental model where each subunit contributes an increment of Vh, Vh(NM)=(NM)(−16.7mV)+(4−NM)(40.5mV), where NM is the number of G375R mutant subunits per channel, 4-NM is the number of WT subunits per channel, Vh(NM) is Vh as a function of NM, and −16.7 and 40.5 mV are the increments of Vh added by each mutant and WT subunit, respectively. Filled red circles are the experimentally observed mean ± SEM values of Vh for the five types of assembled channels from A, and the dashed line indicates the predicted values. Data are for 0 intracellular Ca2+. Histograms of single-channel kinetic parameters have been used previously to investigate the number of β2 regulatory subunits per BK channel (Wang et al., 2002).
Figure 5.

Five different types of functional BK channels are observed following injection of a 1:1 mixture of mutant and WT cRNA into Xenopus oocytes. (A) Histograms of Vh values of single-channels from Fig. 4. WT channels expressed after injection of only WT cRNA are indicated by black bars, and G375R homotetrameric mutant channels expressed after injection of only G375R cRNA are indicated by blue bars. The 33 assembled channels expressed after a 1:1 injection of mutant and WT cRNA are indicated by red bars. The one assembled channel in the cluster of black bars is consistent with a channel comprised of four WT subunits. The four assembled channels within the blue bars are consistent with channels comprised of four mutant subunits. The 28 assembled channels with Vh values falling between those of the blue bars and black bars are consistent with hybrid (heterotetrameric) channels assembled from mixed subunits. The 28 hybrid channels appear to cluster into three groups with mean ± SEM Vh values of 1.4 ± 4.3 mV for the 7 channels in the left hybrid group, 59.6 ± 2.6 mV for the 11 channels in the middle hybrid group, and 96.4 ± 2.2 mV for the 10 channels in the right hybrid group. All Vh comparisons of the three hybrid groups were significantly different, P < 0.0001, two-tailed t test. The single assembled channel that fell within the WT (black) group had a Vh of 151.3 mV, which was significantly different from the 96.4 mV hybrid group, P < 0.0001, one sample two-tailed t test. The group of four hybrid channels that fell within the homotetrameric (blue) group had a Vh of −75.0 ± 22.0 mV which was significantly different from the 1.4 mV hybrid group, P = 0.0019, two-tailed t test. Hypothesized subunit compositions of the three apparent clusters of hybrid channels are depicted by schematics, where black circles indicate WT subunits and blue circles indicate G375R mutant subunits. Subunit structures for the homotetrameric mutant and WT channels are also indicated. (B) The experimentally observed percentages for each of the five types of 33 assembled channels are listed for comparison to the theoretical percentages from Fig. 1. (C)Vh for the five types of assembled channels is approximated by a linear incremental model where each subunit contributes an increment of Vh, Vh(NM)=(NM)(−16.7mV)+(4−NM)(40.5mV), where NM is the number of G375R mutant subunits per channel, 4-NM is the number of WT subunits per channel, Vh(NM) is Vh as a function of NM, and −16.7 and 40.5 mV are the increments of Vh added by each mutant and WT subunit, respectively. Filled red circles are the experimentally observed mean ± SEM values of Vh for the five types of assembled channels from A, and the dashed line indicates the predicted values. Data are for 0 intracellular Ca2+. Histograms of single-channel kinetic parameters have been used previously to investigate the number of β2 regulatory subunits per BK channel (Wang et al., 2002).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal