1. Effects of Temperature on Heteromeric Kv11.1a/1b and Kv11.3 Channels
Maike Mauerhöfer, Christiane K. Bauer 
Biophysical Journal 
Volume 111, Issue 3, Pages (August 2016)
DOI: /j.bpj
Copyright © 2016 Biophysical Society Terms and Conditions

2. Figure 1
Temperature effects on the voltage dependence of Kv11.1a/1b activation. Membrane currents were recorded in CHO cells transiently expressing Kv11.1a/1b channels. (A) Representative Kv11.1a/1b current traces recorded from different cells at 21°C and 35°C. From a holding potential of −80 mV, channel activation was assessed by 4-s test pulses between −90 mV and +40 mV with 10 mV increments, followed by a constant pulse to −110 mV (see pulse diagram). (B) I-V relationship of outward current amplitudes measured at the end of the depolarizing test pulses at 21°C, 30°C, and 35°C. Data are means ± SEM of absolute amplitudes (upper panel) or current amplitudes normalized to the individual maximum outward current (lower panel). The number of experiments is given in brackets. The inset shows mean maximal outward current densities determined in matched experiments (data set A) at 21°C (−10 mV; n = 17) and 35°C (−10 mV; n = 17); asterisks indicate significant differences between the unpaired data (∗∗∗p < 0.001). (C) Peak tail current amplitudes plotted against the potential of the preceding test pulse. Data were normalized to the maximum tail current amplitudes, averaged, and fitted with a Boltzmann function to yield activation curves. The fit parameters of the 4-s isochronal activation curves were V0.5 = −29.3 mV and k = 7.7 mV at 21°C, V0.5 = −33.1 mV and k = 6.7 mV at 30°C, and V0.5 = −39.6 mV and k = 6.6 mV at 35°C. In part of the experiments at the recording temperature of 21°C, an additional activation protocol was applied using depolarizing test pulses of 12 s duration (open circles and dashed fit line; V0.5 = −34.4 mV, k = 6.0 mV). To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

3. Figure 2
Temperature effects on fully activated Kv11.1a/1b currents and steady-state inactivation. (A) Representative families of fully activated Kv11.1a/1b currents recorded from different cells at 21°C and 35°C. Membrane currents were elicited by 1-s depolarizations to +40 mV, followed by variable 1-s test pulses from +40 to −120 mV with 10 mV decrements and a final step to −100 mV (see pulse protocol). (B) To obtain the fully activated I-V relationship, maximal current amplitudes during the test pulses were plotted as a function of the test pulse potential. Data from individual experiments were normalized to the inward current amplitude obtained upon the test pulse to −100 mV before averaging. Data points are means ± SEM, and the number of experiments is given in brackets. (C) Normalized current amplitudes of the experiments shown in (B) were used to calculate the relative conductance as a measure of the voltage dependence of Kv11.1a/1b steady-state inactivation. Data were normalized to the conductance value at −100 mV before averaging. Lines denote fits of a Boltzmann equation to the averaged conductance-voltage data (21°C: V0.5 = −45.4 mV, k = −29.2 mV; 35°C: V0.5 = −20.1 mV, k = −25.5 mV). To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

4. Figure 3
Effect of temperature and voltage on the time course of Kv11.1a/1b channel activation. (A) Representative Kv11.1a/1b currents recorded from different cells at 21°C and 30°C using envelope-of-tails protocols. Please note the different timescales. The pulse protocols consisted of depolarizing pulses of increasing duration (2–2300 ms at 21°C, and 1–440 ms at 30°C), followed by a hyperpolarization to −100 mV to elicit tail currents. Four different potentials were used for the depolarizing pulse in every experiment, and the examples show an overlay of current traces recorded with test pulses to 20 mV (black traces) and 60 mV (gray traces). (B) Normalized peak tail current amplitudes were averaged and plotted against the duration of the preceding depolarizing pulse. Data points obtained with longer pulse durations were fitted with single exponential functions, yielding values for the time constant of activation and a delay. Data are means ± SEM from experiments performed at 21°C (n = 8) or 30°C (n = 12). (C) Mean values (± SEM) of the delay and the time constant of activation as a function of the test pulse potential. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

5. Figure 4
Temperature effects on the time course of Kv11.1a/1b channel inactivation, recovery from inactivation, and deactivation. (A) Typical examples of Kv11.1a/1b current traces recorded at 21°C and 35°C with triple pulse protocols designed to study inactivation rates. Please note the different timescales. Starting from a holding potential of −80 mV, the potential was stepped for 500 ms to +80 mV (P1) to maximally activate and inactivate the channels. Subsequent short P2 pulses to −140 mV (10 ms at 21°C and 2 ms at 35°C) to open the Kv11.1 channels due to fast recovery from inactivation were followed by variable 300-ms test pulses to potentials between +60 and −20 mV (P3) to reinduce inactivation (see pulse protocol). (B) Means (± SEM) of time constants of inactivation (open symbols) and time constants of recovery from inactivation (solid symbols) were plotted versus the test pulse potential; the number of experiments is given in brackets. Time constants of inactivation were obtained from monoexponential fits to the decay phase of the current traces during the P3 pulse as shown in (A). Time constants of recovery from inactivation and deactivation were obtained from experiments with fully activated Kv11.1a/1b channels as shown in Fig. 2 A. (C) Voltage and temperature dependence of Kv11.1a/1b channel deactivation. Time constants of fast and slow deactivation, and the relative contribution of the fast deactivating current component as a function of the test pulse potential are shown. The number of experiments is given in brackets; asterisks indicate significant differences (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

6. Figure 5
Temperature effects on Kv11.3 channel activation. Membrane currents were recorded in CHO cells transiently expressing Kv11.3 channels. (A) Examples of Kv11.3 current traces from two different cells using 4-s (21°C) or 1-s (30°C) depolarizing test pulses. Please note the different timescales. Test pulses varied between −100 mV and +40 mV with 10 mV increments and were followed by a constant pulse to −100 mV to elicit tail currents (see pulse diagram). (B) I-V relationship of outward current amplitudes measured at the end of 4-s depolarizing test pulses at 21°C, 30°C, and 35°C. Data are means ± SEM of absolute amplitudes (upper panel) or current amplitudes normalized to the individual maximum outward current (lower panel). The number of experiments is given in brackets in the symbol legend. The inset shows the mean maximal outward current densities determined in these experiments. Asterisks indicate a significant difference between the unpaired data obtained at 35°C and 21°C (∗∗p < 0.01). (C) Charge of the tail current plotted against the potential of the preceding test pulse. Charge values of individual experiments were normalized to the maximum tail charge, averaged, and fitted with a Boltzmann function. Symbols with a black margin represent means ± SEM from experiments with 4-s test pulses for the three different temperatures. The fit parameters of the 4-s isochronal activation curves are V0.5 = −45.0 mV and k = 7.2 mV at 21°C, V0.5 = −38.2 mV and k = 9.5 mV at 30°C, and V0.5 = −33.5 mV and k = 11.4 mV at 35°C. At the recording temperature of 30°C, additional experiments were performed with 1-s test pulses (squares without margin; V0.5 = −40.4 mV, k = 8.8 mV). The number of experiments is given in brackets. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

7. Figure 6
Temperature effects on fully activated Kv11.3 currents and steady-state inactivation. (A) Representative families of fully activated Kv11.3 currents recorded in two different cells at 21°C and 30°C. Membrane currents were elicited by 500-ms depolarizing pulses to +80 mV followed by variable 250-ms test pulses between +80 and −120 mV with 10 mV decrements, and a final step to +80 mV (see pulse protocol). (B) To obtain the fully activated I-V relationship, maximal current amplitudes during the variable test pulses were plotted as a function of the test pulse potential. Data from individual experiments were normalized to the inward current amplitude obtained upon the test pulse to −100 mV before averaging. Data points are means ± SEM, and the number of experiments is given in brackets. (C) Normalized current amplitudes of the experiments shown in (B) were used to calculate the relative maximal conductance as a measure of the voltage dependence of Kv11.3 steady-state inactivation. Lines denote fits of a Boltzmann equation to the conductance data in the voltage range between −30 mV and +80 mV (21°C: V0.5 = −17.7 mV, k = −22.4 mV) or between −20 mV and +80 mV (30°C: V0.5 = −4.4 mV, k = −21.3 mV). (D and E) With a recording temperature of 35°C, Kv11.3 steady-state inactivation was studied in the voltage range between −120 mV and +40 mV (see pulse protocol in D), and experiments were performed at 21°C with the same pulse protocol for comparison. (D) Fully activated I-V relationship. Current amplitudes were normalized to the inward current amplitude obtained upon the test pulse to −100 mV before averaging. Data points are means ± SEM, and the number of experiments is given in brackets. (E) Relative maximal conductance (means ± SEM) plotted as a function of the test pulse potential. Individual data were normalized to the value at −100 mV. Lines denote fits of a Boltzmann equation to the mean conductance data in the voltage range between −30 mV and +40 mV (21°C: V0.5 = −13.6 mV, k = −24.6 mV) or between −20 mV and +40 mV (35°C: V0.5 = 14.7 mV, k = −18.0 mV). To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

8. Figure 7
Effect of temperature and voltage on the time course of Kv11.3 channel activation. (A) Representative Kv11.3 currents recorded from different cells at 21°C and 30°C with envelope-of tails protocols. Please note the different timescales. The pulse protocol consisted of depolarizing pulses of increasing duration (2–354 ms at 21°C and 1–88 ms at 30°C), followed by a hyperpolarization to −100 mV to elicit tail currents. Four different potentials were used for the depolarizing pulse in every experiment, and the examples show an overlay of current traces recorded with test pulses to 0 mV (black traces) and 40 mV (gray traces). (B) Normalized tail charge values were averaged and plotted against the duration of the preceding depolarizing pulse. Data points obtained with longer pulse durations were fitted with single exponential functions, yielding values for the time constant of activation and a delay. Data are means ± SEM from experiments performed at 21°C (n = 12) or 30°C (n = 8). (C) Mean values (± SEM) of the delay and the time constant of activation as a function of the test pulse potential. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

9. Figure 8
Temperature effects on the time course of Kv11.3 channel inactivation, recovery from inactivation, and deactivation. (A) Typical examples of Kv11.3 current traces recorded at 21°C and 30°C with triple pulse protocols designed to study inactivation rates. Starting from a holding potential of −80 mV, the potential was stepped for 500 ms to +80 mV (P1) to maximally activate and inactivate the channels. Subsequent 2-ms P2 pulses to −100 mV to open the Kv11.3 channels were followed by variable 300-ms test pulses to potentials between +60 and −20 mV (P3) to reinduce inactivation (see pulse protocol). (B) Means (± SEM) of time constants of inactivation (open symbols) and time constants of recovery from inactivation (solid symbols) plotted versus test pulse potential; the number of experiments is given in brackets. Time constants of inactivation were obtained from monoexponential fits to the decay phase of the current traces during the P3 pulse using current recordings as shown in (A). Time constants of recovery from inactivation and deactivation were obtained from experiments with fully activated Kv11.3 channels as shown in Fig. 6 A. (C) Voltage and temperature dependence of Kv11.3 channel deactivation. Time constants of fast and slow deactivation, and the relative contribution of the fast deactivating current component as a function of the test pulse potential are shown. The number of experiments is given in brackets. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions

10. Figure 9
Comparison of temperature effects on the steady-state and kinetic parameters of Kv11.1a/1b and Kv11.3 channels. (A) Kv11.1a/1b steady-state open probability at 21°C and 35°C as a function of the membrane potential was calculated from mean parameters of the Boltzmann functions describing channel activation and inactivation (see Table 1). (B) Theoretical Kv11.1a/1b window current amplitudes normalized to the maximum current value at 21°C were calculated from the open probability data in (A) without (open symbols) or with (continuous lines) adjustment for a low temperature sensitivity of the single-channel conductance (using a Q10 of 1.3). Solid symbols are the experimentally determined values of the maximal steady-state current density normalized to the mean value at 21°C (means ± SEM; for absolute values and number of experiments, see Fig. 1 B, inset). (C) Kv11.3 steady-state open probability at 21°C, 30°C, and 35°C as a function of the membrane potential was calculated from mean parameters of the Boltzmann functions describing channel activation and inactivation (see Table 2). (D) Theoretical Kv11.3 window current amplitudes normalized to the maximum current value at 21°C were calculated from the open probability data in (C) without (open symbols) or with (continuous lines) adjustment for a low temperature sensitivity of the single-channel conductance (using a Q10 of 1.3). Solid symbols are the experimentally determined values of the maximal steady-state current density normalized to the mean value at 21°C (means ± SEM; for absolute values and number of experiments, see Fig. 5 B, inset). (E) Comparison of Q10 values determined for the Kv11.1a/1b and Kv11.3 gating parameters. Except for Kv11.3 channel activation, the average of the Q10 values obtained for the 9°C and 14°C temperature differences is given (see Tables 1 and 2). (F) Temperature dependence of the Gibbs free energy of activation at 0 mV (ΔG0) for Kv11.1a/1b and Kv11.3 channels (means ± SEM; values and number of experiments are given in Tables 1 and 2; the data point for Kv11.1a/1b at 21°C represents combined experiments of data sets A and B). To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2016 Biophysical Society Terms and Conditions