Rapid Induction of P/C-type Inactivation Is the Mechanism for Acid-induced K+ Current Inhibition

Extracellular acidification is known to decrease the conductance of many voltage-gated potassium channels. In the present study, we investigated the mechanism of H+o-induced current inhibition by taking advantage of Na+ permeation through inactivated channels. In hKv1.5, H+o inhibited open-state Na+ current with a similar potency to K+ current, but had little effect on the amplitude of inactivated-state Na+ current. In support of inactivation as the mechanism for the current reduction, Na+ current through noninactivating hKv1.5-R487V channels was not affected by [H+o]. At pH 6.4, channels were maximally inactivated as soon as sufficient time was given to allow activation, which suggested two possibilities for the mechanism of action of H+o. These were that inactivation of channels in early closed states occurred while hyperpolarized during exposure to acid pH (closed-state inactivation) and/or inactivation from the open state was greatly accelerated at low pH. The absence of outward Na+ currents but the maintained presence of slow Na+ tail currents, combined with changes in the Na+ tail current time course at pH 6.4, led us to favor the hypothesis that a reduction in the activation energy for the inactivation transition from the open state underlies the inhibition of hKv1.5 Na+ current at low pH

A Direct Demonstration of Closed-State Inactivation of K+ Channels at Low pH

Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltage-clamp fluorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fluorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker α-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fluorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fluorescence demonstrated slow unquenching that also reflects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fluorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fluorescence at low pH reflects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K+ or the mutation T449V) prevented fluorescence loss at low pH, and the fluorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fluorescence amplitude (pKa = 5.9) in channels held continuously at −80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fluorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state

Inhibition of KCa2.2 and KCa2.3 channel currents by protonation of outer pore histidine residues

Ion channels are often modulated by changes in extracellular pH, with most examples resulting from shifts in the ionization state of histidine residue(s) in the channel pore. The application of acidic extracellular solution inhibited expressed KCa2.2 (SK2) and KCa2.3 (SK3) channel currents, with KCa2.3 (pIC50 of ∼6.8) being approximately fourfold more sensitive than KCa2.2 (pIC50 of ∼6.2). Inhibition was found to be voltage dependent, resulting from a shift in the affinity for the rectifying intracellular divalent cation(s) at the inner mouth of the selectivity filter. The inhibition by extracellular protons resulted from a reduction in the single-channel conductance, without significant changes in open-state kinetics or open probability. KCa2.2 and KCa2.3 subunits both possess a histidine residue in their outer pore region between the transmembrane S5 segment and the pore helix, with KCa2.3 also exhibiting an additional histidine residue between the selectivity filter and S6. Mutagenesis revealed that the outer pore histidine common to both channels was critical for inhibition. The greater sensitivity of KCa2.3 currents to protons arose from the additional histidine residue in the pore, which was more proximal to the conduction pathway and in the electrostatic vicinity of the ion conduction pathway. The decrease of channel conductance by extracellular protons was mimicked by mutation of the outer pore histidine in KCa2.2 to an asparagine residue. These data suggest that local interactions involving the outer turret histidine residues are crucial to enable high conductance openings, with protonation inhibiting current by changing pore shape

Scaling up of highly active antiretroviral therapy in a rural district of Malawi: an effectiveness assessment.

BACKGROUND: The recording of outcomes from large-scale, simplified HAART (highly active antiretroviral therapy) programmes in sub-Saharan Africa is critical. We aimed to assess the effectiveness of such a programme held by Médecins Sans Frontières (MSF) in the Chiradzulu district, Malawi. METHODS: We scaled up and simplified HAART in this programme since August, 2002. We analysed survival indicators, CD4 count evolution, virological response, and adherence to treatment. We included adults who all started HAART 6 months or more before the analysis. HIV-1 RNA plasma viral load and self-reported adherence were assessed on a subsample of patients, and antiretroviral resistance mutations were analysed in plasma with viral loads greater than 1000 copies per mL. Analysis was by intention to treat. FINDINGS: Of the 1308 patients who were eligible, 827 (64%) were female, the median age was 34.9 years (IQR 29.9-41.0), and 1023 (78%) received d4T/3TC/NVP (stavudine, lamivudine, and nevirapine) as a fixed-dose combination. At baseline, 1266 individuals (97%) were HAART-naive, 357 (27%) were at WHO stage IV, 311 (33%) had a body-mass index of less than 18.5 kg/m2, and 208 (21%) had a CD4 count lower than 50 cells per muL. At follow-up (median 8.3 months, IQR 5.5-13.1), 967 (74%) were still on HAART, 243 (19%) had died, 91 (7%) were lost to follow-up, and seven (0.5%) discontinued treatment. Low body-mass index, WHO stage IV, male sex, and baseline CD4 count lower than 50 cells per muL were independent determinants of death in the first 6 months. At 12 months, the probability of individuals still in care was 0.76 (95% CI 0.73-0.78) and the median CD4 gain was 165 (IQR 67-259) cells per muL. In the cross-sectional survey (n=398), 334 (84%) had a viral load of less than 400 copies per mL. Of several indicators measuring adherence, self-reported poor adherence (<80%) in the past 4 days was the best predictor of detectable viral load (odds ratio 5.4, 95% CI 1.9-15.6). INTERPRETATION: These data show that large numbers of people can rapidly benefit from antiretroviral therapy in rural resource-poor settings and strongly supports the implementation of such large-scale simplified programmes in Africa

Fluorescence-Tracking of Activation Gating in Human ERG Channels Reveals Rapid S4 Movement and Slow Pore Opening

Background: hERG channels are physiologically important ion channels which mediate cardiac repolarization as a result of their unusual gating properties. These are very slow activation compared with other mammalian voltage-gated potassium channels, and extremely rapid inactivation. The mechanism of slow activation is not well understood and is investigated here using fluorescence as a direct measure of S4 movement and pore opening. Methods and Findings: Tetramethylrhodamine-5-maleimide (TMRM) fluorescence at E519 has been used to track S4 voltage sensor movement, and channel opening and closing in hERG channels. Endogenous cysteines (C445 and C449) in the S1–S2 linker bound TMRM, which caused a 10 mV hyperpolarization of the VK of activation to 227.562.0 mV, and showed voltage-dependent fluorescence signals. Substitution of S1–S2 linker cysteines with valines allowed unobstructed recording of S3–S4 linker E519C and L520C emission signals. Depolarization of E519C channels caused rapid initial fluorescence quenching, fit with a double Boltzmann relationship, F-VON, with VK,1 = 237.861.7 mV, and VK,2 = 43.567.9 mV. The first phase, VK,1, was,20 mV negative to the conductance-voltage relationship measured from ionic tail currents (G-VK = 218.361.2 mV), and relatively unchanged in a non-inactivating E519C:S620T mutant (V K = 234.461.5 mV), suggesting the fast initial fluorescence quenching tracked S4 voltage sensor movement. The second phase of rapid quenching was absent in the S620T mutant. The E519C fluorescence upon repolarizatio

Pathogenic variants in glutamyl-tRNAGln amidotransferase subunits cause a lethal mitochondrial cardiomyopathy disorder.

Mitochondrial protein synthesis requires charging mt-tRNAs with their cognate amino acids by mitochondrial aminoacyl-tRNA synthetases, with the exception of glutaminyl mt-tRNA (mt-tRNAGln). mt-tRNAGln is indirectly charged by a transamidation reaction involving the GatCAB aminoacyl-tRNA amidotransferase complex. Defects involving the mitochondrial protein synthesis machinery cause a broad spectrum of disorders, with often fatal outcome. Here, we describe nine patients from five families with genetic defects in a GatCAB complex subunit, including QRSL1, GATB, and GATC, each showing a lethal metabolic cardiomyopathy syndrome. Functional studies reveal combined respiratory chain enzyme deficiencies and mitochondrial dysfunction. Aminoacylation of mt-tRNAGln and mitochondrial protein translation are deficient in patients' fibroblasts cultured in the absence of glutamine but restore in high glutamine. Lentiviral rescue experiments and modeling in S. cerevisiae homologs confirm pathogenicity. Our study completes a decade of investigations on mitochondrial aminoacylation disorders, starting with DARS2 and ending with the GatCAB complex

Characterization of Multiple Ion Channels in Cultured Human Cardiac Fibroblasts

Background: Although fibroblast-to-myocyte electrical coupling is experimentally suggested, electrophysiology of cardiac fibroblasts is not as well established as contractile cardiac myocytes. The present study was therefore designed to characterize ion channels in cultured human cardiac fibroblasts. Methods and Findings: A whole-cell patch voltage clamp technique and RT-PCR were employed to determine ion channels expression and their molecular identities. We found that multiple ion channels were heterogeneously expressed in human cardiac fibroblasts. These include a big conductance Ca2+-activated K+ current (BKCa) in most (88%) human cardiac fibroblasts, a delayed rectifier K+ current (IKDR) and a transient outward K+ current (Ito) in a small population (15 and 14%, respectively) of cells, an inwardly-rectifying K+ current (IKir) in 24% of cells, and a chloride current (ICl) in 7% of cells under isotonic conditions. In addition, two types of voltage-gated Na+ currents (INa) with distinct properties were present in most (61%) human cardiac fibroblasts. One was a slowly inactivated current with a persistent component, sensitive to tetrodotoxin (TTX) inhibition (INa.TTX, IC50 = 7.8 nM), the other was a rapidly inactivated current, relatively resistant to TTX (INa.TTXR, IC50 = 1.8 μM). RT-PCR revealed the molecular identities (mRNAs) of these ion channels in human cardiac fibroblasts, including KCa.1.1 (responsible for BKCa), Kv1.5, Kv1.6 (responsible for IKDR), Kv4.2, Kv4.3 (responsible for Ito), Kir2.1, Kir2.3 (for IKir), Clnc3 (for ICl), NaV1.2, NaV1.3, NaV1.6, NaV1.7 (for INa.TTX), and NaV1.5 (for INa.TTXR). Conclusions: These results provide the first information that multiple ion channels are present in cultured human cardiac fibroblasts, and suggest the potential contribution of these ion channels to fibroblast-myocytes electrical coupling. © 2009 Li et al.published_or_final_versio

Localization of uPAR and MMP-9 in lipid rafts is critical for migration, invasion and angiogenesis in human breast cancer cells

<p>Abstract</p> <p>Background</p> <p>uPAR and MMP-9, which play critical roles in tumor cell invasion, migration and angiogenesis, have been shown to be associated with lipid rafts.</p> <p>Methods</p> <p>To investigate whether cholesterol could regulate uPAR and MMP-9 in breast carcinoma, we used MβCD (methyl beta cyclodextrin, which extracts cholesterol from lipid rafts) to disrupt lipid rafts and studied its effect on breast cancer cell migration, invasion, angiogenesis and signaling.</p> <p>Results</p> <p>Morphological evidence showed the association of uPAR with lipid rafts in breast carcinoma cells. MβCD treatment significantly reduced the colocalization of uPAR and MMP-9 with lipid raft markers and also significantly reduced uPAR and MMP-9 at both the protein and mRNA levels. Spheroid migration and invasion assays showed inhibition of breast carcinoma cell migration and invasion after MβCD treatment. <it>In vitro </it>angiogenesis studies showed a significant decrease in the angiogenic potential of cells pretreated with MβCD. MβCD treatment significantly reduced the levels of MMP-9 and uPAR in raft fractions of MDA-MB-231 and ZR 751 cells. Phosphorylated forms of Src, FAK, Cav, Akt and ERK were significantly inhibited upon MβCD treatment. Increased levels of soluble uPAR were observed upon MβCD treatment. Cholesterol supplementation restored uPAR expression to basal levels in breast carcinoma cell lines. Increased colocalization of uPAR with the lysosomal marker LAMP1 was observed in MβCD-treated cells when compared with untreated cells.</p> <p>Conclusion</p> <p>Taken together, our results suggest that cholesterol levels in lipid rafts are critical for the migration, invasion, and angiogenesis of breast carcinoma cells and could be a critical regulatory factor in these cancer cell processes mediated by uPAR and MMP-9.</p

Transcriptome dynamics of a broad host-range cyanophage and its hosts

Cyanobacteria are highly abundant in the oceans and are constantly exposed to lytic viruses. The T4-like cyanomyoviruses are abundant in the marine environment and have broad host-ranges relative to other cyanophages. It is currently unknown whether broad host-range phages specifically tailor their infection program for each host, or employ the same program irrespective of the host infected. Also unknown is how different hosts respond to infection by the same phage. Here we used microarray and RNA-seq analyses to investigate the interaction between the Syn9 T4-like cyanophage and three phylogenetically, ecologically and genomically distinct marine Synechococcus strains: WH7803, WH8102 and WH8109. Strikingly, Syn9 led a nearly identical infection and transcriptional program in all three hosts. Different to previous assumptions for T4-like cyanophages, three temporally regulated gene expression classes were observed. Furthermore, a novel regulatory element controlled early-gene transcription, and host-like promoters drove middle gene transcription, different to the regulatory paradigm for T4. Similar results were found for the P-TIM40 phage during infection of Prochlorococcus NATL2A. Moreover, genomic and metagenomic analyses indicate that these regulatory elements are abundant and conserved among T4-like cyanophages. In contrast to the near-identical transcriptional program employed by Syn9, host responses to infection involved host-specific genes primarily located in hypervariable genomic islands, substantiating islands as a major axis of phage-cyanobacteria interactions. Our findings suggest that the ability of broad host-range phages to infect multiple hosts is more likely dependent on the effectiveness of host defense strategies than on differential tailoring of the infection process by the phage