Huntington's disease (HD) is an inherited neurodegenerative disorder caused by an expanded stretch of CAG trinucleotide repeats that results in neuronal dysfunction and death. Here, the HD consortium reports the generation and characterization of 14 induced pluripotent stem cell (iPSC) lines from HD patients and controls. Microarray profiling revealed CAG expansion-associated gene expression patterns that distinguish patient lines from controls, and early onset versus late onset HD. Differentiated HD neural cells showed disease associated changes in electrophysiology, metabolism, cell adhesion, and ultimately cell death for lines with both medium and longer CAG repeat expansions. The longer repeat lines were however the most vulnerable to cellular stressors and BDNF withdrawal using a range of assays across consortium laboratories. The HD iPSC collection represents a unique and well-characterized resource to elucidate disease mechanisms in HD and provides a novel human stem cell platform for screening new candidate therapeutics.

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Hydrogen sulfide (H(2)S) is produced endogenously in many types of mammalian cells. Evidence is now accumulating to suggest that H(2)S is an endogenous signalling molecule, with a variety of molecular targets, including ion channels. Here, we describe the effects of H(2)S on the large conductance, calcium-sensitive potassium channel (BK(Ca)). This channel contributes to carotid body glomus cell excitability and oxygen-sensitivity. The experiments were performed on HEK 293 cells, stably expressing the human BK(Ca) channel alpha subunit, using patch-clamp in the inside-out configuration. The H(2)S donor, NaSH (100microM-10 mM), inhibited BK(Ca) channels in a concentration-dependent manner with an IC(50) of ca. 670microM. In contrast to the known effects of CO donors, the H(2)S donor maximally decreased the open state probability by over 50% and shifted the half activation voltage by more than +16mV. In addition, although 1 mM KCN completely suppressed CO-evoked channel activation, it was without effect on the H(2)S-induced channel inhibition, suggesting that the effects of CO and H(2)S were non-competitive. RT-PCR showed that mRNA for both of the H(2)S-producing enzymes, cystathionine-beta-synthase and cystathionine-gamma-lyase, were expressed in HEK 293 cells and in rat carotid body. Furthermore, immunohistochemistry was able to localise cystathionine-gamma-lyase to glomus cells, indicating that the carotid body has the endogenous capacity to produce H(2)S. In conclusion, we have shown that H(2)S and CO have opposing effects on BK(Ca)channels, suggesting that these gases have separate modes of action and that they modulate carotid body activity by binding at different motifs in the BK(Ca)alphasubunit.

Carbon monoxide (CO) is a potent activator of large conductance, calcium-dependent potassium (BK Ca) channels of vascular myocytes and carotid body glomus cells or when heterologously expressed. Using the human BK Ca channel alpha1-subunit (hSlo1; KCNMA1) stably and transiently expressed in human embryonic kidney 293 cells, the mechanism and structural basis of channel activation by CO was investigated in inside-out, excised membrane patches. Activation by CO was concentration dependent (EC50 approximately 20 microM), rapid, reversible, and evoked a shift in the V 0.5 of -20 mV. CO evoked no changes in either single channel conductance or in deactivation rate but augmented channel activation rate. Activation was independent of the redox state of the channel, or associated compounds/protein partners, and was partially dependent on [Ca2+]i in the physiological range (100-1,000 nM). Importantly, CO "super-stimulated" BK Ca activity even in saturating [Ca2+]i. Single or double mutation of two histidine residues previously implicated in CO sensing did not suppress CO activation but replacing the S9-S10 module of the C-terminal of Slo1 with that of Slo3 completely prevented the action of CO. These findings show that a motif in the S9-S10 part of the C-terminal is essential for CO activation and suggest that this gas transmitter activates the BK Ca channel by redox-independent changes in gating.

The large conductance, voltage- and calcium-activated potassium channel, BK(Ca), is a known target for the gasotransmitter, carbon monoxide (CO). Activation of BK(Ca) by CO modulates cellular excitability and contributes to the physiology of a diverse array of processes, including vascular tone and oxygen-sensing. Currently, there is no consensus regarding the molecular mechanisms underpinning reception of CO by the BK(Ca). Here, employing voltage-clamped, inside-out patches from HEK293 cells expressing single, double and triple cysteine mutations in the BK(Ca) α-subunit, we test the hypothesis that CO regulation is conferred upon the channel by interactions with cysteine residues within the RCK2 domain. In physiological [Ca(2+)](i), all mutants carrying a cysteine substitution at position 911 (C911G) demonstrated significantly reduced CO sensitivity; the C911G mutant did not express altered Ca(2+)-sensitivity. In contrast, histidine residues in RCK1 domain, previously shown to ablate CO activation in low [Ca(2+)](i), actually increased CO sensitivity when [Ca(2+)](i) was in the physiological range. Importantly, cyanide, employed here as a substituent for CO at potential metal centres, occluded activation by CO; this effect was freely reversible. Taken together, these data suggest that a specific cysteine residue in the C-terminal domain, which is close to the Ca(2+) bowl but which is not involved in Ca(2+) activation, confers significant CO sensitivity to BK(Ca) channels. The rapid reversibility of CO and cyanide binding, coupled to information garnered from other CO-binding proteins, suggests that C911 may be involved in formation of a transition metal cluster which can bind and, thereafter, activate BK(Ca).

The S4 region of the Drosophila Shaker voltage-gated K' channel has been proposed to function as a voltage-sensor. We have synthesised a peptide corresponding to this S4 region. Structural studies on the S4 peptide were conducted using Fourier transform infrared (FTIR) spectroscopy. Spectra were obtained for the peptide dissolved in aqueous solution, in trifluoroethanol solvent and also after reconstitution into lipid bilayers and micelles. The peptide in trifluoroethanol adopts an a-helical conformation which is in good agreement with the results of a recent 2D NMR study on the structure of a S4 peptide corresponding to the rat brain sodium channel [(1989) FEBS Lett. 257, 113-l 171. A predominantly a-helical structure is also observed when the S4 peptide is present in aqueous lysophosphatidylcholine micelles, in dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol lipid bilayers. In contrast to this, the S4 peptide in aqueous solution is in a random coil conformation. The coil-to-helix transition observed for the S4 peptide upon its transfer from aqueous solution to lipid membrane indicates that it has a high degree of conformational flexibility and can undergo large changes in its structure in response to its environment. This may have important implications for its role in the voltage activation process during which the S4 peptide has been postulated to, at least partially, move from a lipid bilayer to an aqueous extracellular media [(1992) Biophys J. 62, 238-2501. The results of our study lend support to such a model.

Minimal potassium channel protein (minK) is a potassium channel protein consisting of 130 amino acids, possessing just one putative transmembrane domain. In this study we have synthesized a peptide with the amino acid sequence RDDSKLEALYILMVLGFFGFFTLGIMLSYI, containing the putative transmembrane region of minK, and analysed its secondary structure by using Fourier-transform IR and CD spectroscopy. The peptide was virtually insoluble in aqueous buffer, forming intermolecular beta-sheet aggregates. On attempted incorporation of the peptide into phospholipid membranes with a method involving dialysis, the peptide adopted a predominantly intermolecular beta-sheet conformation identical with that of the peptide in aqueous buffer, in agreement with a previous report [Horvàth, Heimburg, Kovachev, Findlay, Hideg and Marsh, (1995) Biochemistry 34, 3893-3898]. However, by using an alternative method of incorporating the peptide into phospholipid membranes we found that the peptide adopted a predominantly alpha-helical conformation, a finding consistent with various proposed structural models. These observed differences in secondary structure are due to artifacts of aggregation of the peptide before incorporation into lipid.

Various cardiorespiratory diseases (e.g. congestive heart failure, emphysema) result in systemic hypoxia and patients consequently demonstrate adaptive cellular responses which predispose them to conditions such as pulmonary hypertension and stroke. Central to many affected excitable tissues is activity of large conductance, Ca 2؉ -activated K ؉ (maxiK) channels. We have studied maxiK channel activity in HEK293 cells stably co-expressing the most widely distributed of the human ␣-and ␤-subunits that constitute these channel following maneuvers which mimic severe hypoxia. ] i could sustain an acute hypoxic inhibitory response. Chronic hypoxia caused no change in ␣-subunit immunoreactivity with Western blotting but evoked a 3-fold increase in ␤-subunit expression. These observations were fully supported by immunocytochemistry, which also suggested that chronic hypoxia augmented ␣/␤-subunit colocalization at the plasma membrane. Using a novel nuclear run-on assay and RNase protection we found that chronic hypoxia did not alter mRNA production rates or steady-state levels, which suggests that this important environmental cue modulates maxiK channel function via post-transcriptional mechanisms.Crucial to the cellular and physiological response to acute perturbation of systemic and/or pulmonary O 2 levels is the rapid inhibition of K ϩ channels by hypoxia (see Ref. 1 for recent review). Thus, acute modulation of ion channel activity is central to the homeostatic mechanisms that underlie chemosensing in carotid body (2-4), neuroepithelial body (5, 6) (and its immortalized cellular counterpart, H146 cells, Ref. 7) and (8 -11) systemic vascular smooth muscle (12). Although somewhat controversial, ion channel inhibition has also been implicated in hypoxic vasoconstriction in the pulmonary circulation (13).In addition, such O 2 sensitivity is believed to play a significant role in modulation of excitability in several cellular components of the mammalian nervous system (14 -17).Although O 2 -sensitive tissues express a wide variety of channel types, central to the cellular mechanism of acute O 2 sensing in several is hypoxic suppression of large conductance Ca 2ϩ -activated K ϩ (maxiK) channels. Indeed, hypoxic inhibition of native maxiK channel activity has been demonstrated in carotid body (4, 18, 19), pulmonary arteriolar smooth muscle (20), chromaffin cells (21), and central neurons (20,22). Although their contribution to carotid body, chromaffin cell, and central neuronal function is well supported, some controversy still surrounds their involvement in pulmonary vasoconstriction (15) and there is good evidence for both delayed rectifier (23) and tandem P domain K ϩ channels in the response (24); the latter observation is fully supported by our recent reports of O 2 sensitivity of the recombinant human tandem P domain channels, hTASK1 (25), and hTASK3 (26).Tissue specificity notwithstanding, we have recently demonstrated at the single channel level that a recombinant human maxiK channel can be rapidly and reversibly in...