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Journal Articles

Prepulse facilitation of the honeybee CaV4 channel is produced by a shift in channel activation and requires an intact inactivation sequence

Thierry Cens, Matthieu Rousset, Claudine Menard, Mohamed Chahine, Claude Collet, Jean-Christophe Sandoz, Alain Chavanieu, Sebastien Estaran, Jean-Baptiste Thibaud, Patrick Bois, Pierre Charnet
Journal: Journal of General Physiology
J Gen Physiol (2026) 158 (2): e202413712.
https://doi.org/10.1085/jgp.202413712
Published: 10 February 2026
View article titled, Prepulse facilitation of the honeybee CaV4 channel is produced by a shift in channel activation and requires an intact inactivation sequence
Open the PDF for Prepulse facilitation of the honeybee CaV4 channel is produced by a shift in channel activation and requires an intact inactivation sequence in another window
Images
CaV4 voltage-dependent facilitation. (A) Ba2+ current traces showing the effect of a positive conditioning pP on the current recorded at voltage pulses of different amplitudes. The potentials for the control (P1) and the test pulses (P2) were −20, 0, or +20 mV. The pulse duration was 160 ms, and the test pulse was preceded by a strong 40-ms-long conditioning depolarization (pP) to +80 mV. The time between two successive trains of depolarization was 30 s. The superimposed red, green, and blue top traces are the responses of a non-injected oocyte to the protocol with P1 and P2 voltages of −20, 0, or +20 mV, respectively. The colored bottom traces in red, green, and blue are the responses of an oocyte injected with the CaV4 RNA to the same protocol. I1 and I2 are the peak current amplitudes measured during the P1 and P2 pulses, respectively. (B) Averaged facilitation was calculated as the ratio of the current amplitude of the second pulse (I2, after the strong depolarization) over the current amplitude of the first pulse (I1) for pulses at −20, 0, or +20 mV (in red, green, and blue, respectively), recorded in Ba2+ (dark color) or in Ca2+-containing (hatched color) extracellular solutions. The numbers in the bars indicate the number of oocytes recorded in each condition. The unpaired Student’s P values for the statistical significance of the effect of the pP at −20, 0, and +20 mV (with the Holm correction for multiple comparison) were, respectively, 0.049 (*), 0.007 (**), and 0.547 (ns). (C) Time-to-peak values calculated at −20, 0, or +20 mV (same color code as in A), without (dark color) and with (hatched color) a pP. The paired Student’s P values for the statistical significance of the effect of the pP at −20, 0, and +20 were (after Holm correction), respectively, 4.6 × 10−7 (***), 5.3 × 10−4 (***), and 7.6 × 10−4 (***).

Ca V 4 voltage-dependent facilitation. (A) Ba2+ cu...

in Prepulse facilitation of the honeybee CaV4 channel is produced by a shift in channel activation and requires an intact inactivation sequence > Journal of General Physiology
Published: 10 February 2026
Figure 1. Ca V 4 voltage-dependent facilitation. (A) Ba2+ current traces showing the effect of a positive conditioning pP on the current recorded at voltage pulses of different amplitudes. The potentials for the control (P1) and the test More about this image found in Ca V 4 voltage-dependent facilitation. (A) Ba2+ cu...
Images
Influence of pP on the I–V curve parameters. (A) Schematic of the protocol used to extract the I–V curve parameters. 400-ms-long voltage steps from −80 to +40 mV were given before (in black, P1) or 4 ms after (in red, P2) a pP to +80 mV. (B) The relative Ba2+ current amplitudes (Rel. Cur.), recorded during P1, (black squares) or during P2 (red circles), were plotted against the voltage amplitude of the pulses. I–V curves were normalized to the peak of the I–V curve for each channel and fitted using Eq. 1 (see Materials and methods). Parameter values for these curves are displayed on the bottom: -pP: I–V curves measured during P1; +pP: I–V curves measured during P2. Note the negative shift of Eact and the increase in kact value. (C) Superimposition of the voltage dependence of facilitation (I2/I1, left Y axis) and activation curves, with (in red) or without (in blue) a pP (right Y axis, using the activation parameters Eact and kact obtained from the fit of the I–V curves). The X axis is the amplitude of the voltage pulses P1 or P2.

Influence of pP on the I–V curve parameters. (A) Schematic of the protocol...

in Prepulse facilitation of the honeybee CaV4 channel is produced by a shift in channel activation and requires an intact inactivation sequence > Journal of General Physiology
Published: 10 February 2026
Figure 2. Influence of pP on the I–V curve parameters. (A) Schematic of the protocol used to extract the I–V curve parameters. 400-ms-long voltage steps from −80 to +40 mV were given before (in black, P1) or 4 ms after (in red, P2) a pP to +80 More about this image found in Influence of pP on the I–V curve parameters. (A) Schematic of the protocol...
Images
CaV4 Ba2+current facilitation as a function of voltage and time. (A) Left: The voltage protocol was similar to that shown in Fig. 1 but with the strong conditioning pulse of variable amplitude from −10 to +80 mV (in red) in 10 mV increments. Exemplar Ba2+ current traces recorded during this protocol are displayed. Right: Ba2+ current facilitation (I2/I1) was then plotted as a function of the conditioning pulse amplitude. (B) Left: The voltage protocol was similar to that shown in Fig. 1 but with the strong conditioning pulse of variable duration from 10 to 90 ms in 10-ms increments. Exemplar Ba2+ current traces are shown on the bottom. Right: Ba2+ current facilitation, (I2/I1), plotted as a function of the conditioning pulse duration from 10 to 100 ms (in red). In this case, I2/I1 values were normalized to the I2/I1 value with a pP duration of 10 ms (n[I2/I1]). (C) Left: The voltage protocol was similar to that shown in Fig. 1 but with an interpulse between the strong pre-depolarization (at +80 mV) and P2 of variable duration from 4 to 50 ms in 5-ms increments. Exemplar Ba2+ current traces are shown on the bottom. Right: Current facilitation (I2/I1) as a function of the interpulse duration from 4 to 50 ms (in red).

Ca V 4 Ba 2+ current facilitation as a function o...

in Prepulse facilitation of the honeybee CaV4 channel is produced by a shift in channel activation and requires an intact inactivation sequence > Journal of General Physiology
Published: 10 February 2026
Figure 3. Ca V 4 Ba 2+ current facilitation as a function of voltage and time. (A) Left: The voltage protocol was similar to that shown in Fig. 1 but with the strong conditioning pulse of variable amplitude from −10 to +80 mV (in red) in More about this image found in Ca V 4 Ba 2+ current facilitation as a function o...
Images
Influence of an IFM-homologous sequence on the CaV4 Ba2+current facilitation. (A) Schematic of a CaV4 mutant in which the MFLT sequence, which is homologous to the IFMT sequence found in the loop between domains III and IV of NaV channels, was changed to AAAA. This mutant was named CaV4(6). (B) When the pP protocol used in Fig. 1 A was applied to oocytes expressing the CaV4(6) channel, the Ba2+ currents recorded during depolarizations of −20, 0, or +20 mV displayed no or a low facilitation (see Fig. 5). (C) Bar graph showing current facilitation (I2/I1 ratios) recorded with the mutated CaV4(6) channels (hatched colored bars) at different voltages. Results obtained in Fig. 1 for the CaV4 WT channel are shown for comparison (fully colored bars, same color code as in Fig. 1 A). A Student’s t test was used to assess the statistical significance (after Holm correction for multiple comparison) of the differences between the averaged facilitation of CaV4 and CaV4(6) at −20, 0, and +20 mV. P values are, respectively, 1.04 × 10–6 (***), 0.044 (*), and 5.2 × 10–6 (***).

Influence of an IFM-homologous sequence on the Ca V 4 Ba ...

in Prepulse facilitation of the honeybee CaV4 channel is produced by a shift in channel activation and requires an intact inactivation sequence > Journal of General Physiology
Published: 10 February 2026
Figure 4. Influence of an IFM-homologous sequence on the Ca V 4 Ba 2+ current facilitation. (A) Schematic of a CaV4 mutant in which the MFLT sequence, which is homologous to the IFMT sequence found in the loop between domains III and IV of More about this image found in Influence of an IFM-homologous sequence on the Ca V 4 Ba ...
Images
Ba2+current facilitation as a function of voltage and time for the CaV4(6) mutant. (A–C) Voltage-dependent (A) and time-dependent (B and C) regulation of facilitation (see Fig. 3, A–C, for the description of these protocols) were recorded from oocytes expressing the CaV4(6) channel in which the III–IV loop has been mutated (see text and Fig. 4). Relationships between facilitation and the pP amplitude (A), the pP duration (B), or the interpulse duration (C) are shown. In panel B, I21/I1 values were normalized to the I2/I1 value with a pP duration of 10 ms, as in Fig. 3 B, and labeled n(I2/I1). In C, the time constants of the kinetics of recovery from facilitation were calculated from the best fit to a monoexponential decay yielding values of 13.5 ± 0.7 ms and 25.4 ± 6.3 ms for CaV4 and CaV4(6), respectively. In each case, the Ba2+ current traces are shown on the left, and the effects on facilitation are visualized on graphs shown on the right in blue. The results obtained with the WT CaV4 in Fig. 3 are shown in green.

Ba 2+ current facilitation as a function of voltage and time f...

in Prepulse facilitation of the honeybee CaV4 channel is produced by a shift in channel activation and requires an intact inactivation sequence > Journal of General Physiology
Published: 10 February 2026
Figure 5. Ba 2+ current facilitation as a function of voltage and time for the Ca V 4(6) mutant. (A–C) Voltage-dependent (A) and time-dependent (B and C) regulation of facilitation (see Fig. 3, A–C , for the description of these protocols) More about this image found in Ba 2+ current facilitation as a function of voltage and time f...
Images
Current facilitation does not modify the single-channel conductance. (A and B) In cell-attached patches, using a 100 mM Ba2+ pipet solution, single-channel CaV4 Ba2+ current traces were recorded during 100 depolarizations from −100 mV to −20 mV (A) or to 0 mV (B), either without (top) or with (middle) a pP to +80 mV. The bottom traces represent the single-channel currents averaged over these 100 traces, without (in black) and with the strong pP (in red). These currents were recorded on the same patch. (C) Single-channel I–V curves recorded without (black) or with (red) pP (curves were averaged from different patches). Note that the two single-channel conductances, calculated using the first 50 ms of these protocols, were not significantly different, as shown in the inset, in a bar graph (Student’s t test, P = 0.087). (D) Top: Open probabilities (nPopen) calculated for the first 50 ms of the records obtained from different patches at −20 or +20 mV without pP. Bottom: Ratios of the patch nPopen probabilities calculated with or without a pP at −20 or +20 mV. Note that these ratios closely matched those obtained in two electrode voltage clamp (see Fig. 1).

Current facilitation does not modify the single-channel conductance. (A and...

in Prepulse facilitation of the honeybee CaV4 channel is produced by a shift in channel activation and requires an intact inactivation sequence > Journal of General Physiology
Published: 10 February 2026
Figure 6. Current facilitation does not modify the single-channel conductance. (A and B) In cell-attached patches, using a 100 mM Ba2+ pipet solution, single-channel CaV4 Ba2+ current traces were recorded during 100 depolarizations from −100 mV More about this image found in Current facilitation does not modify the single-channel conductance. (A and...
Images
Absence of frequency-dependent facilitation for CaV4. Top: The oocytes were stimulated at a frequency of 5 Hz with −20 mV depolarizations. Bottom: Graph illustrating the changes in the current’s amplitude recorded over a 12-min period using this protocol. The inset shows the first (black) and last (red) Ba2+ current traces that were recorded during this series.

Absence of frequency-dependent facilitation for Ca V 4. Top: ...

in Prepulse facilitation of the honeybee CaV4 channel is produced by a shift in channel activation and requires an intact inactivation sequence > Journal of General Physiology
Published: 10 February 2026
Figure 7. Absence of frequency-dependent facilitation for Ca V 4. Top: The oocytes were stimulated at a frequency of 5 Hz with −20 mV depolarizations. Bottom: Graph illustrating the changes in the current’s amplitude recorded over a 12-min More about this image found in Absence of frequency-dependent facilitation for Ca V 4. Top: ...
Journal Articles

AMPK-mediated HCN4 channel phosphorylation contributes to age-related intrinsic bradycardia

Luca M.G. Palloni, Nicole Sarno, Caterina Azzoni, Nicol Furia, Matteo E. Mangoni, Alessandro Porro, Teresa Neeman, Andrea Saponaro, Gerhard Thiel, Anna Moroni, Dario DiFrancesco
Journal: Journal of General Physiology
J Gen Physiol (2026) 158 (2): e202513873.
https://doi.org/10.1085/jgp.202513873
Published: 06 February 2026
View article titled, AMPK-mediated HCN4 channel phosphorylation contributes to age-related intrinsic bradycardia
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Includes: Supplementary data
Images
AMPK activation inhibits Ifin female and male mouse pacemaker cells.(A–D) Whole-cell recordings of If in 3-mo-old female (A and B) and 3-mo-old male (C and D) mouse SAN cells in control (black) and after 4-h treatment with AMPK activator (AICAR 1 mM) (red). Data are obtained from N = 3 females and N = 3 males. (A and C) Representative current traces (left) and activation curves (right). Best fitting with the Boltzmann equation yielded the following values: (A) V1/2 = −91.0 ± 1.67; s = 8.2 ± 0.5 mV (n = 14) and V1/2 = −89.5 ± 1.69 mV; s = 7.5 ± 0.4 mV (n = 13) (n.s.); (C) V1/2 = −88.9 ± 1.76; s = 9.2 ± 0.5 mV (n = 12) and V1/2 = −88.2 ± 1.65 mV; s = 9.1 ± 0.7 mV (n = 15) (n.s.), for control and AICAR-treated cells, respectively. (B and D) Steady-state I/V relations; bar graphs on the left show the distributions of current densities at −125 mV. Predicted means ± SEM assuming GLMM (Gamma distribution) were (B) −35.4 ± 4.03 (n = 14) and −24.2 ± 2.87 pA/pF (n = 13) (P = 0.0982, n.s.); (D) −44.6 ± 5.49 (n = 12) and −25.2 ± 2.77 pA/pF (n = 15) (P = 0.0030, **) for control and AICAR-treated cells, respectively. The difference in current density in female vs. males did not reach significance (P = 0.05067). Significance P values were adjusted with Tukey’s test assuming four groups. Details of statistical analysis are shown in Table S1.

AMPK activation inhibits I f in female and male mouse pacemake...

in AMPK-mediated HCN4 channel phosphorylation contributes to age-related intrinsic bradycardia > Journal of General Physiology
Published: 06 February 2026
Figure 1. AMPK activation inhibits I f in female and male mouse pacemaker cells. (A–D) Whole-cell recordings of If in 3-mo-old female (A and B) and 3-mo-old male (C and D) mouse SAN cells in control (black) and after 4-h treatment with AMPK More about this image found in AMPK activation inhibits I f in female and male mouse pacemake...
Images
AMPK activation reduces membrane expression of HCN4 channels in HEK293 cells. Whole-cell recordings from HEK293T cells transfected with hHCN4 in control (black) and after 4-h treatment with the AMPK activator AICAR (1 mM) (red). (A) Representative current traces (left) and activation curves (right). Best fitting with the Boltzmann equation yielded V1/2 = −76.1 ± 1.3 (n = 18) and −75.0 ± 1.6 mV (n = 16) (unpaired t test, P = 0.6105, n.s.); s = 11.8 ± 0.8 and 11.6 ± 0.6 mV, in control and AICAR-treated cells, respectively. (B) Steady-state I/V relations; bar graphs on the left show the distributions of current densities at −130 mV. Mean ± SEM values were −95.3 ± 12.6 (n = 36) and −38.9 ± 5.29 pA/pF (n = 34) in control and AICAR-treated cells, respectively (GLM Gamma, P < 0.0001, ****). Details of statistical analysis are shown in Table S2.

AMPK activation reduces membrane expression of HCN4 channels in HEK293 cell...

in AMPK-mediated HCN4 channel phosphorylation contributes to age-related intrinsic bradycardia > Journal of General Physiology
Published: 06 February 2026
Figure 2. AMPK activation reduces membrane expression of HCN4 channels in HEK293 cells. Whole-cell recordings from HEK293T cells transfected with hHCN4 in control (black) and after 4-h treatment with the AMPK activator AICAR (1 mM) (red). (A) More about this image found in AMPK activation reduces membrane expression of HCN4 channels in HEK293 cell...
Images
Removal of AMPK activation abolishes its action. (A and B) Top: Representative traces of current density recorded from HEK293F cells. Data were obtained from day-matched cells in control conditions or after treatment. (A) Bottom: Mean fully activated I/V curves normalized to capacitance measured under control conditions (black) and after 4-h incubation with either AICAR 1 mM (red) or Compound C 30 µM (blue); normalized conductance values from linear fitting were 1.09, 0.49, and 1.21 nS/pF, respectively. (B) Bottom: Similar set of measurements made under control conditions (black), and after 4-h incubation with either AICAR 1 mM (red) or the combination AICAR 1 mM + Compound C 30 µM (blue); normalized conductance values were 0.98, 0.53, and 0.99 nS/pF, respectively. See Tables 1 and S2 for statistical data analysis.

Removal of AMPK activation abolishes its action. (A and B) Top: Representa...

in AMPK-mediated HCN4 channel phosphorylation contributes to age-related intrinsic bradycardia > Journal of General Physiology
Published: 06 February 2026
Figure 3. Removal of AMPK activation abolishes its action. (A and B) Top: Representative traces of current density recorded from HEK293F cells. Data were obtained from day-matched cells in control conditions or after treatment. (A) Bottom: More about this image found in Removal of AMPK activation abolishes its action. (A and B) Top: Representa...
Images
Involvement of hHCN4 serine 1157 in AMPK-mediated channel modulation. (A and B) In A and B, upper panels are typical current records, and lower panels are fully activated I/V relations, normalized to cell capacitance, from HEK293F cells in control conditions (black) and after AICAR treatment (red). Data from cells transfected with wild-type hHCN4 (left in A and B) are compared with data from cells transfected with S1157A or S1157D mutant channels (right in A and B, respectively). Linear fitting of I/V curves yielded the following normalized conductance values (nS/pF): (A) 1.06, 0.52 (left) and 1.04, 1.01 (right); (B) 1.02, 0.54 (left) and 1.08, 1.05 (right) for wild-type and AICAR-treated cells, respectively. In each experiment, control and mutant channels data are from the same day-matched transfection protocol. Statistical analysis and data comparisons are shown in Tables 1 and S2.

Involvement of hHCN4 serine 1157 in AMPK-mediated channel modulation. (A an...

in AMPK-mediated HCN4 channel phosphorylation contributes to age-related intrinsic bradycardia > Journal of General Physiology
Published: 06 February 2026
Figure 4. Involvement of hHCN4 serine 1157 in AMPK-mediated channel modulation. (A and B) In A and B, upper panels are typical current records, and lower panels are fully activated I/V relations, normalized to cell capacitance, from HEK293F More about this image found in Involvement of hHCN4 serine 1157 in AMPK-mediated channel modulation. (A an...
Images
hHCN4 serine 1158 alone does not directly contribute to AMPK-mediated channel modulation. (A and B) In A and B, upper panels are typical current records, and lower panels are fully activated I/V relations, normalized to cell capacitance, from HEK293F cells in control conditions (black) and after AICAR treatment (red). Data from cells transfected with wild-type hHCN4 (left in A and B) are compared with data from cells transfected with S1158A or S1158D mutant channels (right in A and B, respectively). Linear fitting of I/V curves yielded the following normalized conductance values (nS/pF): (A) 1.08, 0.70 (left) and 1.27, 0.80 (right); (B) 1.10, 0.50 (left) and 1.02, 0.59 (right) for wild-type and AICAR-treated cells, respectively. Statistical analysis and data comparisons are shown in Tables 1 and S2.

hHCN4 serine 1158 alone does not directly contribute to AMPK-mediated chann...

in AMPK-mediated HCN4 channel phosphorylation contributes to age-related intrinsic bradycardia > Journal of General Physiology
Published: 06 February 2026
Figure 5. hHCN4 serine 1158 alone does not directly contribute to AMPK-mediated channel modulation. (A and B) In A and B, upper panels are typical current records, and lower panels are fully activated I/V relations, normalized to cell More about this image found in hHCN4 serine 1158 alone does not directly contribute to AMPK-mediated chann...
Images
Loss of AMPK action on double mutant S1157D/S1158D. Representative current records (top) and fully activated I/V relations (bottom) normalized to cell capacitance, from HEK293F cells in control conditions (black) and after AICAR treatment (red). While cells transfected with wild-type hHCN4 (left) show a standard response to AICAR treatment, cells transfected with S1157D/S1158D double-mutant channels in day-matched experiments (right) have a reduced basal current and are unresponsive to AICAR. Linear fitting of I/V curves yielded the following normalized conductance values (nS/pF): 1.20, 0.63 (left) and 0.68, 0.60 (right) for wild-type and AICAR-treated cells, respectively. Statistical analysis and data comparisons are shown in Tables 1 and S2.

Loss of AMPK action on double mutant S1157D/S1158D. Representative current...

in AMPK-mediated HCN4 channel phosphorylation contributes to age-related intrinsic bradycardia > Journal of General Physiology
Published: 06 February 2026
Figure 6. Loss of AMPK action on double mutant S1157D/S1158D. Representative current records (top) and fully activated I/V relations (bottom) normalized to cell capacitance, from HEK293F cells in control conditions (black) and after AICAR More about this image found in Loss of AMPK action on double mutant S1157D/S1158D. Representative current...
Images
Age dependence of AMPK action of Ifin pacemaker cells.(A–D) Whole-cell recordings of If in 3-mo-old (young) (A and B) and 24-mo-old (old) (C and D) mouse SAN cells in control (black) and after 4-h treatment with AMPK activator (AICAR 1 mM) (red). Data are obtained from N = 3 young and N = 4 old mice. (A and C) Representative current traces (left) and activation curves (right). Best fitting yielded the following values (mV): (A) V1/2 = −89.9 ± 0.81 (n = 21) and −87.8 ± 0.88 (n = 18) (n.s.); s = 8.0 ± 0.4 and 7.4 ± 0.3, for controls and AICAR-treated cells, respectively; (C) V1/2 = −89.5 ± 0.78 (n = 23) and −90.3 ± 0.97 (n = 15) (n.s.); s = 8.0 ± 0.4 and 8.5 ± 0.7 for control and AICAR-treated cells, respectively. (B and D) I/V relations; bar graphs on the left show the distributions of current densities at −125 mV. Mean ± SEM values were (pA/pF) as follows: (B) −42.8 ± 5.34 (n = 21) and −27.9 ± 3.48 (n = 21) (P = 0.0037, **); (D) −25.4 ± 2.91 (n = 23) and −26.5 ± 3.37 (n = 16) (P = 0.9894, n.s.), for control and AICAR-treated cells, respectively. The difference between current densities from old and young mice in control conditions was significant (P = 0.0112, *). Details of statistical analysis are shown in Table S1.

Age dependence of AMPK action of I f in pacemaker cells. (A–D...

in AMPK-mediated HCN4 channel phosphorylation contributes to age-related intrinsic bradycardia > Journal of General Physiology
Published: 06 February 2026
Figure 7. Age dependence of AMPK action of I f in pacemaker cells. (A–D) Whole-cell recordings of If in 3-mo-old (young) (A and B) and 24-mo-old (old) (C and D) mouse SAN cells in control (black) and after 4-h treatment with AMPK activator More about this image found in Age dependence of AMPK action of I f in pacemaker cells. (A–D...
Images
AMPK is constitutively activated in old, but not in young, mice.(A–D) Whole-cell recordings of If in 3-mo-old (young) (A and B) and 24-mo-old (old) (C and D) mouse SAN cells in control (black) and after 4-h treatment with AMPK inhibitor (Compound C 30 µM for 4 h) (blue). Data are obtained from N = 3 young and N = 5 old mice. (A and C) Representative current traces (left) and activation curves (right). Best fitting yielded the following values (mV): (A) V1/2 = −86.2 ± 1.30 (n = 18) and −86.4 ± 1.42 (n = 14) (n.s.); s = 8.3 ± 0.4 and 10.2 ± 0.7; (C) V1/2 = −83.6 ± 1.04 (n = 24) and −82.5 ± 1.19 (n = 17) (n.s.); s = 8.7 ± 0.3 and 9.6 ± 0.4, for control and Compound C–treated cells, respectively. (B and D) I/V relations; bar graphs on the left show the distributions of current densities at −125 mV. Mean ± SE values (pA/pF) were as follows: (B, young) −45.3 ± 6.94 (n = 19) and −44.1 ± 7.24 (n = 16) (P = 0.9986, n.s.); and (D, old) −29.1 ± 3.55 (n = 27) and −47.1 ± 6.37 (n = 20) (P = 0.0110, *), for control and Compound C–treated cells, respectively. Current densities from young mice in control conditions and from old mice after Compound C treatment did not differ significantly (P = 0.9975, n.s.). Details of statistical analysis are shown in Table S1.

AMPK is constitutively activated in old, but not in young, mice. (A–D) Wh...

in AMPK-mediated HCN4 channel phosphorylation contributes to age-related intrinsic bradycardia > Journal of General Physiology
Published: 06 February 2026
Figure 8. AMPK is constitutively activated in old, but not in young, mice. (A–D) Whole-cell recordings of If in 3-mo-old (young) (A and B) and 24-mo-old (old) (C and D) mouse SAN cells in control (black) and after 4-h treatment with AMPK More about this image found in AMPK is constitutively activated in old, but not in young, mice. (A–D) Wh...
Journal Articles

Coincidence detection supported by electrical synapses is shaped by the D-type K+ current

Antonella Dapino, Sebastián Curti
Journal: Journal of General Physiology
Series: Ion Channels in Health and Disease
J Gen Physiol (2026) 158 (2): e202513883.
https://doi.org/10.1085/jgp.202513883
Published: 30 January 2026
View article titled, Coincidence detection supported by electrical synapses is shaped by the D-type K+ current
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Includes: Supplementary data
Images
Characterization of IDsensitivity to 4-AP in MesV neurons. (A) Top: Timeline of experimental procedure in which recorded cells were exposed to increasing extracellular concentrations of 4-AP (0.03, 0.3, 3, 30, and 300 μM) every 10 min starting from the control condition (see Materials and methods). Middle: Representative membrane current recordings in response to a series of 50 ms voltage steps from 0 to 70 mV in steps of +5 mV, starting from a holding potential of −70 mV (bottom) control (first panel from left) and 4-AP (3 μM, second panel from left). To isolate the ID, current recordings in 4-AP were subtracted from those obtained in control conditions (third panel from left). (B) Plot of the maximum ID as a function of membrane voltage from the recordings shown in A. (C) Activation curve of the ID constructed from the data shown in B. Fits to a Boltzmann function (continuous trace) is superimposed on the experimental data (round symbols), and the vertical dashed line indicates the Vhalf value. (D) Representative recordings of the isolated ID obtained employing different concentrations of 4-AP in the same neuron. (E) Magnitude of the ID blocked as a function of the 4-AP concentration for the population of recorded neurons. Superimposed are the individual values (light blue circles) and the corresponding averages (dark blue circles; 0.3 μM: 1.67 ± 1.09 nA [SD], n = 9 cells, N = 5 mice; 3 μM: 4.93 ± 1.64 nA [SD], n = 9 cells, N = 5 mice; 30 μM: 8.88 ± 3.02 nA [SD], n = 13 cells, N = 6 mice; 60 μM: 11.75 ± 2.75 nA [SD], n = 6 cells, N = 3 mice; 100 μM: 9.35 ± 3.19 nA [SD], n = 7 cells, N = 3 mice; 300 μM: 10.34 ± 3.56 nA [SD], n = 7 cells, N = 3 mice). Also superimposed is the fit to a Hill equation. (F) Percentage of ID blockade as a function of 4-AP concentration obtained from data shown in E. Superimposed is the fit to a Hill equation (base: 12.42 ± 10.2%; max: 101.84 ± 11.3%; rate: 1.13 ± 0.83%; xhalf [IC50]: 4.45 ± 2.69 μM). Dashed vertical lines indicate the value of the IC50 (red) and the concentration used to evaluate the contribution of the ID to the excitability, coupling strength, and coincidence detection in the present study (black). (G) Vhalf values of the ID at each 4-AP concentration employed were determined as shown in C. Superimposed are the individual values (light blue circles) and the corresponding averages (dark blue circles; 0.3 μM: −26.05 ± 9.04 mV [SD], n = 9 cells, N = 5 mice; 3 μM: −32.24 ± 5.72 mV [SD], n = 9 cells, N = 5 mice; 30 μM: −33.24 ± 3.18 mV [SD], n = 13 cells, N = 6 mice; 60 μM: −32.06 ± 2.63 mV [SD], n = 6 cells, N = 3 mice; 100 μM: −31.72 ± 2.81 mV [SD], n = 7 cells, N = 3 mice; 300 μM: −30.33 ± 2.95 mV [SD], n = 7 cell, N = 3 mice; P = 0.0512, one-way ANOVA).

Characterization of I D sensitivity to 4-AP in MesV neurons. (...

in Coincidence detection supported by electrical synapses is shaped by the D-type K+ current > Journal of General Physiology
Published: 30 January 2026
Figure 1. Characterization of I D sensitivity to 4-AP in MesV neurons. (A) Top: Timeline of experimental procedure in which recorded cells were exposed to increasing extracellular concentrations of 4-AP (0.03, 0.3, 3, 30, and 300 μM) every 10 More about this image found in Characterization of I D sensitivity to 4-AP in MesV neurons. (...
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IDcontributes to the passive and active electrophysiological properties of MesV neurons. (A) Superimposed membrane voltage responses (above) to depolarizing and hyperpolarizing current pulses (below, Im Cell) from a MesV neuron, in control (left), and after bath application of 10 μM 4-AP (right). Values at the left of membrane voltage traces denote RMP in this and subsequent figures. (B) Plot of the number of spikes evoked by current pulses of 200 ms in duration as a function of the injected current intensity for the same neuron as in A, before (black symbols) and after addition of 4-AP (blue symbols). Each data set was fitted to a linear function (superimposed) whose slope represents the neuron’s excitability (firing gain) in each condition. Coupled cells were pulsed independently (unloaded condition; see text). (C) Firing gain measured as shown in B, for the population of recorded MesV neurons in control (black symbols) and in the presence of 4-AP (10 μM; blue symbols) (control: 3.19 ± 4.67 spikes/nA [SD]; 4-AP: 10.50 ± 15.02 spikes/nA [SD]; n = 57 cells, N = 17 mice; P = 2.27 × 10−5, paired two-tailed t test). (D) RMP measured before (black symbols) and after (blue symbols) addition of 10 μM 4-AP (control: −53.12 ± 3.72 mV [SD]; 4-AP: −50.01 ± 3.13 mV [SD]; n = 45 cells, N = 11 mice; P = 3.85 × 10−9, paired two-tailed t test). (E) Rin before (black symbols) and after addition of 4-AP (10 μM; blue symbols) (control: 87.89 ± 21.79 MΩ [SD]; 4-AP: 108.4 ± 27.80 MΩ [SD]; n = 39 cells, N = 14 mice; P = 3.41 × 10−8, paired two-tailed t test). (F) Superimposed membrane voltage responses of a MesV neuron (above) to a hyperpolarizing current pulse (below, Im Cell), in control (black trace) and after addition of 10 μM 4-AP (blue trace). These responses were fitted to a single exponential function (orange traces) to estimate the membrane time constant (see Materials and methods). (G) Membrane time constant estimated as shown in F for the population of recorded MesV neurons before (black symbols) and after addition of 4-AP (blue symbols) (control: 8.79 ± 4.31 ms [SD], 4-AP: 10.63 ± 5.93 ms [SD]; n = 48 cells, N = 14 mice; P = 0.0159, paired two-tailed t test). Horizontal bars in C, D, E, and G represent population averages. *P < 0.05; ****P < 0.0001.

I D contributes to the passive and active electrophysiological...

in Coincidence detection supported by electrical synapses is shaped by the D-type K+ current > Journal of General Physiology
Published: 30 January 2026
Figure 2. I D contributes to the passive and active electrophysiological properties of MesV neurons. (A) Superimposed membrane voltage responses (above) to depolarizing and hyperpolarizing current pulses (below, Im Cell) from a MesV neuron, in More about this image found in I D contributes to the passive and active electrophysiological...

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