Figure 4. Determination of Kv2.1:Kv6.4 heteromer stoichiometry by TIRF photobleaching assay. (A) Example TIRF images from an oocyte expressing GFP-Kv2.1 before (left) and after photobleaching (middle). Spots that bleached to background during the photobleaching period (blue circles) were analyzed for number of bleaching steps. Bleach-resistant spots (yellow circles) or spots that bleached but did not have a stable baseline (magenta) were not included in the analysis. Example fluorescence traces for spots in the movie bleaching in one (I), two (II), three (III) and four (IV) steps are shown at the right margin. Fluorescent spots (circled) bleachable in discrete steps were observed only in oocytes expressing GFP-tagged channels. (B) Example TIRF images and fluorescence traces before and after photobleaching for Kv2.1 coexpressed with GFP-Kv6.4, labeled as in A. Only spots with one or two bleaching steps were observed. (C) Frequency distribution of bleaching steps for channel spots in oocytes expressing GFP-Kv2.1 (Kv2.1 homomers). Only spots bleaching in one to four steps were observed. Assuming all channels are tetramers with four GFPs, a binomial fit of the distribution (squares) estimates the probability of detecting GFP fluorescence and bleaching at 69% in our experimental setup. (D) Frequency distribution of bleaching steps for channel spots in oocytes coexpressing GFP-Kv2.1 and Kv6.4 compared with GFP-Kv2.1 alone. The addition of Kv6.4 RNA significantly reduces the frequency of four steps from 22% to 11% and reduces the frequency of three steps from 42% to 32%. There is a corresponding increase in the frequency of two steps from 25% to 39% and an increase in the frequency of one steps from 12% to 17% (P < 0.05, Fisher’s exact test). This shift in step distribution confirms detection of Kv2.1:Kv6.4 heteromer formation. (E) Frequency distribution of bleaching steps for channel spots in oocytes injected with Kv2.1:GFP-Kv6.4 in an ∼1:50 ratio to maximize heteromer formation. Only spots bleaching in one to two steps were observed, and 81% bleach in one step. (F) Relative frequency of 3:1R and 2:2R Kv2.1:GFP-Kv6.4 heteromers calculated from the bleaching step distribution using the 69% GFP detection probability determined for Kv2.1 homomers as described in C..
Figure 4.

Determination of Kv2.1:Kv6.4 heteromer stoichiometry by TIRF photobleaching assay. (A) Example TIRF images from an oocyte expressing GFP-Kv2.1 before (left) and after photobleaching (middle). Spots that bleached to background during the photobleaching period (blue circles) were analyzed for number of bleaching steps. Bleach-resistant spots (yellow circles) or spots that bleached but did not have a stable baseline (magenta) were not included in the analysis. Example fluorescence traces for spots in the movie bleaching in one (I), two (II), three (III) and four (IV) steps are shown at the right margin. Fluorescent spots (circled) bleachable in discrete steps were observed only in oocytes expressing GFP-tagged channels. (B) Example TIRF images and fluorescence traces before and after photobleaching for Kv2.1 coexpressed with GFP-Kv6.4, labeled as in A. Only spots with one or two bleaching steps were observed. (C) Frequency distribution of bleaching steps for channel spots in oocytes expressing GFP-Kv2.1 (Kv2.1 homomers). Only spots bleaching in one to four steps were observed. Assuming all channels are tetramers with four GFPs, a binomial fit of the distribution (squares) estimates the probability of detecting GFP fluorescence and bleaching at 69% in our experimental setup. (D) Frequency distribution of bleaching steps for channel spots in oocytes coexpressing GFP-Kv2.1 and Kv6.4 compared with GFP-Kv2.1 alone. The addition of Kv6.4 RNA significantly reduces the frequency of four steps from 22% to 11% and reduces the frequency of three steps from 42% to 32%. There is a corresponding increase in the frequency of two steps from 25% to 39% and an increase in the frequency of one steps from 12% to 17% (P < 0.05, Fisher’s exact test). This shift in step distribution confirms detection of Kv2.1:Kv6.4 heteromer formation. (E) Frequency distribution of bleaching steps for channel spots in oocytes injected with Kv2.1:GFP-Kv6.4 in an ∼1:50 ratio to maximize heteromer formation. Only spots bleaching in one to two steps were observed, and 81% bleach in one step. (F) Relative frequency of 3:1R and 2:2R Kv2.1:GFP-Kv6.4 heteromers calculated from the bleaching step distribution using the 69% GFP detection probability determined for Kv2.1 homomers as described in C..

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal