XBART52995
PLoS One.
January 1, 2017;
12
(1):
e0171213.
KChIP2 genotype dependence of transient outward current (Ito) properties in cardiomyocytes isolated from male and female mice.
Waldschmidt L
,
Junkereit V
,
Bähring R
.
Abstract
The transient outward current (Ito) in
cardiomyocytes is largely mediated by Kv4 channels associated with Kv Channel Interacting Protein 2 (
KChIP2). A knockout model has documented the critical role of
KChIP2 in Ito expression. The present study was conducted to characterize in both sexes the dependence of Ito properties, including current magnitude, inactivation kinetics, recovery from inactivation and voltage dependence of inactivation, on the number of functional
KChIP2 alleles. For this purpose we performed wholecell patchclamp experiments on isolated
left ventricular
cardiomyocytes from male and female mice which had different
KChIP2 genotypes; i.e., wildtype (KChIP2+/+), heterozygous knockout (KChIP2+/) or complete knockout of
KChIP2 (
KChIP2/). We found in both sexes a
KChIP2 gene dosage effect (i.e., a proportionality between number of alleles and phenotype) on Ito magnitude, however, concerning other Ito properties, KChIP2+/ resembled KChIP2+/+. Only in the total absence of
KChIP2 (
KChIP2/) we observed a slowing of Ito kinetics, a slowing of recovery from inactivation and a negative shift of a portion of the voltage dependence of inactivation. In a minor fraction of
KChIP2/
myocytes Ito was completely lost. The distinct
KChIP2 genotype dependences of Ito magnitude and inactivation kinetics, respectively, seen in
cardiomyocytes were reproduced with twoelectrode voltageclamp experiments on Xenopus oocytes expressing Kv4.2 and different amounts of
KChIP2. Our results corroborate the critical role of
KChIP2 in controlling Ito properties. They demonstrate that the Kv4.2/
KChIP2 interaction in
cardiomyocytes is highly dynamic, with a clear
KChIP2 gene dosage effect on Kv4 channel surface expression but not on inactivation gating.
PubMed ID:
28141821
PMC ID:
PMC5283746
Article link:
PLoS One.
Genes referenced:
kcnip2
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Article Images:
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Fig 1. Isolation of Ito amplitude and inactivation kinetics.Wholecell patchclamp 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. Prepulseinactivationsubtraction method to isolate Ito in a male wildtype (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 singleexponential 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 doubleexponential (2exp, top) and a tripleexponential 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 ttest); i.e. the tripleexponential fit was more accurate. D. Mean time constants obtained by fitting the decay of isolated Ito traces with a singleexponential function (τs, empty bar) and by fitting the compound outward current decay with a tripleexponential 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 tripleexponential fitting (A1, A2 and A3, grey bars). The amplitude of the noninactivating current component (A0, right grey bar) was obtained by fitting P/n leaksubtracted current traces (not shown).


Fig 2. Multiexponential 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 noninactivating 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 tripleexponential function. In some KChIP2/ myocytes (7 out of 26, blue, slow decay) a doubleexponential function was sufficient. B. Mean time constants (τ1, τ2 and τ3) obtained with a tripleexponential 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 doubleexponential 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 tripleexponential (A1, A2 and A3) and doubleexponential fitting (A2 and A3, separate bars on the right), and mean amplitudes of the corresponding noninactivating 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+/; oneway 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 doublepulse 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 doublepulse 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 doubleexponential function. Recovery kinetics for KChIP2+/+ and KChIP2+/ were virtually identical. Lines without symbols represent singleexponential 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 doubleexponential function to adequately fit the recovery kinetics for all KChIP2 genotypes. C. Mean recovery time constants (τrec1 and τrec2) obtained by doubleexponential fitting (** significantly different from both KChIP2+/+ and KChIP2+/; oneway 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 Boltzmannfunctions. The voltage dependences for KChIP2+/+ and KChIP2+/ were virtually identical. Lines without symbols represent single Boltzmannfunctions 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 Boltzmannfunctions 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 Boltzmannfunctions. 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+/; oneway ANOVA).


Fig 5. Comparison of outward current inactivation properties in male and female wildtype myocytes.For the compound outward current in male and female wildtype (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 noninactivating current level). For both sexes KChIP2+/+ outward current decay kinetics were best described by a tripleexponential function. Note the slightly faster decay kinetics in the female myocyte. B. Mean time constants obtained with a tripleexponential function (τ1, τ2 and τ3). C. Mean amplitudes of the individual time constants obtained by tripleexponential fitting (A1, A2 and A3) and the amplitude of the noninactivating current component (A0). D. Mean total amplitude of the compound outward current (AΣ). E. Recovery data fitted by a doubleexponential function. Note that for female KChIP2+/+ myocytes the recovery from inactivation was apparently faster. F. Mean recovery time constants (τrec1 and τrec2) obtained by doubleexponential fitting. G. Data describing the voltage dependence of inactivation fitted with the sum of two Boltzmannfunctions. 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 Boltzmannfunctions (¶, unpaired Student’s ttest).


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 noninactivating 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 tripleexponential function. In some KChIP2/ myocytes (3 out of 17, blue, slow decay) a doubleexponential function was sufficient. B. Mean time constants obtained with a tripleexponential 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 doubleexponential 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 tripleexponential (A1, A2 and A3) and doubleexponential fitting (A2 and A3, separate bars on the right) and mean amplitudes of the corresponding noninactivating 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+/; oneway 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 doubleexponential functions. Lines without symbols represent singleexponential 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 doubleexponential 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 Boltzmannfunctions. Lines without symbols represent single Boltzmannfunctions 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 Boltzmannfunctions. 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+/; oneway ANOVA.


Fig 8. Expression of Kv4.2 and KChIP2 in Xenopus oocytes.Twoelectrode voltageclamp 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 doubleexponential function (Kv4.2/KChIP2mediated currents with 0–12.8 ng KChIP2 cRNA coinjected: black) and based on the mean τ1 values from tripleexponential 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 singleexponential function. Black lines without symbols: Singleexponential 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: Singleexponential 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 singleexponential 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 singleexponential 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).
