Activity-Dependent Facilitation of CaV1.3 Calcium Channels Promotes KCa3.1 Activation in Hippocampal Neurons

Abstract

Ca_V1 L-type calcium channels are key to regulating neuronal excitability, with the range of functional roles enhanced by interactions with calmodulin, accessory proteins, or CaMKII that modulate channel activity. In hippocampal pyramidal cells, a prominent elevation of Ca_V1 activity is apparent in late channel openings that can last for seconds following a depolarizing stimulus train. The current study tested the hypothesis that a reported interaction among Ca_V1.3 channels, the scaffolding protein densin, and CaMKII could generate a facilitation of channel activity that outlasts a depolarizing stimulus. We found that Ca_V1.3 but not Ca_V1.2 channels exhibit a long-duration calcium-dependent facilitation (L-CDF) that lasts up to 8 s following a brief 50 Hz stimulus train, but only when coexpressed with densin and CaMKII. To test the physiological role for Ca_V1.3 L-CDF, we coexpressed the intermediate-conductance KCa3.1 potassium channel, revealing a strong functional coupling to Ca_V1.3 channel activity that was accentuated by densin and CaMKII. Moreover, the Ca_V1.3-densin-CaMKII interaction gave rise to an outward tail current of up to 8 s duration following a depolarizing stimulus in both tsA-201 cells and male rat CA1 pyramidal cells. A slow afterhyperpolarization in pyramidal cells was reduced by a selective block of Ca_V1 channels by isradipine, a CaMKII blocker, and siRNA knockdown of densin, and spike frequency increased upon selective block of Ca_V1 channel conductance. The results are important in revealing a Ca_V1.3-densin-CaMKII interaction that extends the contribution of Ca_V1.3 calcium influx to a time frame well beyond a brief input train.SIGNIFICANCE STATEMENT Ca_V1 L-type calcium channels play a key role in regulating the output of central neurons by providing calcium influx during repetitive inputs. This study identifies a long-duration calcium-dependent facilitation (L-CDF) of Ca_V1.3 channels that depends on the scaffolding protein densin and CaMKII and that outlasts a depolarizing stimulus by seconds. We further show a tight functional coupling between Ca_V1.3 calcium influx and the intermediate-conductance KCa3.1 potassium channel that promotes an outward tail current of up to 8 s following a depolarizing stimulus. Tests in CA1 hippocampal pyramidal cells reveal that a slow AHP is reduced by blocking different components of the Ca_V1.3-densin-CaMKII interaction, identifying an important role for Ca_V1.3 L-CDF in regulating neuronal excitability.

Keywords: CDF; CaV1.2; CaV1.3; KCa3.1; densin; facilitation.

Figure 1.

A Ca_V1.3, densin, and CaMKII interaction generates a prolonged facilitation of calcium current. A, Schematic diagram of step protocols to test Ca_V1 channel response to a 10 pulse 50 Hz train of depolarizing stimuli in tsA-201 cells in B–D. B–D, Whole-cell recordings of Ca_V1.3 current evoked by a step to 0 mV before (black trace) and after (red traces) a 10 pulse 50 Hz train of steps to 40 mV from a holding potential of −70 mV. The first test response was evoked at a delay of 20 ms and all subsequent records at 500 ms increments. B, C, Ca_V1.3 expressed alone exhibits no facilitation following a stimulus train (B), but a pronounced late facilitation after the stimulus train when coexpressed with densin and CaMKII (C). D, Cotransfecting densin–siRNA-2 blocks facilitation after the stimulus train (D). E, No late facilitation is detected in cells expressing Ca_V1.3, a short isoform Ca_V1.3s, or Ca_V1.3 with either CaMKII or densin. F, Ca_V1.3 expressed in combination with densin and CaMKII exhibits a pronounced late facilitation that lasts up to 8 s following the stimulus train. Facilitation is reduced upon coexpressing a mutant CaMKII construct that cannot undergo autophosphorylation (CaMKII-T286A) or coexpressing either of two densin siRNAs. G, Recordings of Ca_V1.3 or Ca_V1.3 coexpressed with densin and CaMKII in the presence of 2 mm Ba²⁺ indicate a brief voltage-dependent facilitation at 20 ms following a stimulus train but no late facilitation, identifying the CA_V1.3–densin–CaMKII interactions in F as an extended form of CDF. H, No late facilitation is detected in cells expressing Ca_V1.2 or Ca_V1.2 in combination with CaMKII or densin and CaMKII. I, Whole-cell calcium current in rat CA1 pyramidal cells recorded in the presence of blockers against all Ca_V channels except L-type (1 μm ω-conotoxin, 200 nm ω-agatoxin, 200 nm SNX-482, and 1 μm TTA-P2). Superimposed traces show L-type current evoked by a 100 ms ramp command tested with or without a preceding 50 Hz stimulus train of square wave steps (200 ms) to 60 mV at a delay of 2.5 s. Bar plots show the mean facilitation of calcium current evoked by a ramp command in the presence or absence of a preceding 50 Hz stimulus train square wave steps (200 ms) to either 40 or 60 mV. Facilitation is expressed as a percentage of the calcium current recorded in the absence of the 50 Hz train (100%, Cont.; p < 0.05, one-way ANOVA followed by post hoc Student's paired t test). Cell numbers are shown in brackets. *p < 0.05; **p < 0.01.

Figure 2.

L-type calcium and KCa3.1 potassium channels functionally couple. A, B, Whole-cell currents of Ca_V1.2 (A) and Ca_V1.3 (B) calcium channels expressed in tsA-201 cells exhibit pronounced calcium-dependent inactivation. Ca_V1 channels were coexpressed with α2δ1 and β1b accessory subunits. Coexpression of KCa3.1 channels with Ca_V1.2 (A) or Ca_V1.3 (B) result in an inward Ca²⁺ current followed by KCa3.1-mediated potassium current. C, The current–voltage plots for KCa3.1 channels closely follow the voltage dependency of corresponding Ca_V1 calcium channel isoforms. Outward currents were evoked from a holding potential of −70 mV and measured at 1 s. D, E, Representative traces of KCa3.1 currents blocked by internal perfusion of 1 μm TRAM-34 (D) or 5 mm EGTA (E) over a 5–7 min time period. Numbers of cells are denoted in brackets in C.

Figure 3.

Ca_V1.3 does not associate with KCa3.1 at a level that supports FRET. Fluorescence confocal images of tsA-201 cells expressing constructs of KCa3.1-GFP (donor molecule) and Ca_V1.3-mKate (acceptor molecule) excited with a laser at 457 or 561 nm, respectively. Plots show the excitation line (vertical line) and the associated average emission spectra for each condition obtained from 20–30 cells from three to five independent experiments. A, B, Excitation at 457 nm of cells expressing KCa3.1-GFP and Ca_V1.3-mKate results in only a GFP emission spectra, indicating no FRET. C, In comparison, the expression of KCa3.1-GFP and mKate-CaM as a positive control revealed FRET, as indicated by an emission spectra for mKate (peaks, ∼630 nm; Fig. 3C) when excited at 457 nm. D, Excitation of cells expressing KCa3.1-GFP and mKate-CaM at 561 nm returns only mKate spectra. Scale bars, 20 μm.

Figure 4.

Ca_V1.3 channels evoke a long outward tail current when coexpressed with KCa3.1. A, A long outward tail current is preferentially evoked in tsA-201 cells coexpressing Ca_V1.3 and KCa3.1 channels. Inset of a magnified view of tail currents reveals a graded activation of the tail current in direct relation to the duration of a step command. However, no tail currents are detected in cells expressing KCa3.1 in isolation (bottom records). B, Coexpression of Ca_V1.3 and KCa3.1 along with densin and CaMKII reveals larger outward tail currents of up to 6 s in response to a 100 ms depolarizing step compared with cells expressing Ca_V1.3 and KCa3.1 alone. Larger tail currents are not observed in cells expressing the autophosphorylation mutant CaMKII-T286A along with densin, Ca_V1.3, and KCa3.1. C, Outward tail currents recorded in Ca_V1.2- and KCa3.1-expressing cells are comparatively small in amplitude and are not augmented by the coexpression of densin and CaMKII. D, Bar plots of the mean area and duration of tail current activated by a 100 ms step. All values are normalized to the preceding inward peak calcium current. Ca_V1.3 channels are more effective than Ca_V1.2 in generating an outward tail current that is further augmented by densin and autophosphorylated CaMKII. A one-way ANOVA followed by post hoc unpaired Student's t test was performed between the Ca_V1.3 groups for area (p < 0.05) and duration (p < 0.05) and an unpaired Student's t test between Ca_V1.2 groups (NS). NS, Not significant. *p < 0.05; **p < 0.01. Cell numbers are shown in brackets in D.

Figure 5.

L-type channels play a key role in evoking the CA1 pyramidal cell IsAHP. A–C, The effects of L-type calcium channel blockers on KCa3.1 whole-cell current recorded in tsA-201 cells with 1 μm calcium in the electrode and evoked by a ramp command as shown in B. Bar plots show a differential block of KCa3.1 current by L-type blockers (A). Representative recordings of KCa3.1 current in B and C illustrate a complete block by 1 μm TRAM-34 but no effect of 500 nm isradipine, a dose sufficient to provide substantial block of whole-cell L-type calcium current in tsA-201 cells expressing either Ca_V1.2 or Ca_V1.3 cDNA or in CA1 pyramidal cells (C). D, E, The IsAHP in rat CA1 hippocampal pyramidal cells evoked by a depolarizing step command of 200 ms to 40 mV. The IsAHP is only slightly reduced by a medium containing blockers against all Ca_V channels except L-type (1 μm ω-conotoxin MVIIC, 200 nm ω-agatoxin IVA, 200 nm SNX-482, 1 μm TTA-P2). The remaining presumed L-type channel-activated IsAHP is significantly reduced by 1 μm TRAM-34. Recordings in the bottom part of D are from a separate cell in which 500 nm isradipine was applied in the presence of the non-L-type Ca_V channel blockers, revealing a significant reduction of IsAHP. *p < 0.05; **p < 0.01; ***p < 0.001. NS, Not significant. Cell numbers are shown in brackets in A, C, and E.

Figure 6.

L-type channels control hippocampal CA1 cell excitability by modulating spike accommodation and the sAHP. A, B, Current-evoked spike discharge in rat CA1 hippocampal pyramidal cells in the presence of 100 nm apamin and 10 μm XE-991 to block SK and K_V7 potassium channels, respectively. Bath application of 500 nm isradipine reduced the interspike interval and increased the total spike frequency and number. C, Whole-cell recordings of sAHP in CA1 pyramidal cells evoked by a suprathreshold 5 pulse, 50 Hz stimulus train to stratum radiatum inputs indicates that the sAHP is reduced by 500 nm israpidine. Inset shows in another cell that isradipine does not block the smaller sAHP that follows a subthreshold train of synaptic inputs. D, Bar plots of the mean values of sAHP indicate a significant reduction of sAHP by 500 nm isradipine when the sAHP is evoked by suprathreshold but not subthreshold stratum radiatum inputs (C). *p < 0.05, Wilcoxon signed rank test. Cell numbers are shown in brackets in B and D.

Figure 7.

The IsAHP in hippocampal neurons is regulated by densin and CaMKII. A, Whole-cell recordings of IsAHP evoked by a series of brief pulses to 40 mV in dissociated hippocampal pyramidal neurons in the presence of 100 nm apamin and 10 μm XE-991 to block SK and Kv7 potassium channels, respectively. Pulse trains consisted of 10 5 ms pulses at 50 Hz. Inset shows an enlarged view of the early component of the IsAHP recorded under the indicated conditions. B, The area of the IsAHP is significantly reduced in cells pretreated with either of two siRNAs directed against the postsynaptic scaffolding protein densin compared with nontransfected cells (control) or with cells transfected with a universal control siRNA (one-way ANOVA; p < 0.001). C, Representative Western blot of protein levels of densin or the loading control α-tubulin prepared from lysates of tsA-201 cells expressing densin-GFP with either of two forms of densin siRNA. Proteins were detected using antibodies against GFP or α-tubulin and the density was quantified (using ImageJ) with mean percentages relative to cells expressing only densin-GFP shown above each lane (n = 3; one-way ANOVA, p < 0.001). The levels of densin are greatly reduced in tsA-201 cells cotransfected with either siRNA-1 or siRNA-2 compared with controls. D, Percentage reduction of densin mRNA levels in cultured hippocampal neurons transfected with either of two densin siRNAs compared with cells transfected with control siRNA. The C_t values of densin mRNA from each group are normalized with the C_t value of β-actin as the internal control. Both siRNAs significantly reduced densin mRNA levels in hippocampal neurons compared with neurons transfected with control siRNA (n = 3; one-way ANOVA, p < 0.01). E, Whole-cell recordings of IsAHP in rat CA1 pyramidal cells evoked in a medium containing blockers against all Ca_V channels except L-type (1 μm ω-conotoxin MVIIC, 200 nm ω-agatoxin IVA, 200 nm SNX-482, and 1 μm TTA-P2). The remaining presumed L-type channel-activated IsAHP is reduced by the CaMKII inhibitors KN-62 and AIP. KN-62 (10 μm) was bath applied, and AIP (20 μm) was internally infused through the electrode. F, Bar plots of mean values for IsAHP area in CA1 pyramidal cells indicate a significant reduction upon the application of CaMKII inhibitors KN-62 or AIP relative to traces obtained before the drug application (as in E; paired Student's t test). *p < 0.05; **p < 0.01; ***p < 0.001. NS, Not significant. Cell numbers are shown in brackets in B and F.