Pratap Meera

A calcium switch for the functional coupling between α (hslo) and β subunits (KV, Caβ) of maxi K channels

K V , Ca β subunit dramatically increases the apparent calcium sensitivity of the α subunit of MaxlK channels when probed in the micromolar [Ca2+]i range. Analysis in a wide range of [Ca2+]i revealed that this functional coupling is exquisitely modulated by [Ca2+]i. Ca2+ ions switch MaxiK α+β complex into a functionally coupled state at concentrations beyond resting [Ca2+]i. At [Ca2+] ≤ 100 nM, MaxiK activity becomes independent of Ca2+, is purely voltage‐activated, and its functional coupling with its β subunit is released. The functional switch develops at [Ca2+]i that occur during cellular excitation, providing the molecular basis of how MaxiK channels regulate smooth muscle excitability and neurotransmitter release.

Molecular constituents of maxi KCa channels in human coronary smooth muscle: predominant α+β subunit complexes

1 Human large‐conductance voltage‐ and calcium‐sensitive K+ (maxi KCa) channels are composed of at least two subunits: the pore‐forming subunit, α, and a modulatory subunit, β. Expression of the β subunit induces dramatic changes in α subunit function. It increases the apparent Ca2+ sensitivity and it allows dehydrosoyasaponin I (DHS‐I) to upregulate the channel. 2 The functional coupling of maxi KCa channel α and β subunits in freshly dissociated human coronary smooth muscle cells was assessed. To distinguish maxi KCa currents modulated by the β subunit, we examined (a) their apparent Ca2+ sensitivity, as judged from the voltage necessary to half‐activate the channel (V1/2), and (b) their activation by DHS‐I. 3 In patches with unitary currents, the majority of channels were half‐activated near –85 mV at 18 μm Ca2+, a value similar to that obtained when the human KCa channel α (HSLO) and β (HKVCaβ) subunits are co‐expressed. A small number of channels half‐activated around 0 mV, suggesting the activity of the α subunit alone. 4 The properties of macroscopic currents were consistent with the view that most pore‐forming α subunits were coupled to β subunits, since the majority of currents had values for V1/2 near to –90 mV, and currents were potentiated by DHS‐I. 5 We conclude that in human coronary artery smooth muscle cells, most maxi KCa channels are composed of α and β subunits. The higher Ca2+ sensitivity of maxi KCa channels, resulting from their coupling to β subunits, suggests an important role of this channel in regulating coronary tone. Their massive activation by micromolar Ca2+ concentrations may lead to a large hyperpolarization causing profound changes in coronary blood flow and cardiac function.

Molecular basis for the high THIP/gaboxadol sensitivity of extrasynaptic GABAA receptors

Extrasynaptic GABA(A) receptors (eGABARs) allow ambient GABA to tonically regulate neuronal excitability and are implicated as targets for ethanol and anesthetics. These receptors are thought to be heteropentameric proteins made up of two α subunits-either α4 or α6-two β2 or β3 subunits, and one δ subunit. The GABA analog 4,5,6,7-tetrahydroisoxazolo (5,4-c)pyridin-3(-ol) (THIP) has been proposed as a selective ligand for eGABARs. Behavioral and in vitro studies suggest that eGABARs have nanomolar affinity for THIP; however, all published studies on recombinant versions of eGABARs report micromolar affinities. Here, we examine THIP sensitivity of native eGABARs on cerebellar neurons and on reconstituted GABARs in heterologous systems. Concentration-response data for THIP, obtained from cerebellar granule cells and molecular layer interneurons in wild-type and δ subunit knockout slices, confirm that submicromolar THIP sensitivity requires δ subunits. In recombinant experiments, we find that δ subunit coexpression leads to receptors activated by nanomolar THIP concentrations (EC(50) of 30-50 nM for α4β3δ and α6β3δ), a sensitivity almost 1,000-fold higher than receptors formed by α4/6 and β3 subunits. In contrast, γ2 subunit expression significantly reduces THIP sensitivity. Even when δ subunit cDNA or cRNA was supplied in excess, high- and low-sensitivity THIP responses were often apparent, indicative of variable mixtures of low-affinity αβ and high-affinity αβδ receptors. We conclude that δ subunit incorporation into GABARs leads to a dramatic increase in THIP sensitivity, a defining feature that accounts for the unique behavioral and neurophysiological properties of THIP.

The splicing regulator Rbfox2 is required for both cerebellar development and mature motor function

The Rbfox proteins (Rbfox1, Rbfox2, and Rbfox3) regulate the alternative splicing of many important neuronal transcripts and have been implicated in a variety of neurological disorders. However, their roles in brain development and function are not well understood, in part due to redundancy in their activities. Here we show that, unlike Rbfox1 deletion, the CNS-specific deletion of Rbfox2 disrupts cerebellar development. Genome-wide analysis of Rbfox2(-/-) brain RNA identifies numerous splicing changes altering proteins important both for brain development and mature neuronal function. To separate developmental defects from alterations in the physiology of mature cells, Rbfox1 and Rbfox2 were deleted from mature Purkinje cells, resulting in highly irregular firing. Notably, the Scn8a mRNA encoding the Na(v)1.6 sodium channel, a key mediator of Purkinje cell pacemaking, is improperly spliced in RbFox2 and Rbfox1 mutant brains, leading to highly reduced protein expression. Thus, Rbfox2 protein controls a post-transcriptional program required for proper brain development. Rbfox2 is subsequently required with Rbfox1 to maintain mature neuronal physiology, specifically Purkinje cell pacemaking, through their shared control of sodium channel transcript splicing.

Changes in Purkinje cell firing and gene expression precede behavioral pathology in a mouse model of SCA2

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominantly inherited disorder, which is caused by a pathological expansion of a polyglutamine (polyQ) tract in the coding region of the ATXN2 gene. Like other ataxias, SCA2 most overtly affects Purkinje cells (PCs) in the cerebellum. Using a transgenic mouse model expressing a full-length ATXN2Q127-complementary DNA under control of the Pcp2 promoter (a PC-specific promoter), we examined the time course of behavioral, morphologic, biochemical and physiological changes with particular attention to PC firing in the cerebellar slice. Although motor performance began to deteriorate at 8 weeks of age, reductions in PC number were not seen until after 12 weeks. Decreases in the PC firing frequency first showed at 6 weeks and paralleled deterioration of motor performance with progression of disease. Transcription changes in several PC-specific genes such as Calb1 and Pcp2 mirrored the time course of changes in PC physiology with calbindin-28 K changes showing the first small, but significant decreases at 4 weeks. These results emphasize that in this model of SCA2, physiological and behavioral phenotypes precede morphological changes by several weeks and provide a rationale for future studies examining the effects of restoration of firing frequency on motor function and prevention of future loss of PCs.

Antisense oligonucleotide therapy for spinocerebellar ataxia type 2

There are no disease-modifying treatments for adult human neurodegenerative diseases. Here we test RNA-targeted therapies in two mouse models of spinocerebellar ataxia type 2 (SCA2), an autosomal dominant polyglutamine disease. Both models recreate the progressive adult-onset dysfunction and degeneration of a neuronal network that are seen in patients, including decreased firing frequency of cerebellar Purkinje cells and a decline in motor function. We developed a potential therapy directed at the ATXN2 gene by screening 152 antisense oligonucleotides (ASOs). The most promising oligonucleotide, ASO7, downregulated ATXN2 mRNA and protein, which resulted in delayed onset of the SCA2 phenotype. After delivery by intracerebroventricular injection to ATXN2-Q127 mice, ASO7 localized to Purkinje cells, reduced cerebellar ATXN2 expression below 75% for more than 10 weeks without microglial activation, and reduced the levels of cerebellar ATXN2. Treatment of symptomatic mice with ASO7 improved motor function compared to saline-treated mice. ASO7 had a similar effect in the BAC-Q72 SCA2 mouse model, and in both mouse models it normalized protein levels of several SCA2-related proteins expressed in Purkinje cells, including Rgs8, Pcp2, Pcp4, Homer3, Cep76 and Fam107b. Notably, the firing frequency of Purkinje cells returned to normal even when treatment was initiated more than 12 weeks after the onset of the motor phenotype in BAC-Q72 mice. These findings support ASOs as a promising approach for treating some human neurodegenerative diseases.

Modulation of the skeletal muscle sodium channel α-subunit by the β1 -subunit

Co‐expression of cloned sodium channel β1 ‐subunit with the rat skeletal muscle‐subunit (αμI) accelerated the macroscopic current decay, enhanced the current amplitude, shifted the steady state inactivation curve to more negative potentials and decreased the time required for complete recovery from inactivation. Sodium channels expressed from skeletal muscle mRNA showed a similar behaviour to that observed from , indicating that β1 restores ‘physiological’ behaviour. Northern blot analysis revealed that the Na+ channel β1‐subunit is present in high abundance (about 0.1%) in rat heart, brain and skeletal muscle, and the hybridization with untranslated region of the ‘brain’ β1 cDNA to skeletal muscle and heart mRNA indicated that the diffferent Na + channel α‐subunits in brain, skeletal muscle and heart may share a common β1 ‐subunit.

Alcohol- and Alcohol Antagonist-Sensitive Human GABAA Receptors: Tracking δ Subunit Incorporation into Functional Receptors

GABAA receptors (GABAARs) have long been a focus as targets for alcohol actions. Recent work suggests that tonic GABAergic inhibition mediated by extrasynaptic δ subunit-containing GABAARs is uniquely sensitive to ethanol and enhanced at concentrations relevant for human alcohol consumption. Ethanol enhancement of recombinant α4β3δ receptors is blocked by the behavioral alcohol antagonist 8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester (Ro15-4513), suggesting that EtOH/Ro15-4513-sensitive receptors mediate important behavioral alcohol actions. Here we confirm alcohol/alcohol antagonist sensitivity of α4β3δ receptors using human clones expressed in a human cell line and test the hypothesis that discrepant findings concerning the high alcohol sensitivity of these receptors are due to difficulties incorporating δ subunits into functional receptors. To track δ subunit incorporation, we used a functional tag, a single amino acid change (H68A) in a benzodiazepine binding residue in which a histidine in the δ subunit is replaced by an alanine residue found at the homologous position in γ subunits. We demonstrate that the δH68A substitution confers diazepam sensitivity to otherwise diazepam-insensitive α4β3δ receptors. The extent of enhancement of α4β3δH68A receptors by 1 μM diazepam, 30 mM EtOH, and 1 μM β-carboline-3-carboxy ethyl ester (but not 1 μM Zn2+ block) is correlated in individual recordings, suggesting that δ subunit incorporation into recombinant GABAARs varies from cell to cell and that this variation accounts for the variable pharmacological profile. These data are consistent with the notion that δ subunit-incorporation is often incomplete in recombinant systems yet is necessary for high ethanol sensitivity, one of the features of native δ subunit-containing GABAARs.

Chapter 8 Calcium-Activated Potassium Channels in Muscle and Brain

Publisher Summary This chapter discusses the characterization of the two subfamilies that can account for the variety of Ca 2+- sensitive K + channels: large conductance (BK) and small conductance (SK) channels. BK channels are characterized by their high single-channel conductance, their dual response to voltage and Ca 2+ , and their blockade by nanomolar concentrations of iberiotoxin and micromolar concentrations of TEA. Heterologously expressed α-subunit cDNA clones mirror these features. Structurally, they are characterized by unique N- and C-terminal sequences, implicated in β-subunit and Ca 2+ regulation, appended to a six-transmembrane structure typical of voltage-gated ion channels. SK channels are characterized by their small conductance, their voltage independence, and their Ca 2+ -dependent activation. A family of this class of K + channels has been recently cloned. BK and different types of SK channels are often coexpressed within the same native cell and seem to be coupled to specific Ca 2+ sources. Specific Ca 2+ channel blockers can abolish certain types of calcium-activated K channels without affecting others.

Cellular and circuit mechanisms underlying spinocerebellar ataxias

Degenerative ataxias are a common form of neurodegenerative disease that affect about 20 individuals per 100,000. The autosomal dominant spinocerebellar ataxias (SCAs) are caused by a variety of protein coding mutations (single nucleotide changes, deletions and expansions) in single genes. Affected genes encode plasma membrane and intracellular ion channels, membrane receptors, protein kinases, protein phosphatases and proteins of unknown function. Although SCA‐linked genes are quite diverse they share two key features: first, they are highly, although not exclusively, expressed in cerebellar Purkinje neurons (PNs), and second, when mutated they lead ultimately to the degeneration of PNs. In this review we summarize ataxia‐related changes in PN neurophysiology that have been observed in various mouse knockout lines and in transgenic models of human SCA. We also highlight emerging evidence that altered metabotropic glutamate receptor signalling and disrupted calcium homeostasis in PNs form a common, early pathophysiological mechanism in SCAs. Together these findings indicate that aberrant calcium signalling and profound changes in PN neurophysiology precede PN cell loss and are likely to lead to cerebellar circuit dysfunction that explains behavioural signs of ataxia characteristic of the disease.