Dynamic conformational changes of extracellular S5–P linkers in the hERG channel

The hERG channel has an unusually long 'S5–P linker' (residues 571–613) that lines the outer mouth of the pore. Previously, we have shown that residues along this S5–P linker are critical for the fast-inactivation process and K⁺ selectivity of the hERG channel. Here we used several approaches to probe the structure of this S5–P linker and its interactions with other domains of the hERG channel. Circular dichroism and NMR analysis of a synthetic hERG S5–P linker peptide suggested that this linker is quite dynamic: its central region (positions 583–593) can be unstructured or helical, depending on whether it is immersed in an aqueous phase or in contact with a hydrophobic environment. Cysteine introduced into positions 583–597 of the S5–P linker can form intersubunit disulphide bonds, and at least four of them (at 584, 585, 588 and 589) can form disulphide bonds with counterparts from neighbouring subunits. We propose that the four S5–P linkers in a hERG channel can engage in dynamic conformational changes during channel gating, and interactions between S5–P linkers from neighbouring subunits contribute importantly to channel inactivation.     

Localization of the ergtoxin-1 receptors on the voltage sensing domain of hERG K+ channel by AFM recognition imaging

The inhibition of the human ether-à-go-go-related (hERG) K⁺ channels is the major cause of long QT syndromes inducing fatal cardiac arrhythmias. Ergtoxin 1 (ErgTx1) belongs to scorpion-toxins, which are K⁺ channel-blockers, and binds to hERG channel with 1:1 stoichiometry and high affinity (K _d ∼ 10 nM). Nevertheless, patch-clamp recordings recently demonstrated that ErgTx1 does not establish complete blockade of hERG currents, even at high ErgTx1 concentrations. Such phenomenon is supposed to be consistent with highly dynamic conformational changes of the outer pore domain of hERG. In this study, simultaneous topography and recognition imaging (TREC) on hERG HEK 293 cells was used to visualize binding sites on the extracellular part of hERG channel (on S1–S2 region) for Anti-K_v11.1 (hERG-extracellular-antibody). The recognition maps of hERG channels contained recognition spots, haphazardly distributed and organized in clusters. Recognition images after the addition of ErgTx1 at high concentrations (∼1 μM) revealed subsequent partial disappearance of clusters, indicating that ErgTx1 was bound to the S1–S2 region. These results were supported by AFM force spectroscopy data, showing for the first time that voltage sensing domain (S1–S4) of hERG K⁺ channel might be one of the multiple binding sites of ErgTx1.     

Charges dispersed over the permeation pathway determine the charge selectivity and conductance of a Cx32 chimeric hemichannel

Previous studies have shown that charge substitutions in the amino terminus of a chimeric connexin, Cx32*43E1, which forms unapposed hemichannels in Xenopus oocytes, can result in a threefold difference in unitary conductance and alter the direction and amount of open channel current rectification. Here, we determine the charge selectivity of Cx32*43E1 unapposed hemichannels containing negative and/or positive charge substitutions at the 2nd, 5th and 8th positions in the N-terminus. Unlike Cx32 intercellular channels, which are weakly anion selective, the Cx32*43E1 unapposed hemichannel is moderately cation selective. Cation selectivity is maximal when the extracellular surface of the channel is exposed to low ionic strength solutions implicating a region of negative charge in the first extracellular loop of Cx43 (Cx43E1) in influencing charge selectivity analogous to that reported. Negative charge substitutions at the 2nd, 5th and 8th positions in the intracellular N-terminus substantially increase the unitary conductance and cation selectivity of the chimeric hemichannel. Positive charge substitutions at the 5th position decrease unitary conductance and produce a non-selective channel while the presence of a positive charge at the 5th position and negative charge at the 2nd results in a channel with conductance similar to the parental channel but with greater preference for cations. We demonstrate that a cysteine substitution of the 8th residue in the N-terminus can be modified by a methanthiosulphonate reagent (MTSEA-biotin-X) indicating that this residue lines the aqueous pore at the intracellular entrance of the channel. The results indicate that charge selectivity of the Cx32*43E1 hemichannel can be determined by the combined actions of charges dispersed over the permeation pathway rather than by a defined region that acts as a charge selectivity filter.     

Amino acid residues in the pro region of Escherichia coli heat-stable enterotoxin I that affect efficiency of translocation across the inner membrane.

Escherichia coli heat-stable enterotoxin Ip (STIp), which is a typical extracellular toxin consisting of 18 amino acid residues, is synthesized as a precursor consisting of pre (amino acid residues 1 to 19), pro (amino acid residues 20 to 54), and mature (amino acid residues 55 to 72) regions. Though the pre region functions as a conventional leader peptide that guides the following region to cross the inner membrane, the role of the pro region in the maturation pathway remains to be elucidated. We previously indicated that the sequence from residues 29 to 38 in the pro region increases the efficiency of STI translocation across the inner membrane (H. Yamanaka, Y. Fuke, S. Hitotsubashi, Y. Fujii, and K. Okamoto, Microbiol. Immunol. 37:195-205, 1993). We therefore examined the amino acid residues in the sequence that are responsible for this function. We substituted several amino acid residues in the sequence by means of oligonucleotide-directed site-specific mutagenesis. We then evaluated the effect of the substitution on the efficiency of STI translocation across the inner membrane by determining the enterotoxic activity of the culture supernatant, the amount of a fusion protein consisting of STI and nuclease A released into the periplasm, and the amount of the labeled ST released into the periplasm after pulse-labeling with [35S]cysteine. Substitution of the charged amino acid residues at positions 29 to 31 (K-E-K) with hydrophobic (I-V-L, F-W-F, or F-W-Q) or basic (K-K-K) residues significantly reduced these values in every assay. In contrast, the substitution of these amino acid residues with acidic amino acid residues (E-E-E) increased these values in all assays. This means that the negative charge near position 30 is important for STI to translocate efficiently across the inner membrane. A similar substitution of lysine residues at positions 37 and 38 showed that they are not involved in the translocation of STI across the inner membrane.     

Identification of positive charges situated at the outer mouth of the CFTR chloride channel pore

We have used site-directed mutagenesis and functional analysis to identify positively charged amino acid residues in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel that interact with extracellular anions. Mutation of two positively charged arginine residues in the first extracellular loop (ECL) of CFTR, R104, and R117, as well as lysine residue K335 in the sixth transmembrane region, leads to inward rectification of the current–voltage relationship and decreased single channel conductance. These effects are dependent on the charge of the substituted side chain and on the Cl⁻ concentration, suggesting that these positive charges normally act to concentrate extracellular Cl⁻ ions near the outer mouth of the pore. Side chain charge-dependent effects are mimicked by manipulating charge in situ by mutating these amino acids to cysteine followed by covalent modification with charged cysteine-reactive reagents, confirming the location of these side chains within the pore outer vestibule. State-independent modification of R104C and R117C suggests that these residues are located at the outermost part of the pore. We suggest that ECL1 contributes to the CFTR pore external vestibule and that positively charged amino acid side chains in this region act to attract Cl⁻ ions into the pore. In contrast, we find no evidence that fixed positive charges in other ECLs contribute to the permeation properties of the pore.     

The G604S-hERG mutation alters the biophysical properties and exerts a dominant-negative effect on expression of hERG channels in HEK293 cells

We have recently identified a missense mutation, G604S, in the human ether-a-go-go related gene (hERG) that results in a malignant phenotype in a full pedigree of a Chinese congenital long QT syndrome (LQTS) family. The present study characterized the pathophysiological consequences of the mutation at the cellular level. Mutant G604S-hERG channels were expressed in HEK293 cells using a lipofectamine method. hERG currents were recorded using the voltage clamp technique. The expression of hERG protein was detected by Western blotting, and the subcellular location of hERG channels in cell was analyzed by confocal microscopy. We found that the G604S mutation did not lead to any expression of detectable currents, which was consistent with Western blotting analysis that the G604S-hERG mutation only expressed a band at 135 kDa. When coexpressed with wild-type (WT)-hERG, G604S-hERG exhibited strong dominant-negative current suppression resulting in decreased current density and altered gating properties of the WT-hERG channel, as well as interference with the trafficking of WT-hERG channel protein. In addition, confocal microscopy demonstrated that G604S-hERG subunits could be inserted into the cell membrane when forming heteromultimeric channels with WT-hERG channel subunits. Our results suggest that G604S mutation causes a loss of function in hERG through a strong dominant-negative effect on WT-hERG channel function that caused by impaired trafficking of WT-hERG channels, and further accentuates this suppression by forming heteromultimeric functional channels with WT-hERG subunits.     

H+ ion modulation of C-type inactivation of Shaker K+ channels

 The participation of an extracellular loop in C-type inactivation of voltage-gated K⁺ channels was investigated. A wild-type phenylalanine (at position 425) between the fifth putative transmembrane segment (S5) and the pore region of the Shaker K⁺ channel was mutated to a histidine and the functional consequences of protonating the imidizole group of the histidine were examined. C-type inactivation of both wild-type and mutant channels was sensitive to external pH over the range of 5.2–8. The pH dependence of wild-type channels was characterized by an apparent pK value of 5.0. The inactivation kinetics of F425H mutant channels had a pH dependence with a pK of 5.8 – in addition to the pH dependence of the wild-type channels. Moreover, at pH 7 and 8 the voltage dependence of C-type inactivation kinetics was manifest only in the F425H mutant channels. C-type inactivation in wild-type channels involves a chemical group with a low pK. Taken together, these results suggest that residues located in the extracellular S5-pore loop of the Shaker K⁺ channel participate in C-type inactivation. Received: 30 September 1998 / Received after revision: 8 January 1999 / Accepted: 18 January 1999     

A molecular threading mechanism underlies Jumonji lysine demethylase KDM2A regulation of methylated H3K36

The dynamic reversible methylation of lysine residues on histone proteins is central to chromatin biology. Key components are demethylase enzymes, which remove methyl moieties from lysine residues. KDM2A, a member of the Jumonji C domain-containing histone lysine demethylase family, specifically targets lower methylation states of H3K36. Here, structural studies reveal that H3K36 specificity for KDM2A is mediated by the U-shaped threading of the H3K36 peptide through a catalytic groove within KDM2A. The side chain of methylated K36 inserts into the catalytic pocket occupied by Ni²⁺ and cofactor, where it is positioned and oriented for demethylation. Key residues contributing to K36me specificity on histone H3 are G33 and G34 (positioned within a narrow channel), P38 (a turn residue), and Y41 (inserts into its own pocket). Given that KDM2A was found to also bind the H3K36me3 peptide, we postulate that steric constraints could prevent α-ketoglutarate from undergoing an “off-line”-to-“in-line” transition necessary for the demethylation reaction. Furthermore, structure-guided substitutions of residues in the KDM2A catalytic pocket abrogate KDM2A-mediated functions important for suppression of cancer cell phenotypes. Together, our results deduce insights into the molecular basis underlying KDM2A regulation of the biologically important methylated H3K36 mark.     

The selectivity, voltage-dependence and acid sensitivity of the tandem pore potassium channel TASK-1: contributions of the pore domains

We have investigated the contribution to ionic selectivity of residues in the selectivity filter and pore helices of the P1 and P2 domains in the acid sensitive potassium channel TASK-1. We used site directed mutagenesis and electrophysiological studies, assisted by structural models built through computational methods. We have measured selectivity in channels expressed in Xenopus oocytes, using voltage clamp to measure shifts in reversal potential and current amplitudes when Rb⁺ or Na⁺ replaced extracellular K⁺. Both P1 and P2 contribute to selectivity, and most mutations, including mutation of residues in the triplets GYG and GFG in P1 and P2, made channels non-selective. We interpret the effects of these—and of other mutations—in terms of the way the pore is likely to be stabilised structurally. We show also that residues in the outer pore mouth contribute to selectivity in TASK-1. Mutations resulting in loss of selectivity (e.g. I94S, G95A) were associated with slowing of the response of channels to depolarisation. More important physiologically, pH sensitivity is also lost or altered by such mutations. Mutations that retained selectivity (e.g. I94L, I94V) also retained their response to acidification. It is likely that responses both to voltage and pH changes involve gating at the selectivity filter. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users. K. H. Yuill, P. J. Stansfeld and I. Ashmole contributed equally to this paper.     

Structural and functional characterization of the human formyl peptide receptor ligand-binding region.

The formyl peptide (N-formyl-1-methionyl-1-leucyl-1-phenylalanine [FMLP]) receptor is involved in the activation of neutrophils and their subsequent response to chemotactic N-formylated peptides. Recently, we found that the first extracellular loop closest to the N-terminal end of the FMLP receptor exhibited the strongest ligand binding compared with that shown by other extracellular regions. By constructing amino acid substitutional variants of this domain, we have determined that residues Arg-84 and Lys-85 on this loop play major roles in ligand-binding activity. Furthermore, random rearrangement of the residues of this receptor region demonstrated that the position of these charged amino acids did not affect their involvement in ligand binding, although their presence was essential for this binding to occur. We propose that the portion of the first N-terminal extracellular loop of the FMLP receptor containing residues Arg-84 and Lys-85 contributes significantly to the active site in ligand-receptor binding. We further propose that this binding is not dependent on defined structure but rather that these charged moieties may function as important "contacts" in receptor-ligand interactions.     

The effects of oxidizing and cysteine-reactive reagents on the inward rectifier potassium channels Kir2.3 and Kir1.1

The inwardly rectifying potassium channel Kir2.3 possesses extracellular cysteine residues at positions 113, 140, and 145, as well as at position 79 near the outer membrane boundary. In this study, we have investigated the roles of these extracellular cysteine residues in mediating inhibition of the Kir2.3 channel by the cysteine-reactive reagents para-chloromercuribenzenesulphonate (PCMBS) and thimerosal, and the oxidizing agent hydrogen peroxide (H2O2). We have also compared the effects of these reagents with those on Kir1.1 channels (which do not possess cysteine residues equivalent to 140 and 79 in Kir2.3 channels). Mutant channels were made in which cysteine residues were mutated to serine by site-directed mutagenesis. Wild-type or mutant cRNA was injected into Xenopus oocytes and voltage-clamp recordings made 1-2 days later. Wild-type Kir2.3 currents were significantly inhibited by PCMBS, thimerosal and H2O2. Currents for mutants Kir2.3 C79S and C140S were also inhibited by PCMBS, thimerosal and H2O2. These mutations affected the time course of inhibition by all three reagents. For PCMBS, a slow component of inhibition was absent for the C79S mutation, and a fast component was absent for C140S. For the double mutation C79S/C140S, PCMBS no longer had any effect. For thimerosal, there was a slower time course for C140S, a faster time course for C79S, and a delayed onset for C79S/C140S. For H2O2, the main effect was a delayed onset for the double mutant. The reducing agent dithiothreitol (DTT) reversed the inhibition by both PCMBS and thimerosal of wild-type and mutant currents, but not the inhibition due to H2O2. Finally, wild-type Kir1.1 currents were not significantly inhibited by the applications of either PCMBS or thimerosal, while H2O2 produced small inhibition. The results taken together indicate that inhibition by the cysteine-reactive reagent PCMBS is mediated through cysteine residues 79 and 140 in Kir2.3 channels, with C79 mediating a slow component of inhibition and C140 a faster component, and that both residues are extracellularly exposed. The data indicate that these two cysteine residues are also main sites for inhibition by thimerosal and H2O2 but, unlike for PCMBS, an additional non-extracellular inhibitory site(s) must also be involved.     

A mutation in the extracellular domain of the α7 nAChR reduces calcium permeability

The α7 neuronal nicotinic acetylcholine receptor (nAChR) displays the highest calcium permeability among the different subtypes of nAChRs expressed in the mammalian brain and can impact cellular events including neurotransmitter release, second messenger cascades, cell survival, and apoptosis. The selectivity for cations in nAChRs is thought to be achieved in part by anionic residues which are located on either side of the channel mouth and increase relative cationic concentration. Mutagenesis studies have improved our understanding of the role of the second transmembrane domain and the intracellular loop of the channel in ion selectivity. However, little is known about the influence that the extracellular domain (ECD) plays in ion permeation. In the α7 nAChR, it has been found that the ECD contains a ring of ten aspartates (two per subunit) that is believed to face the lumen of the pore and could attract cations for permeation. Using mutagenesis and a combination of electrophysiology and imaging techniques, we tested the possible involvement of these aspartate residues in the calcium permeability of the rat α7 nAChR. We found that one of these residues (the aspartate at position 44) appears to be essential since mutating it to alanine resulted in a decrease in amplitude for both whole cell and single-channel responses and in the complete disappearance of detectable calcium changes in most cells, which indicates that the ECD of the α7 nAChR plays a key role in calcium permeation.     

Molecular mapping of a site for Cd2+-induced modification of human ether-à-go-go-related gene (hERG) channel activation

Cd²⁺ slows the rate of activation, accelerates the rate of deactivation and shifts the half-points of voltage-dependent activation ( V _0.5,act) and inactivation ( V _0.5,inact) of human ether-à-go-go -related gene (hERG) K⁺ channels. To identify specific Cd²⁺-binding sites on the hERG channel, we mutated potential Cd²⁺-coordination residues located in the transmembrane domains or extracellular loops linking these domains, including five Cys, three His, nine Asp and eight Glu residues. Each residue was individually substituted with Ala and the resulting mutant channels heterologously expressed in Xenopus oocytes and their biophysical properties determined with standard two-microelectrode voltage-clamp technique. Cd²⁺ at 0.5 m m caused a +36 mV shift of V _0.5,act and a +18 mV shift of V _0.5,inact in wild-type channels. Most mutant channels had a similar sensitivity to 0.5 m m Cd²⁺. Mutation of single Asp residues located in the S2 (D456, D460) or S3 (D509) domains reduced the Cd²⁺-induced shift in V _0.5,act, but not V _0.5,inact. Combined mutations of two or three of these key Asp residues nearly eliminated the shift induced by 0.5 m m Cd²⁺. Mutation of D456, D460 and D509 also reduced the comparatively low-affinity effects of Ca²⁺ and Mg²⁺ on V _0.5,act. Extracellular Cd²⁺ modulates hERG channel activation by binding to a coordination site formed, at least in part, by three Asp residues.     

Phosphorylation of serine‐10 of histone H3 shields modified lysine‐9 selectively during mitosis

Post‐translational modifications of histones play important roles in regulating chromatin dynamics and epigenetic inheritance during mitosis. The epigenetic significance and stability of histone H3‐lysine 9 (H3K9) modifications have been well studied in interphase cells, whereas not as much in mitotic cells. Here, we inspected mitosis‐coupled alterations in the global modifications of H3K9. Signals for H3K9 mono‐, di‐methylation and acetylation became invisible as cells entered mitosis in contrast to the pattern observed for H3‐serine 10 phosphorylation (H3S10ph). Treatment with the aurora‐B inhibitor ZM447439 or expression of the dominant negative mutant Aur‐B^K106R resulted in prometaphase chromosomes that lacked signals for H3S10ph but were positive for H3K9 modifications. Trimethylation was the sole K9 modification that remained consistently detectable throughout the cell cycle. This phenomenon was specific for H3K9‐S10, as this pattern was not observed at H3K27‐S28. Methylated H3K27 remained detectable throughout the cell cycle, despite phosphorylation of the adjacent H3S28. Contrastingly, our dot‐blot experiment using synthetic peptides showed that phosphorylation of serine residue basically kept adjacent lysine from antibody access. Together, these results suggest that phosphorylation of serine residue occurs in a selective manner, being influenced by the types of modifications and the nature of neighboring lysine residues.     

Cyclic nucleotide-gated channels: intra- and extracellular accessibility to Cd2+ of substituted cysteine residues within the P-loop

In cyclic nucleotide-gated (CNG) channels from the bovine rod, the pore loop "P-loop", connecting the S5 and S6 transmembrane segments, is formed by the residues R345-S371 (here named R1-S27). It determines channel selectivity and contributes to gating. We have studied its topology, by testing the accessibility to Cd2+ of serially substituted cysteine residues. Channels were expressed in Xenopus oocytes. The accessibility of V4C, S6C, T16C, 117C, T20C, P22C and S27C from the cytoplasmic side of the plasma membrane was tested by applying 1-100 microM Cd2+ to the inner face of inside-out patches, at negative membrane potentials. Under these conditions, the effect of Cd2+ on wild-type channels was negligible. The accessibility of the same residues from the external side of the membrane was tested by measuring CNG current inhibition persisting after wash-out of Cd2+ applied to outside-out patches. T16C and I17C channels were strongly inhibited by Cd2+ from the inside, in the presence of cGMP. The Kd for T16C block was 16 microM. Thus the T16 and I17 residues participate directly in channel function and are accessible from the cytoplasmic side when the channels are open. In contrast, V4C, T20C and P22C residues were only inhibited when 100 microM Cd2+ was applied externally, suggesting that V4C, T20C and P22C face the outer side of the P-loop.     

Selective block of the human 2-P domain potassium channel, TASK-3, and the native leak potassium current, IKSO, by zinc

Background potassium channels control the resting membrane potential of neurones and regulate their excitability. Two-pore-domain potassium (2-PK) channels have been shown to underlie a number of such neuronal background currents. Currents through human TASK-1, TASK-2 and TASK-3 channels expressed in Xenopus oocytes were inhibited by extracellular acidification. For TASK-3, mutation of histidine 98 to aspartate or alanine considerably reduced this effect of pH. Zinc was found to be a selective blocker of TASK-3 with virtually no effect on TASK-1 or TASK-2. Zinc had an IC₅₀ of 19.8 μ m for TASK-3, at +80 mV, with little voltage dependence associated with this inhibition. TASK-3 H98A had a much reduced sensitivity to zinc suggesting this site is important for zinc block. Surprisingly, TASK-1 also has histidine in position 98 but is insensitive to zinc block. TASK-3 and TASK-1 differ at position 70 with glutamate for TASK-3 and lysine for TASK-1. TASK-3 E70K also had a much reduced sensitivity to zinc while the corresponding reverse mutation in TASK-1, K70E, induced zinc sensitivity. A TASK-3–TASK-1 concatamer channel was comparatively zinc insensitive. For TASK-3, it is concluded that positions E70 and H98 are both critical for zinc block. The native cerebellar granule neurone (CGN) leak current, IK_SO, is sensitive to block by zinc, with current reduced to 0.58 of control values in the presence of 100 μ m zinc. This suggests that TASK-3 channels underlie a major component of IK_SO. It has recently been suggested that zinc is released from inhibitory synapses onto CGNs. Therefore it is possible that inhibition of IK_SO in cerebellar granule cells by synaptically released zinc may have important physiological consequences.     

A tryptophan residue (W736) in the amino-terminus of the P-segment of domain II is involved in pore formation in Na(v)1.4 voltage-gated sodium channels

Voltage-gated Na channels comprise four homologous domains each consisting of six transmembrane segments (S1-S6) linked by loops. The linkers between segments S5 and S6 in each domain (P-loops), denoted as SS1-SS2, form the pore of the channel. It is believed that the SS1 region of the P-loops dips into, while the SS2 region exits out of the membrane. We have reported previously that residues A728 and D730 (in SS1 of domain II) contribute to the external vestibule of the pore of the rat skeletal muscle Na channel (Na(v)1.4). In this study, we examined the role of a conserved neighbouring tryptophan residue at position 736 (W736) in the pore formation. The W736 residue of Na(v)1.4 was replaced by a cysteine using site-directed mutagenesis. Complementary RNAs encoding the wild-type and mutant channels were injected into Xenopus laevis oocytes and macroscopic Na(+) currents measured using the two-microelectrode voltage-clamp technique. The W736C mutant showed increased channel sensitivity to externally applied Cd(2+) and methanethiosulphonate-ethyltrimethylammonium (MTSET). Furthermore, micromolar concentrations of Cd(2+) reduced single-channel current amplitude in the Na(v)1.4/W736C mutant without affecting its voltage dependence. However, only small differences in tetrodotoxin and micro-conotoxin GIIIA affinity were observed between the wild-type and mutant channels. Replacing Na(+) with other cations - K(+), Li(+), Cs(+) or NH(4)(+) - did not change the ion permeation sequence of the Na(v)1.4/W736C mutant channel. The results suggest that W736 contributes to the formation of the pore, close to the mouth of the channel, but is not part of the selectivity filter.     

Dihydropyridines modulate K+-evoked amino acid and adenosine release from cerebellar neuronal cultures

Partial depolarization of primary cerebellar neuronal cultures with K+ evoked the release of aspartate, glutamate, adenosine, serine, taurine, gamma-aminobutyric acid (GABA), alanine and proline. The dihydropyridine calcium channel agonist, BAY K 8644, significantly augmented the K+-induced release of adenosine, aspartate, glutamate and GABA, but not that of serine, taurine, alanine or proline. However, in all cases the dihydropyridine antagonist nifedipine decreased this BAY K 8644-enhanced, K+-evoked efflux to below control levels. Neither BAY K 8644 nor nifedipine alone affected basal efflux levels. The phenylalkylamine calcium channel antagonist, verapamil, was ineffective in antagonizing K+-evoked amino acid release except at very high concentration (100 microM). These findings suggest that L-type Ca2+ channels are present in both excitatory (glutamatergic granule cells) and inhibitory (GABAergic stellate and basket cells) neurons in these cultures, and that they appear to be involved in regulating the release of not only neuroactive amino acids, but also some neutral amino acids and adenosine.     

How substrate specificity is imposed on a histone demethylase—lessons from KDM2A

Histone lysine methylation and demethylation regulate histone methylation dynamics, which impacts chromatin structure and function. To read and erase the methylated histone residues, lysine demethylases must specifically recognize the histone sequences and methylated sites and discriminate the degree of these methylations. In this issue of Genes & Development, Cheng and colleagues (pp. 1758–1771) determine a crystal structure of histone lysine demethylase KDM2A that specifically targets lower degrees of H3K36 methylation. The results reveal the structural basis for H3K36 substrate specificity and suggest mechanisms of Lys36 demethylation. This KDM2A–H3K36 complex structure, coupled with functional studies, provides needed insight into the process and regulation of histone demethylation.