Calcium-Dependent Regulation of Neuronal Excitability Is Rescued in Fragile X Syndrome by a Tat-Conjugated N-Terminal Fragment of FMRP.

Fragile X syndrome (FXS) arises from the loss of fragile X messenger ribonucleoprotein (FMRP) needed for normal neuronal excitability and circuit functions. Recent work revealed that FMRP contributes to mossy fiber long-term potentiation by adjusting the Kv4 A-type current availability through interactions with a Cav3-Kv4 ion channel complex, yet the mechanism has not yet been defined. In this study using wild-type and Fmr1 knock-out (KO) tsA-201 cells and cerebellar sections from male Fmr1 KO mice, we show that FMRP associates with all subunits of the Cav3.1-Kv4.3-KChIP3 complex and is critical to enabling calcium-dependent shifts in Kv4.3 inactivation to modulate the A-type current. Specifically, upon depolarization Cav3 calcium influx activates dual-specific phosphatase 1/6 (DUSP1/6) to deactivate ERK1/2 (ERK) and lower phosphorylation of Kv4.3, a signaling pathway that does not function in Fmr1 KO cells. In Fmr1 KO mouse tissue slices, cerebellar granule cells exhibit a hyperexcitable response to membrane depolarizations. Either incubating Fmr1 KO cells or in vivo administration of a tat-conjugated FMRP N-terminus fragment (FMRP-N-tat) rescued Cav3-Kv4 function and granule cell excitability, with a decrease in the level of DUSP6. Together these data reveal a Cav3-activated DUSP signaling pathway critical to the function of a FMRP-Cav3-Kv4 complex that is misregulated in Fmr1 KO conditions. Moreover, FMRP-N-tat restores function of this complex to rescue calcium-dependent control of neuronal excitability as a potential therapeutic approach to alleviating the symptoms of FXS.

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

CaV1 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 CaV1 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 CaV1.3 channels, the scaffolding protein densin, and CaMKII could generate a facilitation of channel activity that outlasts a depolarizing stimulus. We found that CaV1.3 but not CaV1.2 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 CaV1.3 L-CDF, we coexpressed the intermediate-conductance KCa3.1 potassium channel, revealing a strong functional coupling to CaV1.3 channel activity that was accentuated by densin and CaMKII. Moreover, the CaV1.3-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 CaV1 channels by isradipine, a CaMKII blocker, and siRNA knockdown of densin, and spike frequency increased upon selective block of CaV1 channel conductance. The results are important in revealing a CaV1.3-densin-CaMKII interaction that extends the contribution of CaV1.3 calcium influx to a time frame well beyond a brief input train.SIGNIFICANCE STATEMENT CaV1 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 CaV1.3 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 CaV1.3 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 CaV1.3-densin-CaMKII interaction, identifying an important role for CaV1.3 L-CDF in regulating neuronal excitability.

Intermediate conductance calcium-activated potassium channels modulate summation of parallel fiber input in cerebellar Purkinje cells.

Encoding sensory input requires the expression of postsynaptic ion channels to transform key features of afferent input to an appropriate pattern of spike output. Although Ca(2+)-activated K(+) channels are known to control spike frequency in central neurons, Ca(2+)-activated K(+) channels of intermediate conductance (KCa3.1) are believed to be restricted to peripheral neurons. We now report that cerebellar Purkinje cells express KCa3.1 channels, as evidenced through single-cell RT-PCR, immunocytochemistry, pharmacology, and single-channel recordings. Furthermore, KCa3.1 channels coimmunoprecipitate and interact with low voltage-activated Cav3.2 Ca(2+) channels at the nanodomain level to support a previously undescribed transient voltage- and Ca(2+)-dependent current. As a result, subthreshold parallel fiber excitatory postsynaptic potentials (EPSPs) activate Cav3 Ca(2+) influx to trigger a KCa3.1-mediated regulation of the EPSP and subsequent after-hyperpolarization. The Cav3-KCa3.1 complex provides powerful control over temporal summation of EPSPs, effectively suppressing low frequencies of parallel fiber input. KCa3.1 channels thus contribute to a high-pass filter that allows Purkinje cells to respond preferentially to high-frequency parallel fiber bursts characteristic of sensory input.

Calpastatin domain L is a partial agonist of the calmodulin-binding site for channel activation in Cav1.2 Ca2+ channels.

Cav1.2 Ca(2+) channel activity diminishes in inside-out patches (run-down). Previously, we have found that with ATP, calpastatin domain L (CSL) and calmodulin (CaM) recover channel activity from the run-down in guinea pig cardiac myocytes. Because the potency of the CSL repriming effect was smaller than that of CaM, we hypothesized that CSL might act as a partial agonist of CaM in the channel-repriming effect. To examine this hypothesis, we investigated the effect of the competitions between CSL and CaM on channel activity and on binding in the channel. We found that CSL suppressed the channel-activating effect of CaM in a reversible and concentration-dependent manner. The channel-inactivating effect of CaM seen at high concentrations of CaM, however, did not seem to be affected by CSL. In the GST pull-down assay, CSL suppressed binding of CaM to GST fusion peptides derived from C-terminal regions in a competitive manner. The inhibition of CaM binding by CSL was observed with the IQ peptide but not the PreIQ peptide, which is the CaM-binding domain in the C terminus. The results are consistent with the hypothesis that CSL competes with CaM as a partial agonist for the site in the IQ domain in the C-terminal region of the Cav1.2 channel, which may be involved in activation of the channel.

Both N- and C-lobes of calmodulin are required for Ca2+-dependent regulations of CaV1.2 Ca2+ channels.

We investigated the concentration- and Ca(2+)-dependent effects of CaM mutants, CaM(12) and CaM(34), in which Ca(2+)-binding to its N- and C-lobes was eliminated, respectively, on the Ca(V)1.2 Ca(2+) channel by inside-out patch clamp in guinea-pig cardiomyocytes. Both CaM(12) and CaM(34) (0.7-10muM) applied with 3mM ATP produced channel activity after "rundown". Concentration-response curves were bell-shaped, similar to that for wild-type CaM. However, there was no obvious leftward shift of the curves by increasing [Ca(2+)], suggesting that both functional lobes of CaM were necessary for the Ca(2+)-dependent shift. However, channel activity induced by the CaM mutants showed Ca(2+)-dependent decrease, implying a Ca(2+) sensor existing besides CaM. These results suggest that both N- and C-lobes of CaM are required for the Ca(2+)-dependent regulations of Ca(V)1.2 Ca(2+) channels.

Interactions of calmodulin with the multiple binding sites of Cav1.2 Ca2+ channels.

Although calmodulin binding to various sites of the Cav1.2 Ca(2+) channel has been reported, the mechanism of the interaction is not fully understood. In this study we examined calmodulin binding to fragment channel peptides using a semi-quantitative pull-down assay. Calmodulin bound to the peptides with decreasing affinity order: IQ > preIQ > I-II loop > N-terminal peptide. A peptide containing both preIQ and IQ regions (Leu(1599) - Leu(1668)) bound with approximately 2 mol of calmodulin per peptide. These results support the hypothesis that two molecules of calmodulin can simultaneously bind to the C-terminus of the Cav1.2 channel and modulate its facilitatory and inhibitory activities.

FMRP(1-297)-tat restores ion channel and synaptic function in a model of Fragile X syndrome.

Fragile X Syndrome results from a loss of Fragile X Mental Retardation Protein (FMRP). We now show that FMRP is a member of a Cav3-Kv4 ion channel complex that is known to regulate A-type potassium current in cerebellar granule cells to produce mossy fiber LTP. Mossy fiber LTP is absent in Fmr1 knockout (KO) mice but is restored by FMRP(1-297)-tat peptide. This peptide further rapidly permeates the blood-brain barrier to enter cells across the cerebellar-cortical axis that restores the balance of protein translation for at least 24 h and transiently reduces elevated levels of activity of adult Fmr1 KO mice in the Open Field Test. These data reveal that FMRP(1-297)-tat can improve function from the levels of protein translation to synaptic efficacy and behaviour in a model of Fragile X syndrome, identifying a potential therapeutic strategy for this genetic disorder.

A T-type channel-calmodulin complex triggers αCaMKII activation.

Calmodulin (CaM) is an important signaling molecule that regulates a vast array of cellular functions by activating second messengers involved in cell function and plasticity. Low voltage-activated calcium channels of the Cav3 family have the important role of mediating low threshold calcium influx, but were not believed to interact with CaM. We find a constitutive association between CaM and the Cav3.1 channel at rest that is lost through an activity-dependent and Cav3.1 calcium-dependent CaM dissociation. Moreover, Cav3 calcium influx is sufficient to activate αCaMKII in the cytoplasm in a manner that depends on an intact Cav3.1 C-terminus needed to support the CaM interaction. Our findings thus establish that T-type channel calcium influx invokes a novel dynamic interaction between CaM and Cav3.1 channels to trigger a signaling cascade that leads to αCaMKII activation.

Assessing the role of IKCa channels in generating the sAHP of CA1 hippocampal pyramidal cells.

Our previous work reported that KCa3.1 (IKCa) channels are expressed in CA1 hippocampal pyramidal cells and contribute to the slow afterhyperpolarization that regulates spike accommodation in these cells. The current report presents data from single cell RT-PCR that further reveals mRNA in CA1 cells that corresponds to the sequence of an IKCa channel from transmembrane segments 5 through 6 including the pore region, revealing the established binding sites for 4 different IKCa channel blockers. A comparison of methods to internally apply the IKCa channel blocker TRAM-34 shows that including the drug in an electrode from the onset of an experiment is unviable given the speed of drug action upon gaining access for whole-cell recordings. Together the data firmly establish IKCa channel expression in CA1 neurons and clarify methodological requirements to obtain a block of IKCa channel activity through internal application of TRAM-34.

IKCa channels are a critical determinant of the slow AHP in CA1 pyramidal neurons.

Control over the frequency and pattern of neuronal spike discharge depends on Ca2+-gated K+ channels that reduce cell excitability by hyperpolarizing the membrane potential. The Ca2+-dependent slow afterhyperpolarization (sAHP) is one of the most prominent inhibitory responses in the brain, with sAHP amplitude linked to a host of circuit and behavioral functions, yet the channel that underlies the sAHP has defied identification for decades. Here, we show that intermediate-conductance Ca2+-dependent K+ (IKCa) channels underlie the sAHP generated by trains of synaptic input or postsynaptic stimuli in CA1 hippocampal pyramidal cells. These findings are significant in providing a molecular identity for the sAHP of central neurons that will identify pharmacological tools capable of potentially modifying the several behavioral or disease states associated with the sAHP.