|
Figure 2. Voltage-dependent reduction in tail current amplitudes for rEag1 N-terminal chimeras.(A) (Top) Peak tail current amplitudes (arrows) for hERG1-WT (top left) and rEag1-chimera P (top right) current traces shown in Figure 1A. Unlike hERG1-WT, rEag1-chimera P displayed a U-shaped tail I–V curve. (Bottom) Comparison of tail current traces induced by two different test pulses for hERG1-WT (bottom left) and rEag1-chimera P (bottom right). See Figure S2 for more tail current traces of rEag1-chimera P. (B) Steady-state activation curves of various hERG1 (left) and rEag1 (right) constructs. The relative Po was plotted against the corresponding test potential. All data were recorded with 3 mM external KCl. For hERG1 channels, isochronal tail currents were normalized to the corresponding maximum amplitude to obtain relative Po-V curves. For rEag1 channels, steady-state current amplitudes were employed for analyses. For the two rEag1 N-terminal chimeras, tail currents were also used to generate relative Po curves. Data points were fit with one or two Boltzmann equations (solid curves). See Methods and Table 1 for more detail.
|
|
Figure 3. Inactivated rEag1-chimera P channels return to both the open state and the closed state.All data were recorded in 60 mM KCl bath solution. (A) Comparison of tail current traces for rEag1-WT, hERG1-WT, and rEag1-chimera P. Channels were subject to a +60 mV test pulse, followed by the tail potential of either −100 (black lines) or −140 (red lines) mV. For both hERG1-WT and rEag1-chimera P, the inward tail current at −100 mV showed a significant rising phase. Only hERG1-WT, however, displayed the hooked tail current at −140 mV. (B) Inactivation recovery kinetics of hERG1-WT and rEag1-chimera P. Recovery time constants (n = 5–6) at indicated tail potentials were obtained from single exponential fits to the rising phase of inward tail currents. (C) Deactivation kinetics of rEag1-WT, hERG1-WT, and rEag1-chimera P. Deactivation time constants (left) (n = 7–51) at indicated tail potentials were obtained from single exponential fits to the decay phase of inward tail currents. Normalized deactivation time constants (right) were obtained by setting the respective maximal deactivation time constant value for each construct as unity. (D) Peak tail current amplitudes are plotted against corresponding tail potentials (n = 6–14). From a given test pulse potential, channels were subject to a series of different tail potentials. Tail current responses from a total of seven different test pulse potentials are illustrated here. Each of the three constructs exhibited distinctly different tail current-tail potential relationship. See Figure S3 for more tail current traces of rEag1-chimera P.
|
|
Figure 4. The effect of double-pulse protocols on rEag1 and hERG1 channels.(A) (Top) The double-pulse protocol entailed a series of depolarizing prepulses (in 10-mV increments) up to +60 mV and an ensuing test pulse. (Middle) Representative K+ current traces (in 3 mM KCl) induced by double-pulse protocols. The symbols (I) and (S) denote the initial phase and the steady-state phase, respectively, of the K+ currents elicited by the second test pulse. (Bottom) A close inspection of the instantaneous K+ currents (i.e., the initial phase thereof) in response to the second test pulse. Inactivating instantaneous currents were observed in the two rEag1 chimeras only. (B) (Left) Instantaneous K+ current amplitudes (labeled by the arrow “I” in A) were plotted against matching prepulse potentials for each K+ channel construct. (Right) The ratios of instantaneous over steady-state (labeled by the arrow “S” in A) current amplitudes were plotted against prepulse potentials. The data points showing ratios that are larger than unity (dotted line) correspond to the presence of inactivating instantaneous currents in the two rEag1 chimeras.
|
|
Figure 5. The slow phase of inactivation of rEag1 N-terminal chimeras.(A) Representative rEag1-WT K+ current traces (in 3 mM KCl) induced by 10-sec (left) or 40-sec (right) depolarizing test pulses up to +60 mV. The 40-sec current traces are scaled to the same peak amplitude and are vertically dispersed to highlight the fact that rEag1-WT shows no discernible slow inactivation. (B) Representative rEag1-chimera N current traces elicited by 40-sec depolarizing test pulses. The current traces are scaled to the same peak amplitude and are vertically dispersed. rEag1-chimera N shows a small but detectable slow inactivation. (C) Representative rEag1-chimera P current traces in response to 10-sec (left) or 40-sec (center) depolarizing test pulses. The 40-sec current traces are scaled to the same peak amplitude. (Right) Inactivation kinetics of rEag1-chimera P in response to 40-sec pulses. Inactivation time constants (n = 3–4) at indicated test potentials were obtained from single exponential fits.
|
|
Figure 6. Voltage-dependent inactivation of rEag1-Δeag.(A) (Left) Representative rEag1-Δeag K+ current traces (in 3 mM KCl) elicited by depolarizing test pulses up to +60 mV. The holding potential was −110 mV. (Middle, right) Steady-state I–V (n = 10–16) and activation (n = 16–20) curves of rEag1-Δeag. Data points for the relative Po were fit with two Boltzmann equations (solid curve). (B) Representative current traces (in 3 mM KCl) induced by the double-pulse protocol. In response to the second, +30-mV test pulse, inactivating instantaneous currents were observed in rEag1-Δeag channels. (C) Peak tail current amplitudes (arrow) for the current traces shown in A. rEag1-Δeag displayed a U-shaped tail I–V curve. (D) Comparison of rEag1-Δeag tail current traces (in 60 mM KCl) elicited by the tail potentials −90 (black line) and −140 (red line) mV. The test pulse potential preceding the tail potentials was +60 mV. A notable rising phase was only observed in the −90-mV trace. (E) Peak tail current amplitudes (in 60 mM KCl), in response to seven different test pulse potentials, are plotted against corresponding tail potentials (n = 7). rEag1-Δeag exhibited prominent voltage-dependent reduction in peak tail current amplitudes.
|
|
Figure 7. Attenuation of rEag1-Δeag channel inactivation by the rEag1 eag domain fragment.(A) (Left) Schematic representation of the construction of the rEag1 and the hERG1 eag domain fragments (see Methods for more detail). (Right) Protein expression of myc-tagged rEag1-WT, rEag1-Δeag, rEag1 eag domain, and hERG1 eag domain. cDNA for each myc-tagged construct was transfected into HEK293T cells. Proteins in cell lysates were detected by immunoblotting with anti-myc or anti-actin antibodies. The positions of molecular weight markers (in the unit of kDa) are indicated to the left of the blots. (B) Representative K+ current traces (in 3 mM KCl) recorded from oocytes co-expressing rEag1-Δeag with the rEag1 or the hERG1 eag domain fragments in the mRNA molar ratio 1∶10/1∶20. (C) Steady-state I–V (left) (n = 7–16) and activation (right) (n = 10–16) curves of rEag1-Δeag in the presence of the rEag1 or the hERG1 eag domain fragments. (D) Activation (left) and deactivation (right) kinetics of rEag1-Δeag in the absence or presence of rEag1/hERG1 eag domain fragments. Activation (n = 3–5) and deactivation (n = 3–6) time constants were obtained from single exponential fits. (E) Peak tail current amplitudes (in 60 mM KCl) (n = 4–13) of rEag1-Δeag in the presence of the rEag1 or the hERG1 eag domain fragments. Channel inactivation in rEag1-Δeag was notably reduced upon co-expression with the rEag1 eag domain fragment.
|
|
Figure 8. The slow phase of inactivation of rEag1-Δeag.Representative rEag1-Δeag current traces in the absence (A) or presence (B) of the rEag1 eag domain fragment. All data were recorded in 3 mM KCl bath solution. K+ currents were induced by 10-sec (left) or 40-sec (right) depolarizing test pulses up to +60 mV. The current traces for 40-sec pulses are scaled to the same peak amplitude and are vertically dispersed. Co-expression with the rEag1 eag domain fragment accelerated and enhanced the slow phase of inactivation of rEag1-Δeag.
|
|
Figure 9. Voltage-dependent inactivation of rEag1-Y344C.(A) (Far left) Representative rEag1-Y344C K+ current traces (in 3 mM KCl) elicited by test pulses up to +60 mV. The holding potential was −140 mV. (Left) Activation kinetics (n = 4) of rEag1-Y344C. (Right, far right) Steady-state I–V and activation curves (n = 4) of rEag1-Y344C. Data points for the relative Po were fit with two Boltzmann equations (solid curve). (B) Representative current traces (in 3 mM KCl) induced by the double-pulse protocol. In response to the second, +60-mV test pulse, inactivating instantaneous currents were observed in rEag1-Y344C channels. (C) Representative rEag1-Y344C current traces (in 3 mM KCl) induced by 40-sec test pulses. The current traces are scaled to the same peak amplitude and are vertically dispersed. Note the presence of a small but detectable slow inactivation. (D) Peak tail current amplitudes (arrow) for the current traces shown in A. rEag1-Y344C displayed a U-shaped tail I–V curve. (E) Comparison of rEag1-Y344C tail current traces (in 60 mM KCl) elicited by the tail potentials −140 (black line) and −180 (red line) mV. The test pulse potential preceding the tail potentials was +60 mV. A notable rising phase was only observed in the −140-mV trace. (F) Peak tail current amplitudes (in 60 mM KCl), in response to seven different test pulse potentials, are plotted against corresponding tail potentials (n = 7). rEag1-Y344C exhibited substantial voltage-dependent reduction in peak tail current amplitudes.
|
|
Figure 1. Voltage-dependent inactivation of rEag1 N-terminal chimeras.
(A) (Top) Schematic representation of the construction of N-terminal chimeras (see Methods for more detail). (Bottom) Representative K+ current traces recorded from oocytes expressing rEag1-WT, hERG1-WT, rEag1-chimera P, or rEag1-chimera N channels. The bath solution contained 3 mM KCl. Depending on the steady-state voltage dependence properties of different constructs, the holding potential was set at −90, −110, or −130 mV. The pulse protocol comprised depolarizing test pulses (in 10-mV increments) up to +60 mV, followed by a tail potential at −90 (rEag1-WT), −100 (hERG1-WT), −110 (rEag1-chimera P), or −130 (rEag1-chimera N) mV. (
B
) Steady-state I–V curves in response the test pulses for rEag1 (left) and hERG1 (right) N-terminal chimeras (n = 8–19). (
C
) Activation kinetics of rEag1-WT and N-terminal chimeras. Activation time constants (n = 3–6) at indicated potentials were obtained from single exponential fits to the late rising phase of rEag1 currents.
|
