Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative condition characterized by loss of motor neurons in the brain and spinal cord. Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9ORF72 gene are the most common cause of the familial form of ALS (C9-ALS), as well as frontotemporal lobar degeneration and other neurological diseases. How the repeat expansion causes disease remains unclear, with both loss of function (haploinsufficiency) and gain of function (either toxic RNA or protein products) proposed. Here, we report a cellular model of C9-ALS with motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying the C9ORF72 repeat expansion. No significant loss of C9ORF72 expression was observed, and knockdown of the transcript was not toxic to cultured human motor neurons. Transcription of the repeat was increased leading to accumulation of GGGGCC repeat-containing RNA foci selectively in C9-ALS motor neurons. Repeat-containing RNA foci co-localized with hnRNPA1 and Pur-α, suggesting that they may be able to alter RNA metabolism. C9-ALS motor neurons showed altered expression of genes involved in membrane excitability including DPP6, and demonstrated a diminished capacity to fire continuous spikes upon depolarization compared to control motor neurons. Antisense oligonucleotides (ASOs) targeting the C9ORF72 transcript suppressed RNA foci formation and reversed gene expression alterations in C9-ALS motor neurons. These data show that patient-derived motor neurons can be used to delineate pathogenic events in ALS.

Voltage-dependent and calcium-sensitive K ؉ (MaxiK) channels are key regulators of neuronal excitability, secretion, and vascular tone because of their ability to sense transmembrane voltage and intracellular Ca 2؉ . In most tissues, their stimulation results in a noninactivating hyperpolarizing K ؉ current that reduces excitability. In addition to noninactivating MaxiK currents, an inactivating MaxiK channel phenotype is found in cells like chromaffin cells and hippocampal neurons. The molecular determinants underlying inactivating MaxiK channels remain unknown. Herein, we report a transmembrane ␤ subunit (␤2) that yields inactivating MaxiK currents on coexpression with the pore-forming ␣ subunit of MaxiK channels. Intracellular application of trypsin as well as deletion of 19 N-terminal amino acids of the ␤2 subunit abolished inactivation of the ␣ subunit. Conversely, fusion of these N-terminal amino acids to the noninactivating smooth muscle ␤1 subunit leads to an inactivating phenotype of MaxiK channels. Furthermore, addition of a synthetic N-terminal peptide of the ␤2 subunit causes inactivation of the MaxiK channel ␣ subunit by occluding its K ؉ -conducting pore resembling the inactivation caused by the ''ball'' peptide in voltage-dependent K ؉ channels. Thus, the inactivating phenotype of MaxiK channels in native tissues can result from the association with different ␤ subunits. Ca 2ϩ-activated K ϩ channels, also known as BK, MaxiK, or slo channels, are key modulators of cellular excitability. They are characterized by their large single-channel conductance, intrinsic voltage dependence, Ca 2ϩ modulation, and blockade by charybdotoxin (CTX) and iberiotoxin (1-5). In most tissues, MaxiK channels produce noninactivating currents when activated by depolarization and͞or an increase in intracellular Ca 2ϩ . However, in chromaffin cells of the adrenal gland (6) and hippocampal neurons (7), inactivating MaxiK currents are also observed that otherwise resemble their noninactivating counterparts in their biophysical and pharmacological properties. The mechanism of inactivation in MaxiK channels has been investigated in detail in rat chromaffin cells, which express both inactivating and noninactivating MaxiK channels (8). Inactivation is removed by trypsin application to the cytosolic face of the membrane, suggesting the presence of an associated cytosolic inactivating particle (6, 7). A model developed to explain the biophysical and pharmacological properties of inactivating channels in chromaffin cells suggests that these channels are formed by a tetrameric assembly of inactivating and noninactivating subunits. Interestingly, inactivating channels in chromaffin cells are less sensitive to CTX, and heteromeric channels consisting of inactivating and noninactivating isoforms seem to have intermediate toxin sensitivities (9). Identification of a MaxiK channel variant or a subunit capable of producing fast inactivating MaxiK channel currents has so far been elusive (10).Here, we report a human ␤ subunit (␤2) ...

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The pore-forming ␣ subunit of large conductance voltage-and Ca 2؉ -sensitive K (MaxiK) channels is regulated by a ␤ subunit that has two membrane-spanning regions separated by an extracellular loop. To investigate the structural determinants in the pore-forming ␣ subunit necessary for ␤-subunit modulation, we made chimeric constructs between a human MaxiK channel and the Drosophila homologue, which we show is insensitive to ␤-subunit modulation, and analyzed the topology of the ␣ subunit. A comparison of multiple sequence alignments with hydrophobicity plots revealed that MaxiK channel ␣ subunits have a unique hydrophobic segment (S0) at the N terminus. This segment is in addition to the six putative transmembrane segments (S1-S6) usually found in voltage-dependent ion channels. High-conductance voltage-and Ca 2ϩ -sensitive potassium channels are found virtually in all excitable and nonexcitable tissues, with the exception of heart. As sensors of both voltage and intracellular calcium, they are responsible for membrane hyperpolarization, associated with phenomena like repetitive firing, spike shaping, transmitter release, and regulation of vascular and visceral smooth muscle contractility (1-4). Cloning of high-conductance voltage-activated and Ca 2ϩ -sensitive K ϩ (MaxiK) channels revealed that they belong to the S4 superfamily of ion channels (5) but carry a unique C terminus containing four hydrophobic, possibly membrane-spanning regions (S7-S10) with a nonconserved linker between regions S8 and S9 (6-8). The C-terminal region after the nonconserved linker shows the highest sequence conservation between the Drosophila (Dslo) and mammalian clones and includes hydrophobic regions S9 and S10. This region can be expressed as a separate domain and has been proposed to determine the Ca 2ϩ sensitivity of this channel (9). Alternative splicing rather than homologous genes seems to be responsible for the diversity of MaxiK channels (8,10,11).The common features of voltage-dependent K ϩ channels and individual domains of Na ϩ and Ca 2ϩ channels of the S4 superfamily are six putative transmembrane segments with a pore loop between transmembrane segments S5 and S6. The S4 region, which has been shown to move outward during depolarization and activation of these channels (12, 13), carries positive charges that are thought to interact with negative charges in regions S2 and S3 in Shaker K ϩ channels (14). By analyzing sequence alignments and hydrophobicity plots, we show that MaxiK channels may share these features, as initially proposed (7), but carry an additional hydrophobic region (S0) at the N terminus. Our data suggest that this hydrophobic region serves as a type I signal anchor directing the N terminus to the extracellular space.MaxiK channels purified from smooth muscle are tightly associated with an accessory ␤ subunit (15). Purification and cloning of this ␤ subunit revealed that it has two putative membrane-spanning regions and a large extracellular loop with two glycosylation sites (16,17). This ␤ subu...

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.