Figure 5.
ΔF for both labels increases after changing permeation conditions. (Α) ΔF–V data before and after addition of 10 mM CsCl to a bath containing standard 100K solution for spHCNR332C expressing oocytes labeled with ALEXA-488 (left panel) and MTS-TAMRA (right panel). Insets show representative membrane current (upper traces) and ΔF (lower traces) for voltage steps to −160 and +20 mV from 0 mV holding potential and ΔF shown as % of background fluorescence. In both cases, the total ΔF was measured at the end of the test pulse. ΔF–V data were fit with the Boltzmann equation (Eq. 1) (continuous lines). Parameters given in Table 2. ΔF–V data were normalized to control condition (100K superfusion) for n = 9 replicates. (Β) ΔF–V data before and after replacing bath solution with 100Na solution to give 2 mM KCl for spHCNR332C expressing oocytes labeled with ALEXA-488 (left panel) and MTS-TAMRA (right panel). Insets show representative membrane current (upper traces) and ΔF (lower traces) for voltage steps to −160 and +20 mV from 0 mV holding potential and ΔF is shown as percentage of background fluorescence. Data analysis as in A for n = 5 replicates. (C) ΔF–V data for ALEXA-488 labeling showing fast (upper panel) and slow (lower panel) components before and after addition of 10 mM CsCl to the bath as in A. Data were analyzed by fitting ∆Ftotal with a single exponential plus variable offset (see Materials and methods) and normalized with respect to ∆Ftotalmax. A t test performed on the fit parameters ∆Ffast (Table 2) showed no significant difference between the 100K and 100K+Cs parameters (P < 0.05). (D) ΔF–V data for ALEXA-488 labeling showing fast and slow components before and after replacement of 100K with 100Na solution to give 2 mM KCl as in B. Data were analyzed by fitting ∆Ftotal with a single exponential plus variable offset (see Materials and methods) and normalized with respect to ∆Ftotalmax. A t test performed on the fit parameters ∆Ffast (Table 2) showed no significant difference between the 100K and 100Na parameters (P < 0.05).

ΔF for both labels increases after changing permeation conditions. (Α) ΔF–V data before and after addition of 10 mM CsCl to a bath containing standard 100K solution for spHCNR332C expressing oocytes labeled with ALEXA-488 (left panel) and MTS-TAMRA (right panel). Insets show representative membrane current (upper traces) and ΔF (lower traces) for voltage steps to −160 and +20 mV from 0 mV holding potential and ΔF shown as % of background fluorescence. In both cases, the total ΔF was measured at the end of the test pulse. ΔF–V data were fit with the Boltzmann equation (Eq. 1) (continuous lines). Parameters given in Table 2. ΔF–V data were normalized to control condition (100K superfusion) for n = 9 replicates. (Β) ΔF–V data before and after replacing bath solution with 100Na solution to give 2 mM KCl for spHCNR332C expressing oocytes labeled with ALEXA-488 (left panel) and MTS-TAMRA (right panel). Insets show representative membrane current (upper traces) and ΔF (lower traces) for voltage steps to −160 and +20 mV from 0 mV holding potential and ΔF is shown as percentage of background fluorescence. Data analysis as in A for n = 5 replicates. (C) ΔF–V data for ALEXA-488 labeling showing fast (upper panel) and slow (lower panel) components before and after addition of 10 mM CsCl to the bath as in A. Data were analyzed by fitting ∆Ftotal with a single exponential plus variable offset (see Materials and methods) and normalized with respect to ∆Ftotalmax⁠. A t test performed on the fit parameters ∆Ffast (Table 2) showed no significant difference between the 100K and 100K+Cs parameters (P < 0.05). (D) ΔF–V data for ALEXA-488 labeling showing fast and slow components before and after replacement of 100K with 100Na solution to give 2 mM KCl as in B. Data were analyzed by fitting ∆Ftotal with a single exponential plus variable offset (see Materials and methods) and normalized with respect to ∆Ftotalmax⁠. A t test performed on the fit parameters ∆Ffast (Table 2) showed no significant difference between the 100K and 100Na parameters (P < 0.05).

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