Figure S2.
GxTX-594 selectively labels Kv2 proteins on cell surfaces. GxTX-594 labeling was assessed in CHO cells expressing Kv2.1-GFP, Kv2.2-GFP, Kv4.2-GFP, Kv1.5-GFP, and BK-GFP. Each of these channel subtypes were assessed for voltage-dependent outward currents to identify cell surface expression of the K+ channels. CHO cells were not cotransfected with the β subunits for Kv4.2, KChIP2, or Kv1.5, Kvβ2, to assess whether these β subunits interfere with GxTX-594 binding. (A) Exemplar whole-cell voltage clamp recordings of CHO cells expressing Kv2.1-GFP, Kv2.2-GFP, Kv4.2-GFP, Kv1.5-GFP, or BK-GFP. Recordings shown are representative responses to 100-ms steps from −100 mV to −40, 0, and +40 mV. (B) Confocal imaging of fluorescence from live CHO cells transfected with Kv2.1-GFP, Kv2.2-GFP, Kv4.2-GFP, Kv1.5-GFP, or BK-GFP (indicated by row) and labeled with GxTX-594. Confocal imaging plane was >1 μm above the glass-adhered surface. Cells were incubated with 100 nM GxTX-594 and 5 μg/ml WGA-405 and rinsed before imaging. Fluorescence shown corresponds to emission of GFP (column 1); Alexa Fluor 594 (column 2); WGA-405 (column 3); or an overlay of GFP, Alexa Fluor 594, and WGA-405 (column 4). Scale bars, 20 μm. (C) Ratio of fluorescence intensity resulting from excitation of GxTX-594 at 594 nm and GFP at 488 nm. Analysis methods as in Fig. 4 B. Kv2.1, n = 11; Kv2.2, n = 9; Kv4.2, n = 13; Kv1.5, n = 13; and BK, n = 12; n indicates the number of individual cells analyzed in a single dish during a single application of GxTX-594 with the indicated K+ channel-GFP type. Bars represent the mean. Each circle corresponds to a cell. Significant differences were observed between GxTX:GFP ratio for Kv2.1 or Kv2.2 and Kv1.5, Kv4.2, or BK by Mann–Whitney U test (P < 0.0001). The P value to determine significance is adjusted for multiple comparisons using the Bonferroni method, where P < 0.0033 is considered significant, with the caveat that data points under each condition are technical replicates. (D) Pearson correlation coefficients between GxTX-594 and GFP. Same cells as C. Analysis methods as in Fig. 4 C.

GxTX-594 selectively labels Kv2 proteins on cell surfaces. GxTX-594 labeling was assessed in CHO cells expressing Kv2.1-GFP, Kv2.2-GFP, Kv4.2-GFP, Kv1.5-GFP, and BK-GFP. Each of these channel subtypes were assessed for voltage-dependent outward currents to identify cell surface expression of the K+ channels. CHO cells were not cotransfected with the β subunits for Kv4.2, KChIP2, or Kv1.5, Kvβ2, to assess whether these β subunits interfere with GxTX-594 binding. (A) Exemplar whole-cell voltage clamp recordings of CHO cells expressing Kv2.1-GFP, Kv2.2-GFP, Kv4.2-GFP, Kv1.5-GFP, or BK-GFP. Recordings shown are representative responses to 100-ms steps from −100 mV to −40, 0, and +40 mV. (B) Confocal imaging of fluorescence from live CHO cells transfected with Kv2.1-GFP, Kv2.2-GFP, Kv4.2-GFP, Kv1.5-GFP, or BK-GFP (indicated by row) and labeled with GxTX-594. Confocal imaging plane was >1 μm above the glass-adhered surface. Cells were incubated with 100 nM GxTX-594 and 5 μg/ml WGA-405 and rinsed before imaging. Fluorescence shown corresponds to emission of GFP (column 1); Alexa Fluor 594 (column 2); WGA-405 (column 3); or an overlay of GFP, Alexa Fluor 594, and WGA-405 (column 4). Scale bars, 20 μm. (C) Ratio of fluorescence intensity resulting from excitation of GxTX-594 at 594 nm and GFP at 488 nm. Analysis methods as in Fig. 4 B. Kv2.1, n = 11; Kv2.2, n = 9; Kv4.2, n = 13; Kv1.5, n = 13; and BK, n = 12; n indicates the number of individual cells analyzed in a single dish during a single application of GxTX-594 with the indicated K+ channel-GFP type. Bars represent the mean. Each circle corresponds to a cell. Significant differences were observed between GxTX:GFP ratio for Kv2.1 or Kv2.2 and Kv1.5, Kv4.2, or BK by Mann–Whitney U test (P < 0.0001). The P value to determine significance is adjusted for multiple comparisons using the Bonferroni method, where P < 0.0033 is considered significant, with the caveat that data points under each condition are technical replicates. (D) Pearson correlation coefficients between GxTX-594 and GFP. Same cells as C. Analysis methods as in Fig. 4 C.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal