Waldschmidt L , Junkereit V , Bähring R .

	Fig 1. Isolation of Ito amplitude and inactivation kinetics.Whole-cell patch-clamp recordings were taken from individual murine cardiomyocytes isolated from the left ventricular free wall. Outward currents were activated by depolarizing voltage pulses from -80 to +40 mV. Inward sodium currents (not shown) were inactivated by a brief (8 ms) prepulse to -50 mV. Voltage protocols are shown below current traces. A. Prepulse-inactivation-subtraction method to isolate Ito in a male wild-type (KChIP2+/+) myocyte. A fraction of current was optionally inactivated by a 160 ms prepulse to -40 mV. Subtraction of the remaining outward current (2) from the compound outward current (obtained without the 160 ms prepulse, 1) yielded a rapidly inactivating current trace (S), referred to as Ito. The inactivation of the Ito trace was fitted by a single-exponential function. B. Compound outward current recorded from the same myocyte as in A during a 5 s voltage pulse to +40 mV (dotted lines represent zero current). For the same current trace the decay was fitted by a double-exponential (2exp, top) and a triple-exponential function (3exp, bottom). The root mean square (RMS) values of the fits are indicated. C. Corresponding RMS2exp and RMS3exp values obtained for 12 male KChIP2+/+ myocytes. RMS3exp values were significantly smaller than RMS2exp values (§, Student's paired t-test); i.e. the triple-exponential fit was more accurate. D. Mean time constants obtained by fitting the decay of isolated Ito traces with a single-exponential function (τs, empty bar) and by fitting the compound outward current decay with a triple-exponential function (τ1, τ2 and τ3, grey bars). Note the close match between τs and τ1. E. Mean peak amplitude of the isolated Ito traces (empty bar) and amplitudes of the individual time constants obtained by triple-exponential fitting (A1, A2 and A3, grey bars). The amplitude of the non-inactivating current component (A0, right grey bar) was obtained by fitting P/n leak-subtracted current traces (not shown).
	Fig 2. Multi-exponential fitting of compound outward current decay in male myocytes with different KChIP2 genotypes.Outward currents were activated by 5 s voltage pulses from -80 to +40 mV in male KChIP2+/+, KChIP2+/- and KChIP2-/- myocytes. The voltage protocol is shown below the current traces. A. Representative current traces for the different KChIP2 genotypes were normalized to peak and only the inactivating current components are shown (dotted line represents non-inactivating current level). Current decay kinetics in KChIP2+/+ (grey), KChIP2+/- (orange) and most KChIP2-/- myocytes (19 out of 26, blue, fast decay) were best described by a triple-exponential function. In some KChIP2-/- myocytes (7 out of 26, blue, slow decay) a double-exponential function was sufficient. B. Mean time constants (τ1, τ2 and τ3) obtained with a triple-exponential function for male KChIP2+/+ (grey bars), KChIP2+/- (orange bars) and most KChIP2-/- myocytes (19 out of 26, blue bars), and mean time constants obtained with a double-exponential function for 7 out of 26 male KChIP2-/- myocytes (τ2 and τ3, separate blue bars on the right). C. Mean amplitudes of the individual time constants obtained by triple-exponential (A1, A2 and A3) and double-exponential fitting (A2 and A3, separate bars on the right), and mean amplitudes of the corresponding non-inactivating current components (A0, A0). Note the KChIP2 gene dosage effect on A1. D. Mean total amplitudes of the compound outward current (AΣ, AΣ). The KChIP2 gene dosage effect observed for A1 is also reflected in AΣ (* significantly different from KChIP2+/+; ** significantly different from both KChIP2+/+ and KChIP2+/-; one-way ANOVA).
	Fig 3. Recovery from inactivation in male myocytes with different KChIP2 genotypes.Recovery from inactivation at -80 mV was studied in male myocytes using a double-pulse protocol with interpulse durations between 6 ms and 6.14 s (Δt) followed by a brief test pulse (voltage protocol shown below current traces). A. Representative current families, recorded with the double-pulse protocol for the different KChIP2 genotypes (dotted lines represent zero current). B. Relative peak current amplitudes obtained with the brief test pulse were plotted against interpulse duration for the different KChIP2 genotypes (KChIP2+/+: grey, KChIP2+/-: orange, KChIP2-/-: blue), and the data were fitted with a double-exponential function. Recovery kinetics for KChIP2+/+ and KChIP2+/- were virtually identical. Lines without symbols represent single-exponential functions fitted to a fraction of the data points and forced to reach 1 (orange and grey line superimpose). They represent the fast recovery component and emphasize the necessity of a double-exponential function to adequately fit the recovery kinetics for all KChIP2 genotypes. C. Mean recovery time constants (τrec1 and τrec2) obtained by double-exponential fitting (** significantly different from both KChIP2+/+ and KChIP2+/-; one-way ANOVA). D. Mean relative amplitudes of the recovery time constants (Arec1 and Arec2).
	Fig 4. Voltage dependence of inactivation in male myocytes with different KChIP2 genotypes.The voltage dependence of inactivation was studied in male myocytes with a variable prepulse protocol. The prepulse (1 s, between -100 and 0 mV) was followed by a brief test pulse (voltage protocol shown below current traces). A. Representative current families, recorded with the variable prepulse protocol, for the different KChIP2 genotypes. B. Normalized peak current amplitudes obtained with the test pulse (I / Imax) were plotted against prepulse voltage for the different KChIP2 genotypes (KChIP2+/+: grey, KChIP2+/-: orange, KChIP2-/-: blue), and the data were fitted with the sum of two Boltzmann-functions. The voltage dependences for KChIP2+/+ and KChIP2+/- were virtually identical. Lines without symbols represent single Boltzmann-functions fitted to a fraction of the data points and forced to reach 0. They extrapolate the more negative portion of the voltage dependences and emphasize the necessity of the sum of two Boltzmann-functions to adequately fit the voltage dependence of inactivation. C. Mean values of the voltages of halfmaximal inactivation obtained for the two components (V1/21 and V1/22) obtained with the sum of two Boltzmann-functions. Note that V1/21 was shifted to more negative potentials for KChIP2-/-, whereas V1/22 values were similar in all KChIP2 genotypes. D. Mean relative amplitudes of the two components defined by V1/21 and V1/22, respectively (Av1 and Av2); ** significantly different from both KChIP2+/+ and KChIP2+/-; one-way ANOVA).
	Fig 5. Comparison of outward current inactivation properties in male and female wild-type myocytes.For the compound outward current in male and female wild-type (KChIP2+/+) myocytes the kinetics of macroscopic inactivation, the kinetics of recovery from inactivation and the voltage dependence of inactivation were compared. Data from male myocytes are depicted as black symbols, data from female myocytes as empty symbols. A. Representative outward current traces from a male and a female KChIP2+/+ myocyte, respectively, recorded during a 5 s voltage pulse to +40 mV (voltage protocol shown below current traces; dotted line represents non-inactivating current level). For both sexes KChIP2+/+ outward current decay kinetics were best described by a triple-exponential function. Note the slightly faster decay kinetics in the female myocyte. B. Mean time constants obtained with a triple-exponential function (τ1, τ2 and τ3). C. Mean amplitudes of the individual time constants obtained by triple-exponential fitting (A1, A2 and A3) and the amplitude of the non-inactivating current component (A0). D. Mean total amplitude of the compound outward current (AΣ). E. Recovery data fitted by a double-exponential function. Note that for female KChIP2+/+ myocytes the recovery from inactivation was apparently faster. F. Mean recovery time constants (τrec1 and τrec2) obtained by double-exponential fitting. G. Data describing the voltage dependence of inactivation fitted with the sum of two Boltzmann-functions. Note the apparent negative shift of the fitting curve in female relative to male KChIP2+/+ myocytes. H. Mean values for V1/21 and V1/22 obtained with the sum of two Boltzmann-functions (¶, unpaired Student’s t-test).
	Fig 6. Analysis of macroscopic inactivation in female myocytes with different KChIP2 genotypes.Outward currents were activated by 5 s voltage pulses from -80 to +40 mV in female KChIP2+/+, KChIP2+/- and KChIP2-/- myocytes. A. Representative current traces for the different KChIP2 genotypes were normalized to peak and only the inactivating current components are shown (voltage protocol below current traces; dotted line represents non-inactivating current level). Current decay kinetics in KChIP2+/+ (grey), KChIP2+/- (orange) and most KChIP2-/- myocytes (14 out of 17, blue, fast decay) were best described by a triple-exponential function. In some KChIP2-/- myocytes (3 out of 17, blue, slow decay) a double-exponential function was sufficient. B. Mean time constants obtained with a triple-exponential function (τ1, τ2 and τ3) for female KChIP2+/+ (grey bars), KChIP2+/- (orange bars) and most KChIP2-/- myocytes (14 out of 17, blue bars), and mean time constants obtained with a double-exponential function for 3 out of 17 female KChIP2-/- myocytes (τ2 and τ3, separate blue bars on the right). C. Mean amplitudes of the individual time constants obtained by triple-exponential (A1, A2 and A3) and double-exponential fitting (A2 and A3, separate bars on the right) and mean amplitudes of the corresponding non-inactivating current components (A0, A0). Like in male myocytes, there was a gene dosage effect on A1 in female myocytes (* significantly different from KChIP2+/+; ** significantly different from both KChIP2+/+ and KChIP2+/-; one-way ANOVA). D. Mean total amplitudes of the compound outward current (AΣ, AΣ). The KChIP2 gene dosage effect observed for A1 is not reflected in AΣ in female myocytes.
	Fig 7. Recovery from inactivation and voltage dependence of inactivation in female myocytes with different KChIP2 genotypes.Recovery from inactivation and the voltage dependence of inactivation were studied in female KChIP2+/+ (grey symbols), KChIP2+/- (orange symbols) and KChIP2-/- myocytes (blue symbols). A. Recovery plots. Fitting curves represent double-exponential functions. Lines without symbols represent single-exponential functions fitted to a fraction of the data points (the fast component) and forced to reach 1. Note that in female myocytes a moderate apparent slowing of the recovery kinetics was observed for KChIP2+/- compared to KChIP2+/+. B. Mean recovery time constants (τrec1 and τrec2) obtained by fitting the recovery kinetics with a double-exponential function. C. Mean relative amplitudes of recovery time constants for all KChIP2 genotypes (Arec1 and Arec2). D. Inactivation curves. The data were fitted with the sum of two Boltzmann-functions. Lines without symbols represent single Boltzmann-functions fitted to a fraction to the data points (the more negative portion) and forced to reach 0. E. Mean values for V1/21 and V1/22 obtained with the sum of two Boltzmann-functions. Note that V1/22 values are similar in all KChIP2 genotypes. F. Mean relative amplitudes of the voltage dependences defined by V1/21 and V1/22 (Av1 and Av2); * significantly different from KChIP2+/+; ** significantly different from both KChIP2+/+ and KChIP2+/-; one-way ANOVA.
	Fig 8. Expression of Kv4.2 and KChIP2 in Xenopus oocytes.Two-electrode voltage-clamp experiments were performed on individual oocytes following cRNA injection. A. Currents obtained with depolarizing voltage jumps from -80 to +40 mV (voltage protocols below traces) 48 h after cRNA injection. Different amounts of KChIP2 cRNA (ng per oocyte indicated above traces) were coinjected with a fixed amount of Kv4.2 cRNA (1 ng per oocyte). Note the tight correlation between KChIP2 cRNA amount and peak current amplitude. B. Idealized current traces based on the mean fit results obtained with a double-exponential function (Kv4.2/KChIP2-mediated currents with 0–12.8 ng KChIP2 cRNA coinjected: black) and based on the mean τ1 values from triple-exponential fits (i.e., the Ito component) of female myocytes (KChIP2+/+: grey, KChIP2+/-: orange). All currents were normalized to peak. C. Recovery from inactivation at -80 mV obtained for oocytes injected with different amounts of KChIP2 cRNA. Pooled recovery data are shown for oocytes injected with 1 ng Kv4.2 cRNA and 0 ng (empty symbols), 0.2 ng (grey symbols) and 0.4 ng KChIP2 cRNA (black symbols). Kv4.2/KChIP2 recovery kinetics were fitted by a single-exponential function. Black lines without symbols: Single-exponential fits for all other KChIP2 cRNA amounts (0.1, 0.8, 1.6, 3.2, 6.4 and 12.8 ng per oocyte); colored lines without symbols: Single-exponential functions representing the fast recovery component of female myocytes (see Fig 7A; KChIP2+/+: grey, KChIP2+/-: orange). D. Idealized current traces based on the same fitting procedures as the currents in B; black traces: currents obtained after consecutive injection of 1 ng Kv4.2 and 1 ng KChIP2 cRNA into the same oocyte; faster decay: 48 h Kv4.2 expression followed by 5 h KChIP2 expression (48+5); slower decay: 48 h Kv4.2 expression followed by 48 h KChIP2 expression (48+48). Red dotted traces represent the data for 0 and 12.8 ng KChIP2 cRNA from B. E. Recovery from inactivation for the two experimental paradigms (grey symbols: 48+5; black symbols: 48+48). The data were fitted by a single-exponential function. Red dotted lines: single exponential fits to the recovery data obtained with 0 and 12.8 ng KChIP2 cRNA per oocyte). Note the slow recovery component (~ 15%) in the 48+5 data, which was not captured by the single-exponential fit. F. Mean peak current amplitudes from A and from the experiments with consecutive cRNA injection (48+5 and 48+48). G. Inactivation time constants (τinact1 and τinact2) from B (0–12.8 ng KChIP2 cRNA per oocyte) and D (48+5 and 48+48). H. Recovery time constants (τrec) from C (0–12.8 ng KChIP2 cRNA per oocyte) and E (48+5 and 48+48).

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