Functional Coupling of Cav2.3 and BK Potassium Channels Regulates Action Potential Repolarization and Short-Term Plasticity in the Mouse Hippocampus

Figure 1. Cav2.3 knockout (KO) CA1 pyramidal cells are hyperexcitable. (A) Resting membrane potential (Vrest; A1) and cell capacitance (A2), measured after break-in. (B) Input resistance quantified as the slope of an IV-curve for hyperpolarizing current steps from −160 to −20 pA. Insets represent averaged voltage traces for wildtype (WT; black) and KO (red) recordings during current injection. (C) Hyperpolarization activated current IH, measured as sag-percentage from maximum voltage deflection (C1) and as the slope of the rebound potential after the end of a series of hyperpolarizing current injections plotted against the steady-state voltage during the current injection (C2). (D) Firing frequency of action potentials (APs) in CA1 pyramidal neurons of Cav2.3 KO and WT animals, elicited by current steps from Vrest, by injection of positive or negative current in steps of 20 pA. Inset shows example recordings for injections of +200 pA WT (black traces) or Cav2.3 KO (red traces). n = 17 for WT, and 21 for KO. All data shown as mean ± SEM, with Student’s T-Test to probe for significance (n.s. = not significant, *p < 0.05, **p < 0.01).

Figure 2. AP waveform properties from CA1 pyramidal neurons. (A) Average first AP elicited by a +150 pA current injection for WT (black traces) and Cav2.3 KO (red traces) ± SEM. (B–G) Quantification of (A). Firing threshold = first derivative of voltage >0.2, and AP onset = time between current step onset and firing threshold. After-hyperpolarization (AHP) is calculated relative to firing threshold. (H) Example traces showing the first six APs elicited by a +200 pA current injection for WT (black traces) and Cav2.3 KO (red traces). (I–N) Quantification of (H). Instead of AP onset, this analysis shows the inter-spike interval (time between peak voltages) and AHP was calculated as the minimum voltage between two APs. n = 17 for WT, and 19 for KO, all data shown as mean ± SEM, with Student’s T-Test to probe for significance and Holm-Sidak to account for multiple comparisons (n.s. = not significant, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3. AP waveform properties from CA1 pyramidal neurons after NiCl2-block of voltage-gated calcium channels. (A) Average first AP elicited by a +150 pA current injection for WT (black traces) and WT + 200 μM NiCl2 (green traces) ± SEM. (B–G) Quantification of (A). Firing threshold = first derivative of voltage >0.5, and AP onset = time between current step onset and firing threshold. AHP is calculated relative to firing threshold (H–K) AP half-width (H), amplitude (I), and fAHP (J) for the first six APs after a current step to +200 pA. (K) Firing frequency of APs in CA1 pyramidal neurons of WT mice (black) and WT treated with 200 μM NiCl2 (green), elicited by current steps from Vrest, by injection of positive or negative current in steps of 20 pA. n = 17 for WT, and 17 for Ni2+, all data shown as mean ± SEM, with Student’s T-Test to probe for significance and Holm-Sidak to account for multiple comparisons (n.s. = not significant, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 4. AP waveform properties from CA1 pyramidal neurons under pharmacological block of various ion channels [100 nM Iberiotoxin (IbTx) to block BK-channels, [100 nM Apamin to block SK-channels, 1 mM 4-Aminopyridine (4-AP) to block voltage-gated potassium channels]. (A) Quantification of the AP half-width. (B) Quantification of the fast after-hyperpolarization. n = 10–17 for all groups. (C,D) Quantification of the half-width (C) and fAHP (D) of the first six APs after a current injection of 200 pA and after application of potassium channel blockers (C1 and D1 Apamin, C2 and D2 IbTx). (E) Firing frequency of current-step elicited APs in CA1 pyramidal neurons after application of potassium channel blockers (E1 Apamin, E2 IbTx). All data shown as mean ± SEM or as min/max boxplot, with one-way ANOVA to probe for significance and Holm-Sidak to account for multiple comparisons (n.s. = not significant, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5. Representative images and quantification of western blot analysis of calcium- and voltage-gated potassium channels. (A) Total expression of BK and SK2 potassium channels in mouse hippocampus showed no significant differences between WT and Cav2.3-KO mice (WT = 8, KO = 6; p > 0.05). In contrast, in the Cav2.3-KO, the total level of Kv4.2 was significantly increased compared to the WT (WT = 8, KO = 6; p < 0.05). Pooled data were normalized to total beta-actin level. Error bars represent SEM. (B) Native co-IP from mouse hippocampus shows that endogenous Cav2.3 interacts with BK-channel. IgG as nonspecific control (*p < 0.05).

Figure 6. Altered synaptic connectivity between CA1 and subiculum. (A–D) Analysis of spontaneous excitatory postsynaptic currents (sEPSCs) recorded from subicular pyramidal neurons in the presence of Gabazine. (A) Representative traces from WT (black traces) and Cav2.3 KO (red traces) and cumulative probability of sEPSC amplitudes for all recorded events (n > 5,000). Insets represent the average traces over all sEPSCs. (B) Quantification of amplitude and frequency for the average event recorded from each cell per genotype (n = 20). (C) Cumulative distribution and quantification for the rise- and decay-times for the average sEPSCs. (D) Quantification for the charge conveyed with each sEPSC (area under the current trace). (E–H) Experimental setup (E) and analysis (F–H) of paired-pulse facilitation between CA1 and subiculum. (F,G) EPSP amplitude and decay time are not significantly different between WT and KO [analyzed from the first elicited EPSP of the 1,000 ms inter-stimulus intervals (ISIs) pulse]. Insets in (H) represent average traces for WT (black) and Cav2.3 KO (red) for two representative ISI. All data shown as mean ± SEM, with student’s T-Test or two-way ANOVA where appropriate, to…