C-termini of P/Q-type Ca2+ channel alpha1A subunits translocate to nuclei and promote polyglutamine-mediated toxicity

P/Q-type voltage-gated calcium channels are regulated, in part, through the cytoplasmic C-terminus of their alpha1A subunit. Genetic absence or alteration of the C-terminus leads to abnormal channel function and neurological disease. Here, we show that the terminal 60-75 kDa of the endogenous alpha1A C-terminus is cleaved from the full-length protein and is present in cell nuclei. Antiserum to the C-terminus (CT-2) labels both wild-type mouse and human Purkinje cell nuclei, but not leaner mouse cerebellum. Human embryonic kidney cells stably expressing beta3 and alpha2delta subunits and transiently transfected with full-length human alpha1A contain a 75 kDa CT-2 reactive peptide in their nuclear fraction. Primary granule cells transfected with C-terminally Green fluorescent protein (GFP)-tagged alpha1A exhibit GFP nuclear labeling. Nuclear translocation depends partly on the presence of three nuclear localization signals within the C-terminus. The C-terminal fragment bears a polyglutamine tract which, when expanded (Q33) as in spinocerebellar ataxia type 6 (SCA6), is toxic to cells. Moreover, polyglutamine-mediated toxicity is dependent on nuclear localization. Finally, in the absence of flanking sequence, the Q33 expansion alone does not kill cells. These results suggest a novel processing of the P/Q-type calcium channel and a potential mechanism for the pathogenesis of SCA6.     

L-Type calcium channels unmasked by cell-permeant Ca2+ buffer at mouse motor nerve terminals

 The involvement of the different types of voltage-dependent calcium channels (VDCC) in both DM-BAPTA-AM-incubated and EGTA-AM-incubated mature mice levator auris neuromuscular junctions (NMJ) was studied. We evaluated the effects of ω-agatoxin IVA (ω-Aga IVA), nitrendipine and ω-conotoxin GVIA (ω-CgTX) (P/Q-, L- and N-type VDCC blockers, respectively) on perineurial calcium currents (I _Ca) and nerve-evoked transmitter release. The application of ω-Aga IVA (100 nM) drastically reduced perineurial I _Ca (>90%) and nerve-evoked transmitter release (>90% of reduction in quantal content, m) at both DM-BAPTA-AM-incubated and EGTA-AM-incubated NMJ. The L-type VDCC antagonist nitrendipine (10 μM) caused a significant reduction (23±9%, n=5) of perineurial I _Ca at DM-BAPTA-AM-incubated NMJ. In addition, after the block of P/Q-type VDCC with ω-Aga IVA (100 nM), nitrendipine reduced (>90%, n=2) the remaining perineurial I _Ca. Such reduction was not observed at EGTA-AM-incubated NMJ, before or after the total block of P/Q-type VDCC. Moreover, nitrendipine did not significantly reduce the quantal content of DM-BAPTA-AM-incubated NMJ. Finally, the application of ω-CgTX (5 μM) did not significantly affect perineurial I _Ca or nerve-evoked transmitter release at either DM-BAPTA-AM-incubated or EGTA-AM-incubated NMJ. These results show the existence of a nitrendipine-sensitive, L-type component of perineurial I _Ca in DM-BAPTA-AM-incubated NMJ of mature mice. Received: 2 September 1998 / Received after revision:13 October 1998 / Accepted:14 October 1998     

Regional expression of L-type calcium channel subunits during cardiac development.

The contraction of cardiomyocytes is initiated by the entrance of extracellular calcium through specific calcium channels. Within the myocardium, L-type calcium channels are most abundant. In the heart, the main pore-forming subunit is the alpha1C, although there is a larger heterogeneity on auxiliary beta subunits. We have analyzed the distribution pattern of different alpha1C and beta subunits during cardiac development by immunohistochemistry. We observed homogeneous expression of alpha1C and beta subunits within the early tubular heart, whereas regional differences are observed during the late embryogenesis. beta2 and beta4 show differential expression within the embryonic myocardium. alpha1CD1 displays only a transient enhanced expression in the ventricular conduction system. In adult heart, the expression of the different calcium channel subunits analyzed is homogeneous along the entire myocardium except for alpha1CD1 that is practically undetectable. These findings suggest that beta subunits might play a major role in conferring calcium handling heterogeneity within the developing embryonic myocardium, while alpha1C subunits might contribute just transiently.     

Differential distribution of Ca2+-activated potassium channel beta4 subunit in rat brain: immunolocalization in neuronal mitochondria

Large conductance Ca(2+)-activated potassium channels (BK(Ca) channels) are expressed in the plasma membrane of various cell types. Interestingly, recent studies provided evidence for the existence of BK(Ca) channels also in mitochondria. However, the molecular composition of these channels as well as their cellular and tissue distribution is still unknown. The goal of the present study was to find a candidate for the regulatory component of the mitochondrial large conductance calcium activated potassium (mitoBK(Ca)) channel in neurons. A combined approach of Western blot analysis, high-resolution immunofluorescence and immunoelectron microscopy with the use of antibodies directed against four distinct beta subunits demonstrated the presence of the BK(Ca) channel beta4 subunit (KCNMB4) in the inner membrane of neuronal mitochondria in the rat brain and cultured neurons. Within the cell, the expression of beta4 subunit was restricted to a subpopulation of mitochondria. The analysis of beta4 subunit distribution throughout the brain revealed that the highest expression levels occur in the thalamus and the brainstem. Our results suggest that beta4 subunit is a regulatory component of mitochondrial BK(Ca) channels in neurons. These findings may support the perspectives for the neuroprotective role of mitochondrial BK(Ca) channel in specific brain structures.     

KCNMB1 regulates surface expression of a voltage and Ca2+-activated K+ channel via endocytic trafficking signals

Voltage-dependent and calcium-activated K(+) (MaxiK, BK) channels are ubiquitously expressed and have various physiological roles including regulation of neurotransmitter release and smooth muscle tone. Coexpression of the pore-forming alpha (hSlo) subunit of MaxiK channels with a regulatory beta1 subunit (KCNMB1) produces noninactivating currents that are distinguished by high voltage/Ca(2+) sensitivities and altered pharmacology [McManus OB, Helms LM, Pallanck L, Ganetzky B, Swanson R, Leonard RJ (1995) Functional role of the beta subunit of high conductance calcium-activated potassium channels. Neuron 14:645-650; Wallner M, Meera P, Ottolia M, Kaczorowski G, Latorre R, Garcia ML, Stefani E, Toro L (1995) Characterization of and modulation by a beta-subunit of a human maxi K(Ca) channel cloned from myometrium. Receptors Channels 3:185-199]. We now show that beta1 can regulate hSlo traffic as well, resulting in decreased hSlo surface expression. beta1 subunit expressed alone is able to reach the plasma membrane; in addition, it exhibits a distinct intracellular punctated pattern that colocalizes with an endosomal marker. Coexpressing beta1 subunit with hSlo, switches hSlo's rather diffuse intracellular expression to a punctate cytoplasmic localization that overlaps beta1 expression. Furthermore, coexpressed beta1 subunit reduces steady-state hSlo surface expression. Site-directed mutagenesis underscores a role of a putative endocytic signal at the beta1 C-terminus in the control of hSlo surface expression. We propose that aside from its well-established role as regulator of hSlo electrical activity, beta1 can regulate hSlo expression levels by means of an endocytic mechanism. This highlights a new beta1 subunit feature that regulates hSlo channels by a trafficking mechanism.     

Human skeletal muscle calcium channel alpha1S is expressed in the basal ganglia: distinctive expression pattern among L-type Ca2+ channels

Voltage-gated calcium channels (VGCCs) are essential molecules for neuronal function. VGCCs consist of five subunits, alpha1, alpha2, beta, gamma, and delta. Among the ten subtypes of the alpha1 subunit (alpha1A-I and S), expression of alpha1S was previously believed to be restricted to the skeletal muscle. We report here, however, that alpha1S is also expressed in human and rat central nervous system. First, we performed PCR screening for VGCC alpha1 subunits in human nervous system using degenerate primers, and identified alpha1S as well as all the eight alpha1 subunits with previously described expression. Intriguingly, alpha1S was selectively localized to the basal ganglia, particularly the caudate nucleus. In situ hybridization showed that alpha1S was expressed in medium-sized caudate neurons. Quantitative analysis using real time RT-PCR revealed a distinct pattern of alpha1S expression among L-type calcium channels. Furthermore, RT-PCR using laser-mediated manipulation of single cells suggested that human alpha1S was coexpressed with ryanodine receptors (RYRs) in GABAergic neurons. Our results suggest the potential relevance of alpha1S to dopaminergic signal transduction and calcium-induced calcium release in caudate neurons.     

A novel molecular inactivation determinant of voltage-gated CaV1.2 L-type Ca2+ channel

The inactivation of voltage-gated L-type Ca(2+) channels (Ca(V)1) regulates Ca(2+) entry and controls intracellular Ca(2+) levels that are essential for cellular activity. The molecular entities implicated in L-channel (Ca(V)1.2) inactivation are not fully identified. Here we show for the first time the functional impact of one of the two highly conserved clusters of six negatively charged glutamates and aspartate (802-807; poly ED motif) at the II-III loop of the alpha 1 subunits of rabbit of Ca(v)1.2, alpha(1)1.2 and alpha(1)1.2 DeltaN60-Delta1733) on voltage-dependent inactivation. Mutation of the poly ED motif to alanine or glutamine/asparagine greatly enhanced voltage-dependent inactivation, shifting the voltage dependence to negative potentials by >50 mV and conferring a neuronal like inactivation kinetics onto Ca(V)1.2. The large shift in the midpoint of inactivation of the steady-state inactivation kinetics was observed also in Ca(2+) or Ba(2+) and was not altered by the beta2A subunit. Missing from the fast inactivating neuronal P/Q (Ca(V)2.1)-, N (Ca(V)2.2)- or R (Ca(V)2.3)-type channels and modulating Ca(V)1.2 inactivation kinetics, the poly ED motif is likely to be a specific L-type Ca(2+) channels inactivating domain. Our results fit a model in which the poly ED either by itself or as part of a larger inactivating motif acts as Ca(V)1.2 specific built-in "stopper." In this model, Ca(V)1 accomplishes a large Ca(2+) influx during depolarization, possibly by the poly ED hindering occlusion at the pore. Furthermore, the selective designed poly ED perhaps clarifies major inactivation differences between L- and non-L-type calcium channels.     

Up-regulation of alpha1D Ca2+ channel subunit mRNA expression in the hippocampus of aged F344 rats

There is growing evidence that alterations in calcium (Ca2+) homeostasis may play a role in processes of brain aging and neurodegeneration. There also is evidence that some of the altered Ca2+ homeostasis in hippocampal neurons may arise from an increased density of L-type voltage sensitive Ca2+ channels (L-VSCC). In the present studies, we tested the possibility that previously observed increases in functional L-VSCC with aging might be related to up-regulated gene/mRNA expression for Ca2+ channel subunits. A significant aging-related increase in mRNA content for the alpha1D subunit of the L-type VSCC was observed in hippocampus of aged F344 rats (25 months old) relative to young (4 months old) and middle-aged animals (13 months old), as assessed by both in situ hybridization analyses (densitometry and grain density) and ribonuclease protection assay (RPA). In RPA analyses, the alpha1C subunit mRNA also showed a significant increase in 25-month-old rats. No age changes were seen in mRNA for the beta1b subunit of VSCC or for GAPDH, a standard control. The clearest increases in alpha1D mRNA expression were observed in subfield CA1, with little or no change seen in dentate gyrus. Although these results alone do not demonstrate that mRNA/gene expression changes contribute directly to changes in functional Ca2+ channels, they clearly fulfill an important prediction of that hypothesis. Therefore, these studies may have important implications for the role of gene expression in aging-dependent alterations in brain Ca2+ homeostasis.     

Roles of alpha(1) and alpha(1)/beta subunits derived from cardiac L-type Ca(2+) channels on voltage-dependent facilitation mechanisms

Strong depolarization pulses facilitate L-type Ca(2+) channels in various cell types including cardiac myocytes. The mechanisms underlying prepulse facilitation are controversial with respect to the requirements for channel subunits, cAMP-dependent protein kinase, and additional anchor proteins. The properties of voltage-dependent facilitation of the L-type Ca(2+) channel was studied in recombinant cardiac alpha(1) subunits with or without cardiac beta subunit, expressed in Chinese hamster fibroblast cells. The magnitude of voltage-dependent I(Ba) facilitation in the alpha(1) subunit channel is dependent on the duration of the prepulse as well as on the interval duration between prepulse and test pulse. The characteristics of this facilitation were not affected by coexpression of the beta subunit. These results indicate that cardiac alpha(1) subunits exhibit voltage-dependent facilitation because of their own intrinsic structure, independent of any other accessory subunit or additional regulatory proteins, and that cardiac beta subunits have no essential regulatory role at the onset or continuance of the voltage-dependent facilitation.     

Expression pattern of voltage-dependent calcium channel subunits in hippocampal inhibitory neurons in mice

Different subtypes of voltage-dependent calcium channels (VDCCs) generate various types of calcium currents that play important role in neurotransmitter release, membrane excitability, calcium transients and gene expression. Well-established differences in the physiological properties and variable sensitivity of hippocampal GABAergic inhibitory neurons to excitotoxic insults suggest that the calcium homeostasis, thus VDCC subunits expression pattern is likely different in subclasses of inhibitory cells. Using double-immunohistochemistry, here we report that in mice: 1) Cav2.1 and Cav3.1 subunits are expressed in almost all inhibitory neurons; 2) subunits responsible for the L-type calcium current (Cav1.2 and Cav1.3) are infrequently co-localized with calretinin inhibitory cell marker while Cav1.3 subunit, at least in part, tends to compensate for the low expression of Cav1.2 subunit in parvalbumin-, metabotropic glutamate receptor 1alpha- and somatostatin-immunopositive inhibitory neurons; 3) Cav2.2 subunit is expressed in the majority of inhibitory neurons except in calbindin-reactive inhibitory cells; 4) Cav2.3 subunit is expressed in the vast majority of the inhibitory cells except in parvalbumin- and calretinin-immunoreactive neurons where the proportion of expression of this subunit is considerably lower. These data indicate that VDCC subunits are differentially expressed in hippocampal GABAergic interneurons, which could explain the diversity in their electrophysiological properties, the existence of synaptic plasticity in certain inhibitory neurons and their vulnerability to stressful stimuli.     

Endocytic trafficking signals in KCNMB2 regulate surface expression of a large conductance voltage and Ca(2+)-activated K+ channel

Large conductance voltage and calcium-activated K(+) channels play critical roles in neuronal excitability and vascular tone. Previously, we showed that coexpression of the transmembrane beta2 subunit, KCNMB2, with the human pore-forming alpha subunit of the large conductance voltage and Ca(2+)-activated K(+) channel (hSlo) yields inactivating currents similar to those observed in hippocampal neurons [Hicks GA, Marrion NV (1998) Ca(2+)-dependent inactivation of large conductance Ca(2+)-activated K(+) (BK) channels in rat hippocampal neurones produced by pore block from an associated particle. J Physiol (Lond) 508 (Pt 3):721-734; Wallner M, Meera P, Toro L (1999b) Molecular basis of fast inactivation in voltage and Ca(2+)-activated K(+) channels: A transmembrane beta-subunit homolog. Proc Natl Acad Sci U S A 96:4137-4142]. Herein, we report that coexpression of beta2 subunit with hSlo can also modulate hSlo surface expression levels in HEK293T cells. We found that, when expressed alone, beta2 subunit appears to reach the plasma membrane but also displays a distinct intracellular punctuated pattern that resembles endosomal compartments. beta2 Subunit coexpression with hSlo causes two biological effects: i) a shift of hSlo's intracellular expression pattern from a relatively diffuse to a distinct punctated cytoplasmic distribution overlapping beta2 expression; and ii) a decrease of hSlo surface expression that surpassed an observed small decrease in total hSlo expression levels. beta2 Site-directed mutagenesis studies revealed two putative endocytic signals at the C-terminus of beta2 that can control expression levels of hSlo. In contrast, a beta2 N-terminal consensus endocytic signal had no effect on hSlo expression levels. Thus, beta2 subunit not only can influence hSlo currents but also has the ability to limit hSlo surface expression levels via an endocytic mechanism. This new mode of beta2 modulation of hSlo may depend on particular coregulatory mechanisms in different cell types.     

N- and P/Q-type Ca2+ channels in adrenal chromaffin cells

Ca2+ is the most ubiquitous second messenger found in all cells. Alterations in [Ca2+]i contribute to a wide variety of cellular responses including neurotransmitter release, muscle contraction, synaptogenesis and gene expression. Voltage-dependent Ca2+ channels, found in all excitable cells (Hille 1992), mediate the entry of Ca2+ into cells following depolarization. Ca2+ channels are composed of a large pore-forming subunit, called the alpha1 subunit, and several accessory subunits. Ten different alpha1 subunit genes have been identified and classified into three families, Ca(v1-3) (Dunlap et al. 1995, Catterall 2000). Each alpha1 gene produces a unique Ca2+ channel. Although chromaffin cells express several different types of Ca2+ channels, this review will focus on the Cav(2.1) and Cav(2.2) channels, also known as P/Q- and N-type respectively (Nowycky et al. 1985, Llinas et al. 1989b, Wheeler et al. 1994). These channels exhibit physiological and pharmacological properties similar to their neuronal counterparts. N-, P/Q and to a lesser extent R-type Ca2+ channels are known to regulate neurotransmitter release (Hirning et al. 1988, Horne & Kemp 1991, Uchitel et al. 1992, Luebke et al. 1993, Takahashi & Momiyama 1993, Turner et al. 1993, Regehr & Mintz 1994, Wheeler et al. 1994, Wu & Saggau 1994, Waterman 1996, Wright & Angus 1996, Reid et al. 1997). N- and P/Q-type Ca2+ channels are abundant in nerve terminals where they colocalize with synaptic vesicles. Similarly, these channels play a role in neurotransmitter release in chromaffin cells (Garcia et al. 2006). N- and P/Q-type channels are subject to many forms of regulation (Ikeda & Dunlap 1999). This review pays particular attention to the regulation of N- and P/Q-type channels by heterotrimeric G-proteins, interaction with SNARE proteins, and channel inactivation in the context of stimulus-secretion coupling in adrenal chromaffin cells.     

Functional interaction of auxiliary subunits and synaptic proteins with Ca(v)1.3 may impart hair cell Ca2+ current properties

We assessed the functional determinants of the properties of L-type Ca(2+) currents in hair cells by co-expressing the pore-forming Ca(V)1.3alpha(1) subunit with the auxiliary subunits beta(1A) and/or alpha(2delta). Because Ca(2+) channels in hair cells are poised to interact with synaptic proteins, we also co-expressed the Ca(V)1.3alpha(1) subunit with syntaxin, vesicle-associated membrane protein (VAMP), and synaptosome associated protein of 25 kDa (SNAP25). Expression of the Ca(V)1.3alpha(1) subunit in human embryonic kidney cells (HEK 293) produced a dihydropyridine (DHP)-sensitive Ca(2+) current (peak current density -2.0 +/- 0.2 pA/pF; n = 11). Co-expression with beta(1A) and alpha(2delta) subunits enhanced the magnitude of the current (peak current density: Ca(V)1.3alpha(1) + beta(1A) = -4.3 +/- 0.8 pA/pF, n = 10; Ca(V)1.3alpha(1) + beta(1A) + alpha(2delta) = -4.1 +/- 0.6 pA/pF, n = 9) and produced a leftward shift of approximately 9 mV in the voltage-dependent activation of the currents. Furthermore, co-expression of Ca(V)1.3alpha(1) with syntaxin/VAMP/SNAP resulted in at least a twofold increase in the peak current density (-4.7 +/- 0.2 pA/pF; n = 11) and reduced the extent of inactivation of the Ca(2+) currents. Botulinum toxin, an inhibitor of syntaxin, accelerated the inactivation profile of Ca(2+) currents in hair cells. Immunocytochemical data also indicated that the Ca(2+) channels and syntaxin are co-localized in hair cells, suggesting there is functional interaction of the Ca(V)1.3alpha(1) with auxiliary subunits and synaptic proteins, that may contribute to the distinct properties of the DHP-sensitive channels in hair cells.     

High-voltage-activated calcium channel messenger RNA expression in the 140-3 neuroblastoma-glioma cell line

Expression of calcium channel alpha1 subunits in oocytes or cell lines has proven to be a powerful method in the analysis of structure-function relations, but these experimental systems are of limited value in the examination of neuron-specific functions such as transmitter release. Cell lines derived from neurons are often capable of these functions, but their intrinsic calcium channel alpha1 subunits are complicating factors in experimental design. We have examined the biophysical and molecular properties of calcium channels in a little studied neuroblastoma-glioma hybrid cell line, 140-3, a close relative of the NG108-15 cell line, to test whether this cell line might serve a role as an expression system for neural mechanisms. This cell was selected as it contains an intact transmitter release mechanism yet secretes little in response to depolarization. Patch-clamp recording revealed only a prominent low-threshold, rapidly inactivating calcium current with a single-channel conductance of approximately 7 pS that was identified as T type. A search for calcium channel alpha1 subunit messenger RNA in the 140-3 cells with three different tests only revealed alpha1C, whereas alpha1A-alpha1C were present in the parent NG108-15 line. We made a particular effort to search for alpha1E, since this subunit has been associated with a low-voltage-activated current. Our findings suggest that, since the principal nerve terminal-associated calcium channels (alpha1A, alpha1B, alpha1E) are absent in the 140-3 cell, this cell line may prove a particularly useful model for the analysis of the role of high-voltage-activated calcium channels in complex functions of neuronal cells.     

Generalized epilepsy with febrile seizures plus-associated sodium channel beta1 subunit mutations severely reduce beta subunit-mediated modulation of sodium channel function

Two novel mutations (R85C and R85H) on the extracellular immunoglobulin-like domain of the sodium channel beta1 subunit have been identified in individuals from two families with generalized epilepsy with febrile seizures plus (GEFS+). The functional consequences of these two mutations were determined by co-expression of the human brain NaV1.2 alpha subunit with wild type or mutant beta1 subunits in human embryonic kidney (HEK)-293T cells. Patch clamp studies confirmed the regulatory role of beta1 in that relative to NaV1.2 alone the NaV1.2+beta1 currents had right-shifted voltage dependence of activation, fast and slow inactivation and reduced use dependence. In addition, the NaV1.2+beta1 current entered fast inactivation slightly faster than NaV1.2 channels alone. The beta1(R85C) subunit appears to be a complete loss of function in that none of the modulating effects of the wild type beta1 were observed when it was co-expressed with NaV1.2. Interestingly, the beta1(R85H) subunit also failed to modulate fast kinetics, however, it shifted the voltage dependence of steady state slow inactivation in the same way as the wild type beta1 subunit. Immunohistochemical studies revealed cell surface expression of the wild type beta1 subunit and undetectable levels of cell surface expression for both mutants. The functional studies suggest association of the beta1(R85H) subunit with the alpha subunit where its influence is limited to modulating steady state slow inactivation. In summary, the mutant beta1 subunits essentially fail to modulate alpha subunits which could increase neuronal excitability and underlie GEFS+ pathogenesis.     

The sodium channel auxiliary subunits beta1 and beta2 are differentially expressed in the spinal cord of neuropathic rats

Neuropathic pain is thought to arise from ectopic discharges at the site of injury within the peripheral nervous system, and is manifest as a general increase in the level of neuronal excitability within primary afferent fibres and their synaptic contacts within the spinal cord. Voltage-activated Na+ channel blockers such as lamotrigine have been shown to be clinically effective in the treatment of neuropathic pain. Na+ channels are structurally diverse comprising a principal a subunit (of which there are variable isoforms) and two auxiliary subunits termed beta1 and beta2. Both beta subunits affect the rates of channel activation and inactivation, and can modify alpha subunit density within the plasma membrane. In addition, these subunits may interact with extracellular matrix molecules to affect growth and myelination of axons. Using in situ hybridization histochemistry we have shown that the expression of the beta1 and beta2 subunits within the dorsal horn of the spinal cord of neuropathic rats is differentially regulated by a chronic constrictive injury to the sciatic nerve. At days 12-15 post-neuropathy, beta1 messenger RNA levels had increased, whereas beta2 messenger RNA levels had decreased significantly within laminae I, II on the ipsilateral side of the cord relative to the contralateral side. Within laminae III-IV beta2 messenger RNA levels showed a small but significant decrease on the ipsilateral side relative to the contralateral side, whilst expression of beta1 messenger RNA remained unchanged. Thus, differential regulation of the individual beta subunit types may (through their distinct influences on Na+ channel function) contribute to altered excitability of central neurons after neuropathic injury.     

Auxiliary Hyperkinetic beta subunit of K+ channels: regulation of firing properties and K+ currents in Drosophila neurons.

Auxiliary Hyperkinetic beta subunit of K+ channels: regulation of firing properties and K+ currents in Drosophila neurons. Molecular analysis and heterologous expression have shown that K+ channel beta subunits regulate the properties of the pore-forming alpha subunits, although how they influence neuronal K+ currents and excitability remains to be explored. We studied cultured Drosophila "giant" neurons derived from mutants of the Hyperkinetic (Hk) gene, which codes for a K+ channel beta subunit. Whole cell patch-clamp recording revealed broadened action potentials and, more strikingly, persistent rhythmic spontaneous activities in a portion of mutant neurons. Voltage-clamp analysis demonstrated extensive alterations in the kinetics and voltage dependence of K+ current activation and inactivation, especially at subthreshold membrane potentials, suggesting a role in regulating the quiescent state of neurons that are capable of tonic firing. Altered sensitivity of Hk currents to classical K+ channel blockers (4-aminopyridine, alpha-dendrotoxin, and TEA) indicated that Hk mutations modify interactions between voltage-activated K+ channels and these pharmacological probes, apparently by changing both the intra- and extracellular regions of the channel pore. Correlation of voltage- and current-clamp data from the same cells indicated that Hk mutations affect not only the persistently active neurons, but also other neuronal categories. Shaker (Sh) mutations, which alter K+ channel alpha subunits, increased neuronal excitability but did not cause the robust spontaneous activity characteristic of some Hk neurons. Significantly, Hk Sh double mutants were indistinguishable from Sh single mutants, implying that the rhythmic Hk firing pattern is conferred by intact Shalpha subunits in a distinct neuronal subpopulation. Our results suggest that alterations in beta subunit regulation, rather than elimination or addition of alpha subunits, may cause striking modifications in the excitability state of neurons, which may be important for complex neuronal function and plasticity.     

Prolongation of hippocampal miniature inhibitory postsynaptic currents in mice lacking the GABA(A) receptor alpha1 subunit

GABA(A) receptors (GABA(A)-Rs) are pentameric structures consisting of two alpha, two beta, and one gamma subunit. The alpha subunit influences agonist efficacy, benzodiazepine pharmacology, and kinetics of activation/deactivation. To investigate the contribution of the alpha1 subunit to native GABA(A)-Rs, we analyzed miniature inhibitory postsynaptic currents (mIPSCs) in CA1 hippocampal pyramidal cells and interneurons from wild-type (WT) and alpha1 subunit knock-out (alpha1 KO) mice. mIPSCs recorded from interneurons and pyramidal cells obtained from alpha1 KO mice were detected less frequently, were smaller in amplitude, and decayed more slowly than mIPSCs recorded in neurons from WT mice. The effect of zolpidem was examined in view of its reported selectivity for receptors containing the alpha1 subunit. In interneurons and pyramidal cells from WT mice, zolpidem significantly increased mIPSC frequency, prolonged mIPSC decay, and increased mIPSC amplitude; those effects were diminished or absent in neurons from alpha1 KO mice. Nonstationary fluctuation analysis of mIPSCs indicated that the zolpidem-induced increase in mIPSC amplitude was associated with an increase in the number of open receptors rather than a change in the unitary conductance of individual channels. These data indicate that the alpha1 subunit is present at synapses on WT interneurons and pyramidal cells, although differences in mIPSC decay times and zolpidem sensitivity suggest that the degree to which the alpha1 subunit is functionally expressed at synapses on CA1 interneurons may be greater than that at synapses on CA1 pyramidal cells.