Genome-wide analysis identifies gain and loss/change of function within the small multigenic insecticidal Albumin 1 family of Medicago truncatula.

BACKGROUND: Albumin 1b peptides (A1b) are small disulfide-knotted insecticidal peptides produced by Fabaceae (also called Leguminosae). To date, their diversity among this plant family has been essentially investigated through biochemical and PCR-based approaches. The availability of high-quality genomic resources for several fabaceae species, among which the model species Medicago truncatula (Mtr), allowed for a genomic analysis of this protein family aimed at i) deciphering the evolutionary history of A1b proteins and their links with A1b-nodulins that are short non-insecticidal disulfide-bonded peptides involved in root nodule signaling and ii) exploring the functional diversity of A1b for novel bioactive molecules. RESULTS: Investigating the Mtr genome revealed a remarkable expansion, mainly through tandem duplications, of albumin1 (A1) genes, retaining nearly all of the same canonical structure at both gene and protein levels. Phylogenetic analysis revealed that the ancestral molecule was most probably insecticidal giving rise to, among others, A1b-nodulins. Expression meta-analysis revealed that many A1b coding genes are silent and a wide tissue distribution of the A1 transcripts/peptides within plant organs. Evolutionary rate analyses highlighted branches and sites with positive selection signatures, including two sites shown to be critical for insecticidal activity. Seven peptides were chemically synthesized and folded in vitro, then assayed for their biological activity. Among these, AG41 (aka MtrA1013 isoform, encoded by the orphan TA24778 contig.), showed an unexpectedly high insecticidal activity. The study highlights the unique burst of diversity of A1 peptides within the Medicago genus compared to the other taxa for which full-genomes are available: no A1 member in Lotus, or in red clover to date, while only a few are present in chick pea, soybean or pigeon pea genomes. CONCLUSION: The expansion of the A1 family in the Medicago genus is reminiscent of the situation described for another disulfide-rich peptide family, the "Nodule-specific Cysteine-Rich" (NCR), discovered within the same species. The oldest insecticidal A1b toxin was described from the Sophorae, dating the birth of this seed-defense function to more than 58 million years, and making this model of plant/insect toxin/receptor (A1b/insect v-ATPase) one of the oldest known.

Effects of aging on the cardiac remodeling induced by chronic high-altitude hypoxia in rat.

Effects of chronic high-altitude hypoxia on the remodeling of right ventricle were examined in three age groups of rats: 2, 6, and 18 mo. The extent of right ventricular (RV) hypertrophy (RVH) showed an age-associated diminution. RV cell size and pericellular fibrosis showed a significant increase in the 2- and 6-mo-old exposed rats but not in the 18-mo-old exposed rats compared with control. A hyperplasic response was underscored in the three exposed age groups but appeared less pronounced in the 18-mo-old rats. A significant decrease in the transient outward potassium current (Ito) density was observed in RV cell only in the 2-mo-old exposed group compared with the control group. In the control group, there was a clear tendency for Ito density to decrease as a function of age. The sustained outward current density was modified neither by the hypoxia condition nor by the age. Neither the cytochrome c oxidase activity nor the heat shock protein 72 content in the RV was altered after hypoxic exposure regardless of age. The norepinephrine content in the RV was significantly decreased in each age group exposed to hypoxia when compared with their age-matched control group. Our findings indicate that the remodeling (at morphological and electrophysiological levels) induced by chronic hypoxia in the RV can be decreased by the natural aging process.

Reversibility of electrophysiological changes induced by chronic high-altitude hypoxia in adult rat heart.

Recent studies indicate that regression of left ventricular hypertrophy normalizes membrane ionic current abnormalities. This work was designed to determine whether regression of right ventricular hypertrophy induced by permanent high-altitude exposure (4,500 m, 20 days) in adult rats also normalizes changes of ventricular myocyte electrophysiology. According to the current data, prolonged action potential, decreased transient outward current density, and increased inward sodium/calcium exchange current density normalized 20 days after the end of altitude exposure, whereas right ventricular hypertrophy evidenced by both the right ventricular weight-to-heart weight ratio and the right ventricular free wall thickness measurement normalized 40 days after the end of altitude exposure. This morphological normalization occurred at both the level of muscular tissue, as shown by the decrease toward control values of some myocyte parameters (perimeter, capacitance, and width), and the level of the interstitial collagenous connective tissue. In the chronic high-altitude hypoxia model, the regression of right ventricular hypertrophy would not be a prerequisite for normalization of ventricular electrophysiological abnormalities.

Effects of calcium channel blockers on cloned cardiac K+ channels IKr and IKs.

Cloned HERG and KvLQT1-IsK K+ channels have been expressed in mammalian cells and assayed as a target for calcium channel blockers. These channels generate the rapid and slow components of the cardiac delayed rectifier K+ current, and mutations can affect them that lead to long QT syndromes. HERG is blocked by bepridil (EC50 = 0.55 microM), verapamil (EC50 = 0.83 microM) and mibefradil (EC50 = 1.43 microM), whereas nitrendipine and diltiazem have negligible effects. Steady-state activation and inactivation parameters are shifted to more negative values in the presence of the blockers. Similarly, KvLQT1-IsK is inhibited by bepridil (EC50 = 10.0 microM) and mibefradil (EC50 = 11.8 microM), whilst being insensitive to nitrendipine, diltiazem or verapamil. This work may help to understand the mechanisms of action of verapamil in certain ventricular tachycardias as well as some of the deleterious adverse cardiac events associated with bepridil and mibefradil.

Increased sodium-calcium exchange current in right ventricular cell hypertrophy induced by simulated high altitude in adult rats.

Ventricular hypertrophy is associated with an increase in action potential (AP) duration which is potentially arrhythmogenic. The implication of the Na-Ca exchange current (I(Na-Ca)) in the lengthening of the AP is controversial. The role of this current in the increased duration of the low plateau of the AP in hypertrophied adult rat ventricular myocytes by simulated chronic high-altitude exposure ( approximately 4500 m) was evaluated. Electrophysiological experiments were carried out on isolated right ventricular myocytes from exposed and control rats with the perforated patch or the conventional whole-cell technique in current or in voltage clamp condition. With the two techniques, a significant increase of the low plateau duration was observed in hypertrophied myocytes as compared to controls. The low plateau in hypertrophied myocytes was depressed when Na was replaced by Li and was no longer recorded when intracellular Ca was buffered with EGTA. Inward tail currents, evoked either on repolarization to -80 mV following a depolarizing pulse to +10 mV or by interrupted AP technique, were greater in hypertrophied than in control myocytes and were abolished when Na was replaced by Li or when intracellular Ca was buffered with EGTA, indicating an increased Na-Ca exchange activity. The Li-sensitive current-voltage curves, obtained by a voltage clamp ramp protocol with an intracellular calcium buffered solution, were not significantly different in both hypertrophied and control myocytes, suggesting no modification in the density of the Na-Ca exchange protein. This was corroborated by the lack of difference in NCX1 mRNA levels between right ventricles from control and exposed rats. We conclude that increased duration of the low plateau of rat ventricular AP in altitude cardiac hypertrophy may be attributed to an increase of the inward I(Na-Ca). This augmented I(Na-Ca)may result from a modification in the intracellular Ca homeostasis.

Opposite effects of halothane on guinea-pig ventricular action potential duration.

Halothane protects the heart against the reperfusion injury observed after an ischemia. In ischemic or anoxic conditions, a large ATP-sensitive K(+) (K(ATP)) conductance is supposed to provide an endogenous protection to the myocardium. In this study, we tested the possibility that halothane acted by modulating this conductance. Isolated guinea-pig cardiomyocytes were successively studied in current clamp and in voltage-clamp conditions. Action potentials regulation by halothane was tested in control conditions and in situations where the K(ATP) channels were activated. In control conditions, halothane decreased action potential duration of myocytes but did not significantly alter the inward rectifying K(+) current. Conversely, halothane lengthened action potential of cells in which the K(ATP) conductance was activated, by inhibiting the K(ATP) current. In ischemic conditions, simultaneous shortening of long action potentials and lengthening of shortened ones would be expected to homogenize the absolute refractory period at the border between normoxic and anoxic zones. This effect, together with a decrease in calcium load, could protect the myocardium against re-entrant arrhythmias.

Novel mutations in KvLQT1 that affect Iks activation through interactions with Isk.

OBJECTIVES: We report the functional expression of four KCNQ1 mutations affecting arginine residues and resulting in Romano-Ward (RW) and the Jervell and Lange-Nielsen (JLN) congenital long QT syndromes. RESULTS: The R539W and R190Q mutations were found in typical RW families with an autosomal dominant transmission. The R243H mutation was found in a compound heterozygous JLN patient who presents with deafness and cardiac symptoms. The fourth mutation, R533W, was a new case of recessive form of the RW syndrome since homozygous carriers experienced syncopes but showed no deafness, whereas the heterozygous carriers were asymptomatic. The R190Q mutation failed to produce functional homomeric channels. The R243H, R533W and R539W mutations induced a positive voltage shift of the channel activation but only when co-expressed with IsK, pointing out the critical role of these positively charged residues in the modulation of the gating properties of KvLQT1 by IsK. The positive shift induced by R533W was merely 15%. This small effect was compatible with the recessive character of the RW phenotype transmission. The average QTc was significantly longer (P < 0.01) in patients carrying mutations inducing a total loss of channel function and those patients were also prone to cardiac adverse symptoms (whether syncopes or sudden death) to a greater extent (62 vs. 21%, P < 0.001). CONCLUSIONS: Novel mutations are described that induce a voltage shift of the channel activation only in the presence of IsK. They appear associated with a milder cardiac phenotype.

The KCNQ2 potassium channel: splice variants, functional and developmental expression. Brain localization and comparison with KCNQ3.

Benign familial neonatal convulsions, an autosomal dominant epilepsy of newborns, are linked to mutations affecting two six-transmembrane potassium channels, KCNQ2 and KCNQ3. We isolated four splice variants of KCNQ2 in human brain. Two forms generate, after transient expression in COS cells, a potassium-selective current similar to the KCNQ1 current. L-735,821, a benzodiazepine molecule which inhibits the KCNQ1 channel activity (EC50 = 0.08 microM), also blocks KCNQ2 currents (EC50 = 1.5 microM). Using in situ hybridization, KCNQ2 and KCNQ3 have been localized within the central nervous system, in which they are expressed in the same areas, mainly in the hippocampus, the neocortex and the cerebellar cortex. During brain development, KCNQ3 is expressed later than KCNQ2.

HERG and KvLQT1/IsK, the cardiac K+ channels involved in long QT syndromes, are targets for calcium channel blockers.

We examined the effects of the calcium channel blockers nitrendipine, diltiazem, verapamil, bepridil, and mibefradil on the cloned HERG and KvLQT1/IsK K+ channels. These channels generate the rapid and slow components of the cardiac delayed rectifier K+ current, and mutations can affect them, which leads to long QT syndromes. When expressed in transfected COS cells, HERG is blocked in a concentration-dependent manner by bepridil (EC50 = 0.55 microM), verapamil (EC50 = 0.83 microM), and mibefradil (EC50 = 1.43 microM), whereas nitrendipine and diltiazem have negligible effects. Steady state activation and inactivation parameters are shifted to more negative values in the presence of the blockers. Similarly, KvLQT1/IsK is inhibited by bepridil (EC50 = 10.0 microM) and mibefradil (EC50 = 11.8 microM), while being insensitive to nitrendipine, diltiazem, or verapamil. These results demonstrate that both cloned K+ channels HERG and KvLQT1/IsK, which represent together the cardiac delayed rectifier K+ current, are sensitive targets to calcium channel blockers. This work may help in understanding the mechanisms of action of verapamil in certain ventricular tachycardia, as well as some of the deleterious adverse cardiac events associated with bepridil.

Involvement of IsK-associated K+ channel in heart rate control of repolarization in a murine engineered model of Jervell and Lange-Nielsen syndrome.

The Jervell and Lange-Nielsen (JLN) syndrome affects the human cardioauditory system, associating a profound bilateral deafness with an abnormally long QT interval on the ECG. It results from mutations in KVLQT1 and ISK genes that encode the 2 subunits forming the K+ channel responsible for the cardiac and inner ear slowly activating component of the delayed rectifier K+ current (IKs). A JLN mouse model that presents typical inner ear defects has been created by knocking out the isk gene (isk-/-). This study specifically reports on the cardiac phenotype counterpart, determined in the whole animal and at mRNAs and cellular levels. Surface ECG recordings of isk-/- mice showed a longer QT interval at slow heart rates, a paradoxical shorter QT interval at fast heart rates, and an overall exacerbated QT-heart rate adaptation compared with wild-type (WT) mice. A 300-ms increase in the heart rate cycle length induces a 309+/-21% increase in the QT duration of the WT mice versus a 500+/-50% in isk-/- mice (P<0.001). It is concluded that the isk gene product and/or IKs, when present, blunts the QT adaptation to heart rate variations and that steeper QT-RR relationships reflect a greater susceptibility to arrhythmias in patients lacking IKs.

Properties of KvLQT1 K+ channel mutations in Romano-Ward and Jervell and Lange-Nielsen inherited cardiac arrhythmias.

Mutations in the delayed rectifier K+ channel subunit KvLQT1 have been identified as responsible for both Romano-Ward (RW) and Jervell and Lange-Nielsen (JLN) inherited long QT syndromes. We report the molecular cloning of a human KvLQT1 isoform that is expressed in several human tissues including heart. Expression studies revealed that the association of KvLQT1 with another subunit, IsK, reconstitutes a channel responsible for the IKs current involved in ventricular myocyte repolarization. Six RW and two JLN mutated KvLQT1 subunits were produced and co-expressed with IsK in COS cells. All the mutants, except R555C, fail to produce functional homomeric channels and reduce the K+ current when co-expressed with the wild-type subunit. Thus, in both syndromes, the main effect of the mutations is a dominant-negative suppression of KvLQT1 function. The JLN mutations have a smaller dominant-negative effect, in agreement with the fact that the disease is recessive. The R555C subunit forms a functional channel when expressed with IsK, but with altered gating properties. The voltage dependence of the activation is strongly shifted to more positive values, and deactivation kinetics are accelerated. This finding indicates the functional importance of a small positively charged cytoplasmic region of the KvLQT structure where two RW and one JLN mutations have been found to take place.

Molecular mechanism and functional significance of the MinK control of the KvLQT1 channel activity.

The very slowly activating delayed rectifier K+ channel IKs is essential for controlling the repolarization phase of cardiac action potentials and K+ homeostasis in the inner ear. The IKs channel is formed via the assembly of two transmembrane proteins, KvLQT1 and MinK. Mutations in KvLQT1 are associated with a long QT syndrome that causes syncope and sudden death and also with deafness. Here, we show a new mode of association between ion channel forming subunits in that the cytoplasmic C-terminal end of MinK interacts directly with the pore region of KvLQT1. This interaction reduces KvLQT1 channel conductance from 7.6 to 0.58 picosiemens. However, because MinK also reveals a large number of previously silent KvLQT1 channels (x 60), the overall effect is a large increase (x 4) in the macroscopic K+ current. Conformational changes associated with the KvLQT1/MinK association create very slow and complex activation kinetics without much alteration in the deactivation process. Changes induced by MinK have an essential regulatory role in the development of this K+ channel activity upon repetitive electrical stimulation with a particular interest in tachycardia.

Reduction of I(Ca,L) and I(to1) density in hypertrophied right ventricular cells by simulated high altitude in adult rats.

The present paper describes the effect of a simulated hypobaric condition (at the altitude of 4500 m) on morphological characteristics and on some ionic currents in ventricular cells of adult rats. According to current data, chronic high-altitude exposure led to mild right ventricular hypertrophy. Increase in right ventricular weight appeared to be due wholly or partly to an enlargement of myocytes. The whole-cell patch-clamp technique was used and this confirmed, by cell capacitance measurement, that chronic high-altitude exposure induced an increase in the size of the right ventricular cells. Hypertrophied cells showed prolongation of action potential (AP). Four ionic currents, playing a role along with many others in the precise balance of inward and outward currents that control the duration of cardiac AP, were investigated. We report a significant decrease in the transient outward (I(to1)) and in the L-type calcium current (I(Ca,L)) densities while there was no significant difference in the delayed rectifier current (I(K)) or in the inward rectifier current (I(K1)) densities in hypertrophied right ventricular cells compared to control cells. At a given potential the decrease in I(to 1) density was relatively more important than the decrease in I(Ca,L) density. In both cell types, all the currents displayed the same voltage dependence. The inactivation kinetics of I(to 1) and I(Ca,L) or the steady-state activation and inactivation relationships were not significantly modified by chronic high-altitude exposure. We conclude that chronic high-altitude exposure induced true right ventricular myocyte hypertrophy and that the decrease in I(to 1) density might account for the lengthened action potential, or have a partial effect.