Abnormal features in mutant cerebellar Purkinje cells lacking junctophilins

Junctional membrane complexes (JMCs) generated by junctophilins are required for Ca(2+)-mediated communication between cell-surface and intracellular channels in excitable cells. Knockout mice lacking neural junctophilins (JP-DKO) show severe motor defects and irregular cerebellar plasticity due to abolished channel crosstalk in Purkinje cells (PCs). To precisely understand aberrations in JP-DKO mice, we further analyzed the mutant PCs. During the induction of cerebellar plasticity via electrical stimuli, JP-DKO PCs showed insufficient depolarizing responses. Immunochemistry detected mild impairment in synaptic maturation and hyperphosphorylation of protein kinase Cgamma in JP-DKO PCs. Moreover, gene expression was slightly altered in the JP-DKO cerebellum. Therefore, the mutant PCs bear marginal but widespread abnormalities, all of which likely cause cerebellar motor defects in JP-DKO mice.     

Enhancement of both long-term depression induction and optokinetic response adaptation in mice lacking delphilin

In the cerebellum, Delphilin is expressed selectively in Purkinje cells (PCs) and is localized exclusively at parallel fiber (PF) synapses, where it interacts with glutamate receptor (GluR) delta2 that is essential for long-term depression (LTD), motor learning and cerebellar wiring. Delphilin ablation exerted little effect on the synaptic localization of GluRdelta2. There were no detectable abnormalities in cerebellar histology, PC cytology and PC synapse formation in contrast to GluRdelta2 mutant mice. However, LTD induction was facilitated at PF-PC synapses in Delphilin mutant mice. Intracellular Ca(2+) required for the induction of LTD appeared to be reduced in the mutant mice, while Ca(2+) influx through voltage-gated Ca(2+) channels and metabotropic GluR1-mediated slow synaptic response were similar between wild-type and mutant mice. We further showed that the gain-increase adaptation of the optokinetic response (OKR) was enhanced in the mutant mice. These findings are compatible with the idea that LTD induction at PF-PC synapses is a crucial rate-limiting step in OKR gain-increase adaptation, a simple form of motor learning. As exemplified in this study, enhancing synaptic plasticity at a specific synaptic site of a neural network is a useful approach to understanding the roles of multiple plasticity mechanisms at various cerebellar synapses in motor control and learning.     

Altered cerebellar function in mice lacking CaV2.3 Ca2+ channel

Voltage-dependent Ca(2+) channels play important roles in cerebellar functions including motor coordination and learning. Since abundant expression of Ca(V)2.3 Ca(2+) channel gene in the cerebellum was detected, we searched for possible deficits in the cerebellar functions in the Ca(V)2.3 mutant mice. Behavioral analysis detected in delayed motor learning in rotarod tests in mice heterozygous and homozygous for the Ca(V)2.3 gene disruption (Ca(V)2.3+/- and Ca(V)2.3-/-, respectively). Electrophysiological analysis of mutant mice revealed perplexing results: deficit in long-term depression (LTD) at the parallel fiber Purkinje cell synapse in Ca(V)2.3+/- mice but apparently normal LTD in Ca(V)2.3-/- mice. On the other hand, the number of spikes evoked by current injection in Purkinje cells under the current-clamp mode decreased in Ca(V)2.3 mutant mice in a gene dosage-dependent manner, suggesting that Ca(V)2.3 channel contributed to spike generation in Purkinje cells. Thus, Ca(V)2.3 channel seems to play some roles in cerebellar functions.     

Profound alterations in the intrinsic excitability of cerebellar Purkinje neurons following neurotoxin 3-acetylpyridine (3-AP)-induced ataxia in rat: new insights into the role of small conductance K+ channels

Alterations in the intrinsic properties of Purkinje cells (PCs) may contribute to the abnormal motor performance observed in ataxic rats. To investigate whether such changes in the intrinsic neuronal excitability could be attributed to the role of Ca(2+)-activated K(+) channels (K(Ca)), whole cell current clamp recordings were made from PCs in cerebellar slices of control and ataxic rats. 3-AP induced profound alterations in the intrinsic properties of PCs, as evidenced by a significant increase in both the membrane input resistance and the initial discharge frequency, along with the disruption of the firing regularity. In control PCs, the blockade of small conductance K(Ca) channels by UCL1684 resulted in a significant increase in the membrane input resistance, action potential (AP) half-width, time to peak of the AP and initial discharge frequency. SK channel blockade also significantly decreased the neuronal discharge regularity, the peak amplitude of the AP, the amplitude of the afterhyperpolarization and the spike frequency adaptation ratio. In contrast, in ataxic rats, both the firing regularity and the initial firing frequency were significantly increased by the blockade of SK channels. In conclusion, ataxia may arise from alterations in the functional contribution of SK channels, to the intrinsic properties of PCs.     

Hypotonicity-induced TRPV4 function in renal collecting duct cells: modulation by progressive cross-talk with Ca2+-activated K+ channels

The mouse cortical collecting duct (CCD) M-1 cells were grown to confluency on coverslips to assess the interaction between TRPV4 and Ca(2+)-activated K(+) channels. Immunocytochemistry demonstrated strong expression of TRPV4, along with the CCD marker, aquaporin-2, and the Ca(2+)-activated K(+) channels, the small conductance SK3 (K(Ca)2.3) channel and large conductance BKα channel (K(Ca)1.1). TRPV4 overexpression studies demonstrated little physical dependency of the K(+) channels on TRPV4. However, activation of TRPV4 by hypotonic swelling (or GSK1016790A, a selective agonist) or inhibition by the selective antagonist, HC-067047, demonstrated a strong dependency of SK3 and BK-α activation on TRPV4-mediated Ca(2+) influx. Selective inhibition of BK-α channel (Iberiotoxin) or SK3 channel (apamin), thereby depolarizing the cells, further revealed a significant dependency of TRPV4-mediated Ca(2+) influx on activation of both K(+) channels. It is concluded that a synergistic cross-talk exists between the TRPV4 channel and SK3 and BK-α channels to provide a tight functional regulation between the channel groups. This cross-talk may be progressive in nature where the initial TRPV4-mediated Ca(2+) influx would first activate the highly Ca(2+)-sensitive SK3 channel which, in turn, would lead to enhanced Ca(2+) influx and activation of the less Ca(2+)-sensitive BK channel.     

Impairment of LTD and cerebellar learning by Purkinje cell-specific ablation of cGMP-dependent protein kinase I

The molecular basis for cerebellar plasticity and motor learning remains controversial. Cerebellar Purkinje cells (PCs) contain a high concentration of cGMP-dependent protein kinase type I (cGKI). To investigate the function of cGKI in long-term depression (LTD) and cerebellar learning, we have generated conditional knockout mice lacking cGKI selectively in PCs. These cGKI mutants had a normal cerebellar morphology and intact synaptic calcium signaling, but strongly reduced LTD. Interestingly, no defects in general behavior and motor performance could be detected in the LTD-deficient mice, but the mutants exhibited an impaired adaptation of the vestibulo-ocular reflex (VOR). These results indicate that cGKI in PCs is dispensable for general motor coordination, but that it is required for cerebellar LTD and specific forms of motor learning, namely the adaptation of the VOR.     

Modulation of endothelial SK3 channel activity by Ca²⁺-dependent caveolar trafficking

Small- and intermediate-conductance Ca(2+)-activated K(+) channels (SK3/Kcnn3 and IK1/Kcnn4) are expressed in vascular endothelium. Their activities play important roles in regulating vascular tone through their modulation of intracellular concentration ([Ca(2+)](i)) required for the production of endothelium-derived vasoactive agents. Activation of endothelial IK1 or SK3 channels hyperpolarizes endothelial cell membrane potential, increases Ca(2+) influx, and leads to the release of vasoactive factors, thereby impacting blood pressure. To examine the distinct roles of IK1 and SK3 channels, we used electrophysiological recordings to investigate IK1 and SK3 channel trafficking in acutely dissociated endothelial cells from mouse aorta. The results show that SK3 channels undergo Ca(2+)-dependent cycling between the plasma membrane and intracellular organelles; disrupting Ca(2+)-dependent endothelial caveolae cycling abolishes SK3 channel trafficking. Moreover, transmitter-induced changes in SK3 channel activity and surface expression modulate endothelial membrane potential. In contrast, IK1 channels do not undergo rapid trafficking and their activity remains unchanged when either exo- or endocytosis is block. Thus modulation of SK3 surface expression may play an important role in regulating endothelial membrane potential in a Ca(2+)-dependent manner.     

Control of electrical activity in central neurons by modulating the gating of small conductance Ca2+-activated K+ channels

In most central neurons, action potentials are followed by an afterhyperpolarization (AHP) that controls firing pattern and excitability. The medium and slow components of the AHP have been ascribed to the activation of small conductance Ca(2+)-activated potassium (SK) channels. Cloned SK channels are heteromeric complexes of SK alpha-subunits and calmodulin. The channels are activated by Ca(2+) binding to calmodulin that induces conformational changes resulting in channel opening, and channel deactivation is the reverse process brought about by dissociation of Ca(2+) from calmodulin. Here we show that SK channel gating is effectively modulated by 1-ethyl-2-benzimidazolinone (EBIO). Application of EBIO to cloned SK channels shifts the Ca(2+) concentration-response relation into the lower nanomolar range and slows channel deactivation by almost 10-fold. In hippocampal CA1 neurons, EBIO increased both the medium and slow AHP, strongly reducing electrical activity. Moreover, EBIO suppressed the hyperexcitability induced by low Mg(2+) in cultured cortical neurons. These results underscore the importance of SK channels for shaping the electrical response patterns of central neurons and suggest that modulating SK channel gating is a potent mechanism for controlling excitability in the central nervous system.     

Interaction of SK(Ca) channels and L-type Ca(2+) channels in catecholamine secretion in the rat adrenal gland

We elucidated the interaction of small-conductance Ca(2+)-activated K(+) (SK(Ca)) channels and L-type Ca(2+) channels in muscarinic receptor-mediated control of catecholamine secretion in the isolated perfused rat adrenal gland. The muscarinic agonist methacholine (10-300 microM) produced concentration-dependent increases in adrenal output of epinephrine and norepinephrine. The SK(Ca) channel blocker apamin (1 microM) enhanced the methacholine-induced catecholamine responses. The facilitatory effect of apamin on the methacholine-induced catecholamine responses was not observed during treatment with the L-type Ca(2+) channel blocker nifedipine (3 microM) or Ca(2+)-free solution. Nifedipine did not affect the methacholine-induced catecholamine responses, but it inhibited the responses during treatment with apamin. The L-type Ca(2+) channel activator Bay k 8644 (1 microM) enhanced the methacholine-induced catecholamine responses, whereas the enhancement of the methacholine-induced epinephrine and norepinephrine responses were prevented and attenuated by apamin, respectively. These results suggest that SK(Ca) channels are activated by muscarinic receptor stimulation, which inhibits the opening of L-type Ca(2+) channels and thereby attenuates adrenal catecholamine secretion.     

Interactions of N-terminal and C-terminal parts of the small conductance Ca2+ activated K+ channel, hSK3.

Ca(2+) activated K(+) channels modulate the afterhyperpolarization in neurons. Using a variety of different techniques we obtained information about the function of N- and C-terminal parts of the Ca(2+)-activated K(+) channel, SK3. By means of the yeast two hybrid technique we found an interaction between N-C and N-N- terminal parts of SK3. The strong N-C and N-N interaction was specific for SK3 and could not be observed for SK1 and SK2. Possibly a homotetrameric assembly of SK3 is favored in tissues were all SK channels are expressed. In addition, the interaction in SK3 was independent of the length of the polymorphic glutamine repeat in the N-terminus of SK3. Electrophysiological investigations showed that expression of amino acids 1-299 of SK3 (SK3N_299) modified the 1-EBIO pharmacology of endogenous SK3 channels in PC12 cells without affecting the Ca(2+)-sensitvity. The activation by 0.5 mM 1-EBIO in cells expressing amino acids 1-299 of SK3 was reduced by 32% in comparison to control experiments. Considering the N-C interaction in yeast, we conclude that the sensitivity of SK3 channels to 1-EBIO was modified by N-C interactions with SK3N_299. Therefore we conclude that N-C interactions influence SK3 channel function.     

Protein kinase CK2 is coassembled with small conductance Ca(2+)-activated K+ channels and regulates channel gating

Small conductance Ca(2+)-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. Here we show that, in addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels.     

Specific enhancement of SK channel activity selectively potentiates the afterhyperpolarizing current I(AHP) and modulates the firing properties of hippocampal pyramidal neurons

SK channels are Ca2+-activated K+ channels that underlie after hyperpolarizing (AHP) currents and contribute to the shaping of the firing patterns and regulation of Ca2+ influx in a variety of neurons. The elucidation of SK channel function has recently benefited from the discovery of SK channel enhancers, the prototype of which is 1-EBIO. 1-EBIO exerts profound effects on neuronal excitability but displays a low potency and limited selectivity. This study reports the effects of DCEBIO, an intermediate conductance Ca2+-activated K+ channel modulator, and the effects of the recently identified potent SK channel enhancer NS309 on recombinant SK2 channels, neuronal apamin-sensitive AHP currents, and the excitability of CA1 neurons. NS309 and DCEBIO increased the amplitude and duration of the apamin-sensitive afterhyperpolarizing current without affecting the slow afterhyperpolarizing current in contrast to 1-EBIO. The potentiation by DCEBIO and NS309 was reversed by SK channel blockers. In current clamp experiments, NS309 enhanced the medium afterhyperpolarization (but not the slow afterhyperpolarization sAHP) and profoundly affected excitability by facilitating spike frequency adaptation in a frequency-independent manner. The potent and specific effect of NS309 on the excitability of CA1 pyramidal neurons makes this compound an ideal tool to assess the role of SK channels as possible targets for the treatment of disorders linked to neuronal hyperexcitability.     

A helical region in the C terminus of small-conductance Ca2+-activated K+ channels controls assembly with apo-calmodulin.

Small conductance Ca(2+)-activated potassium (SK) channels underlie the afterhyperpolarization that follows the action potential in many types of central neurons. SK channels are voltage-independent and gated solely by intracellular Ca(2+) in the submicromolar range. This high affinity for Ca(2+) results from Ca(2+)-independent association of the SK alpha-subunit with calmodulin (CaM), a property unique among the large family of potassium channels. Here we report the solution structure of the calmodulin binding domain (CaMBD, residues 396-487 in rat SK2) of SK channels using NMR spectroscopy. The CaMBD exhibits a helical region between residues 423-437, whereas the rest of the molecule lacks stable overall folding. Disruption of the helical domain abolishes constitutive association of CaMBD with Ca(2+)-free CaM, and results in SK channels that are no longer gated by Ca(2+). The results show that the Ca(2+)-independent CaM-CaMBD interaction, which is crucial for channel function, is at least in part determined by a region different in sequence and structure from other CaM-interacting proteins.     

Ca2+ requirements for cerebellar long-term synaptic depression: role for a postsynaptic leaky integrator

Photolysis of a caged Ca(2+) compound was used to characterize the dependence of cerebellar long-term synaptic depression (LTD) on postsynaptic Ca(2+) concentration ([Ca(2+)](i)). Elevating [Ca(2+)](i) was sufficient to induce LTD without requiring any of the other signals produced by synaptic activity. A sigmoidal relationship between [Ca(2+)](i) and LTD indicated a highly cooperative triggering of LTD by Ca(2+). The duration of the rise in [Ca(2+)](i) influenced the apparent Ca(2+) affinity of LTD, and this time-dependent behavior could be described by a leaky integrator process with a time constant of 0.6 s. A computational model, based on a positive-feedback cycle that includes protein kinase C and MAP kinase, was capable of simulating these properties of Ca(2+)-triggered LTD. Disrupting this cycle experimentally also produced the predicted changes in the Ca(2+) dependence of LTD. We conclude that LTD arises from a mechanism that integrates postsynaptic Ca(2+) signals and that this integration may be produced by the positive-feedback cycle.     

Chronic suppression of STIM1-mediated calcium signaling in Purkinje cells rescues the cerebellar pathology in spinocerebellar ataxia type 2

Distorted neuronal calcium signaling has been reported in many neurodegenerative disorders, including different types of spinocerebellar ataxias (SCAs). Cerebellar Purkinje cells (PCs) are primarily affected in SCAs and the disturbances in the calcium homeostasis were observed in SCA PCs. Our previous results have revealed that 3,5-dihydroxyphenylglycine (DHPG) induced greater calcium responses in SCA2-58Q PC cultures than in wild type (WT) PC cultures. Here we observed that glutamate-induced calcium release in PCs cells bodies is significantly higher in SCA2-58Q PCs from acute cerebellar slices compared to WT PCs of the same age. Recent studies have demonstrated that the stromal interaction molecule 1 (STIM1) plays an important role in the regulation of the neuronal calcium signaling in cerebellar PCs in mice. The main function of STIM1 is to regulate store-operated calcium entry through the TRPC/Orai channels formation to refill the calcium stores in the ER when it is empty. Here we demonstrated that the chronic viral-mediated expression of the small interfering RNA (siRNA) targeting STIM1 specifically in cerebellar PCs alleviates the deranged calcium signaling in SCA2-58Q PCs, rescues the spine loss in these cerebellar neurons, and also improves the motor decline in SCA2-58Q mice. Thus, our preliminary results support the important role of the altered neuronal calcium signaling in SCA2 pathology and also suggest the STIM1-mediated signaling pathway as a potential therapeutic target for treatment of SCA2 patients.     

Lack of kinase regulation of canonical transient receptor potential 3 (TRPC3) channel-dependent currents in cerebellar Purkinje cells

Canonical transient receptor potential (TRPC) channels are widely expressed in the brain and play several roles in development and normal neuronal function. In the cerebellum, Purkinje cell TRPC3 channels underlie the slow excitatory postsynaptic potential observed after parallel fiber stimulation. In these cells TRPC3 channel opening requires stimulation of metabotropic glutamate receptor 1, activation of which can also lead to the induction of long term depression (LTD), which underlies cerebellar motor learning. LTD induction requires protein kinase C (PKC) and protein kinase G (PKG) activation, and although PKC phosphorylation targets are well established, virtually nothing is known about PKG targets in LTD. Because TRPC3 channels are inhibited after phosphorylation by PKC and PKG in expression systems, we examined whether native TRPC3 channels in Purkinje cells are a target for PKG or PKC, thereby contributing to cerebellar LTD. We find that in Purkinje cells, activation of TRPC3-dependent currents is not inhibited by conventional PKC or PKG to any significant extent and that inhibition of these kinases does not significantly impact on TRPC3-mediated currents either. Based on these and previous findings, we propose that TRPC3-dependent currents may differ significantly in their regulation from those overexpressed in expression systems.     

Tamapin,a venom peptide from the Indian red scorpion (Mesobuthus tamulus) that targets small conductance Ca2+-activated K+ channels and afterhyperpolarization currents in central neurons

The biophysical properties of small conductance Ca(2+)-activated K(+) (SK) channels are well suited to underlie afterhyperpolarizations (AHPs) shaping the firing patterns of a conspicuous number of central and peripheral neurons. We have identified a new scorpion toxin (tamapin) that binds to SK channels with high affinity and inhibits SK channel-mediated currents in pyramidal neurons of the hippocampus as well as in cell lines expressing distinct SK channel subunits. This toxin distinguished between the SK channels underlying the apamin-sensitive I(AHP) and the Ca(2+)-activated K(+) channels mediating the slow I(AHP) (sI(AHP)) in hippocampal neurons. Compared with related scorpion toxins, tamapin displayed a unique, remarkable selectivity for SK2 versus SK1 ( approximately 1750-fold) and SK3 ( approximately 70-fold) channels and is the most potent SK2 channel blocker characterized so far (IC(50) for SK2 channels = 24 pm). Tamapin will facilitate the characterization of the subunit composition of native SK channels and help determine their involvement in electrical and biochemical signaling.     

Local calcium release in dendritic spines required for long-term synaptic depression

We have used rats and mice with mutations in myosin-Va to evaluate the range and function of IP3-mediated Ca2+ signaling in dendritic spines. In these mutants, the endoplasmic reticulum and its attendant IP3 receptors do not enter the postsynaptic spines of parallel fiber synapses on cerebellar Purkinje cells. Long-term synaptic depression (LTD) is absent at the parallel fiber synapses of the mutants, even though the structure and function of these synapses otherwise appear normal. This loss of LTD is associated with selective changes in IP3-mediated Ca2+ signaling in spines and can be rescued by photolysis of a caged Ca2+ compound. Our results reveal that IP3 must release Ca2+ locally in the dendritic spines to produce LTD and indicate that one function of dendritic spines is to target IP3-mediated Ca2+ release to the proper subcellular domain.