Figure 8. Kv2.1:Kv6.4 expression ratio... | Rockefeller University Press

$Figure 8. Kv2.1:Kv6.4 expression ratio titrations predict that 2:2R heteromers have negligible conductance for Kv6.4 WT and are conducting for Kv6.4PIPIIV-Kv2.1CT. (A) Example currents from oocytes expressing Kv2.1 alone (left, black) or Kv2.1:Kv6.4 WT (magenta) at 9:1, 1:1, and 1:20 ratios. Kv2.1 cRNA level was kept constant, and currents were recorded at +40 mV following a 4-s prepulse to −120 mV to relieve SSI. Note the dramatic decrease in current size as Kv6.4 cRNA level is increased. Scale bars indicate current amplitude and time. (B) Example currents from a similar titration of Kv2.1 versus Kv6.4-PIPIIV-KV2.1CT; Kv2.1 control is shown in black, and cRNA ratios shown are 3:1, 1:1, and 1:20. More current is readily apparent at the 1:20 ratio for Kv6.4-PIPIIV-KV2.1CT than Kv6.4 WT. (C) Current amplitude versus expression ratio titrations are shown for Kv2.1:Kv6.4 WT (magenta) and Kv2.1:Kv6.4-PIPIIV-KV2.1CT (blue). Expression ratio is given as −log(Kv2.1fraction); 1.0 = a 1:10 Kv2.1:Kv6.4 cRNA ratio. The Kv2.1 cRNA amount remained constant while the Kv6.4 cRNA species amount was varied to achieve the given expression ratio. Data were normalized to the amplitude of control Kv2.1 homomeric currents. Data points show mean ± SEM (n = 10–18 eggs per ratio), and asterisks indicate a significant difference (P < 0.05, t test) between the indicated pairs of data points. (D) Normalized current size ratio for Kv2.1:Kv6.4-PIPIIV-Kv2.1CT versus Kv2.1:Kv6.4 WT increases approximately fourfold as the Kv6.4 species cRNA is increased. Data show mean ± SD and are derived from the data point pairs statistically compared in C. Kv2.1:Kv6.4 ratios were similar but not identical between the titrations, and data are plotted using the average of the Kv2.1:Kv6.4 ratio of the data pair. SD for the ratios was calculated by propagation of error from the original measurements in C. (E) Example single-channel current traces from an on-cell patch from an oocyte expressing Kv2.1 alone. Traces were recorded at −20 mV, 0 mV, and +20 mV; the dashed blue line indicates the average closed baseline, and the dashed magenta lines indicate 1× and 2× the average open-channel amplitude for the trace. (F) Traces labeled as in D for an on-cell patch containing two channels from an oocyte coexpressing Kv2.1 with Kv6.4. In this example, the dashed magenta lines indicate 1× and 2× the average open-channel amplitude of the largest channel recorded in the trace. Arrows point to examples of single- or double-channel openings with smaller than expected amplitudes, indicating a second channel with a lower conductance. (G) Plots of single-channel current amplitude versus voltage are given for Kv2.1 homomers expressed in isolation and for presumed homomers and heteromers from oocytes expressing Kv2.1 with Kv6.4. Single-channel conductance values shown on the graph were calculated from linear fits; n was 6 for the two Kv2.1 homomer measurements and 5 for the Kv2.1:Kv6.4 heteromers. The Kv2.1:Kv6.4 heteromer conductance was significantly smaller (P < 0.05, t test). Note there is a small offset in reversal potential between the experiments conducted on oocytes expressing Kv2.1 alone versus Kv2.1 + Kv6.4 that alters single-channel amplitude but does not interfere with conductance measurements; these experiments were conducted at different times with distinct solution batches. (H) Normalized total current versus heteromeric current for the Kv2.1:Kv6.4 WT coexpression titration shown in C. To determine the amplitude of the heteromeric current, we selectively inactivated heteromers with a 4-s prepulse to −40 mV before measuring current amplitude at +40 mV. The amplitude of the heteromeric current was determined by adjusting the current fraction removed by the −40 mV prepulse by the fractional inactivation determined from SSI analysis (Figure 3 D and Table 1). In this case, the inactivating fraction was multiplied by 1.38 to account for the observation that 27.8% of heteromeric channels are not expected to inactivate during the prepulse. (I) Normalized heteromeric versus total current amplitude for Kv2.1:Kv6.4-PIPIIV-Kv2.1CT; heteromeric fraction was calculated as in H except using a multiplication factor of 1.44 to match the 30.4% pedestal observed for Kv2.1:Kv6.4-PIPIIV-Kv2.1CT in SSI analysis. Note in both titrations the current approaches 100% heteromer at a 1:10 Kv2.1:Kv6.4 species expression ratio (−logKv2.1 fraction = 1).$

Figure 8.

Kv2.1:Kv6.4 expression ratio titrations predict that 2:2R heteromers have negligible conductance for Kv6.4 WT and are conducting for Kv6.4PIPIIV-Kv2.1CT. (A) Example currents from oocytes expressing Kv2.1 alone (left, black) or Kv2.1:Kv6.4 WT (magenta) at 9:1, 1:1, and 1:20 ratios. Kv2.1 cRNA level was kept constant, and currents were recorded at +40 mV following a 4-s prepulse to −120 mV to relieve SSI. Note the dramatic decrease in current size as Kv6.4 cRNA level is increased. Scale bars indicate current amplitude and time. (B) Example currents from a similar titration of Kv2.1 versus Kv6.4-PIPIIV-KV2.1CT; Kv2.1 control is shown in black, and cRNA ratios shown are 3:1, 1:1, and 1:20. More current is readily apparent at the 1:20 ratio for Kv6.4-PIPIIV-KV2.1CT than Kv6.4 WT. (C) Current amplitude versus expression ratio titrations are shown for Kv2.1:Kv6.4 WT (magenta) and Kv2.1:Kv6.4-PIPIIV-KV2.1CT (blue). Expression ratio is given as −log(Kv2.1fraction); 1.0 = a 1:10 Kv2.1:Kv6.4 cRNA ratio. The Kv2.1 cRNA amount remained constant while the Kv6.4 cRNA species amount was varied to achieve the given expression ratio. Data were normalized to the amplitude of control Kv2.1 homomeric currents. Data points show mean ± SEM (n = 10–18 eggs per ratio), and asterisks indicate a significant difference (P < 0.05, t test) between the indicated pairs of data points. (D) Normalized current size ratio for Kv2.1:Kv6.4-PIPIIV-Kv2.1CT versus Kv2.1:Kv6.4 WT increases approximately fourfold as the Kv6.4 species cRNA is increased. Data show mean ± SD and are derived from the data point pairs statistically compared in C. Kv2.1:Kv6.4 ratios were similar but not identical between the titrations, and data are plotted using the average of the Kv2.1:Kv6.4 ratio of the data pair. SD for the ratios was calculated by propagation of error from the original measurements in C. (E) Example single-channel current traces from an on-cell patch from an oocyte expressing Kv2.1 alone. Traces were recorded at −20 mV, 0 mV, and +20 mV; the dashed blue line indicates the average closed baseline, and the dashed magenta lines indicate 1× and 2× the average open-channel amplitude for the trace. (F) Traces labeled as in D for an on-cell patch containing two channels from an oocyte coexpressing Kv2.1 with Kv6.4. In this example, the dashed magenta lines indicate 1× and 2× the average open-channel amplitude of the largest channel recorded in the trace. Arrows point to examples of single- or double-channel openings with smaller than expected amplitudes, indicating a second channel with a lower conductance. (G) Plots of single-channel current amplitude versus voltage are given for Kv2.1 homomers expressed in isolation and for presumed homomers and heteromers from oocytes expressing Kv2.1 with Kv6.4. Single-channel conductance values shown on the graph were calculated from linear fits; n was 6 for the two Kv2.1 homomer measurements and 5 for the Kv2.1:Kv6.4 heteromers. The Kv2.1:Kv6.4 heteromer conductance was significantly smaller (P < 0.05, t test). Note there is a small offset in reversal potential between the experiments conducted on oocytes expressing Kv2.1 alone versus Kv2.1 + Kv6.4 that alters single-channel amplitude but does not interfere with conductance measurements; these experiments were conducted at different times with distinct solution batches. (H) Normalized total current versus heteromeric current for the Kv2.1:Kv6.4 WT coexpression titration shown in C. To determine the amplitude of the heteromeric current, we selectively inactivated heteromers with a 4-s prepulse to −40 mV before measuring current amplitude at +40 mV. The amplitude of the heteromeric current was determined by adjusting the current fraction removed by the −40 mV prepulse by the fractional inactivation determined from SSI analysis (Figure 3 D and Table 1). In this case, the inactivating fraction was multiplied by 1.38 to account for the observation that 27.8% of heteromeric channels are not expected to inactivate during the prepulse. (I) Normalized heteromeric versus total current amplitude for Kv2.1:Kv6.4-PIPIIV-Kv2.1CT; heteromeric fraction was calculated as in H except using a multiplication factor of 1.44 to match the 30.4% pedestal observed for Kv2.1:Kv6.4-PIPIIV-Kv2.1CT in SSI analysis. Note in both titrations the current approaches 100% heteromer at a 1:10 Kv2.1:Kv6.4 species expression ratio (−logKv2.1 fraction = 1).

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