The repolarization phase of cardiac action potential is prone to aberrant excitation that is common in cardiac patients. Here, we demonstrate that this phase is markedly sensitive to Ca 2؉ because of the surprising existence of a Ca 2؉ -activated K ؉ currents in cardiac cells. The current was revealed using recording conditions that preserved endogenous Ca 2؉ buffers. The Ca 2؉ -activated K ؉ current is expressed differentially in atria compared with ventricles. Because of the significant contribution of the current toward membrane repolarization in cardiac myocytes, alterations of the current magnitude precipitate abnormal action potential profiles. We confirmed the presence of a small conductance Ca 2؉ -activated K ؉ channel subtype (SK2) in human and mouse cardiac myocytes using Western blot analysis and reverse transcription-polymerase chain reaction and have cloned SK2 channels from human atria, mouse atria, and ventricles. Because of the marked differential expression of SK2 channels in the heart, specific ligands for Ca 2؉ -activated K ؉ currents may offer a unique therapeutic opportunity to modify atrial cells without interfering with ventricular myocytes.Cardiac action potentials (APs) 1 are shaped predominantly by the interplay between transient inward Na ϩ , Ca 2ϩ , and outward K ϩ currents (1). While the repolarization phase of the AP can be wrought by the kinetics of the principal currents, small and sustained outward currents also define this phase, rendering this region prone to irregular membrane excitation.In humans, delineation of the outward currents that confer the late repolarization phase of the cardiac AP is crucial for our understanding of the etiology of arrhythmias. We provide a novel report that demonstrates that the repolarization phase of cardiac AP shows marked sensitivity toward apamin, an exclusive ligand for a small conductance Ca 2ϩ -activated K ϩ channel (2).Ca 2ϩ -activated K ϩ channels (K Ca ) are present in most neurons and mediate the afterhyperpolarizations following AP (3, 4). However, functional significance of K Ca in the heart has not previously been documented. K Ca channels can be divided into three main subfamilies (3, 5-7). These include the large-conductance Ca 2ϩ -and voltage-activated K ϩ channels (BK), the intermediate-conductance K Ca channels (IK), and the smallconductance K Ca channels (SK), which are sensitive to apamin and scyllatoxin. Among the SK channels, they are encoded by at least three genes, SK1, SK2, SK3 (4, 6), with differential sensitivity toward apamin. SK2 is highly sensitive to apamin, with a half-blocking concentration (IC 50 ) of 60 pmol/liter, whereas SK1 channels are not affected by 100 nmol/liter apamin (2). SK3 channels are intermediate.Here, we report for the first time, the presence of I K,Ca (Ca 2ϩ -activated K ϩ current) in cardiac myocytes that plays a crucial role in cardiac AP profile. Using a combination of electrophysiological recordings and biochemical and molecular biological techniques, we have identified the presence of SK2...

Small-conductance Ca2+-activated K+ channels (SK channels, KCa channels) have been reported in excitable cells, where they aid in integrating changes in intracellular Ca2+ with membrane potential. We recently reported for the first time the functional existence of SK2 (KCa2.2) channels in human and mouse cardiac myocytes. Here, we report cloning of SK1 (KCa2.1) and SK3 (KCa2.3) channels from mouse atria and ventricles using RT-PCR. Full-length transcripts and their variants were detected for both SK1 and SK3 channels. Variants of mouse SK1 channel (mSK1) differ mainly in the COOH-terminal structure, affecting a portion of the sixth transmembrane segment (S6) and the calmodulin binding domain (CaMBD). Mouse SK3 channel (mSK3) differs not only in the number of polyglutamine repeats in the NH2 terminus but also in the intervening sequences between the polyglutamine repeats. Full-length cardiac mSK1 and mSK3 show 99 and 91% nucleotide identity with those of mouse colon SK1 and SK3, respectively. Quantification of SK1, SK2, and SK3 transcripts between atria and ventricles was performed using real-time quantitative RT-PCR from single, isolated cardiomyocytes. SK1 transcript was found to be more abundant in atria compared with ventricles, similar to the previously reported finding for SK2 channel. In contrast, SK3 showed similar levels of expression in atria and ventricles. Together, our data are the first to indicate the presence of the three different isoforms of SK channels in heart and the differential expression of SK1 and SK2 in mouse atria and ventricles. Because of the marked differential expression of SK channel isoforms in heart, specific ligands for Ca2+-activated K+ currents may offer a unique therapeutic opportunity to modify atrial cells without interfering with ventricular myocytes.

The biosynthetic gene cluster for the enediyne antitumor antibiotic neocarzinostatin (NCS) was localized to 130 kb continuous DNA from Streptomyces carzinostaticus ATCC15944 and confirmed by gene inactivation. DNA sequence analysis of 92 kb of the cloned region revealed 68 open reading frames (ORFs), 47 of which were determined to constitute the NCS cluster. Sequence analysis of the genes within the NCS cluster suggested dNDP-D-mannose as a precursor for the deoxy aminosugar, revealed two distinct type I polyketide synthases (PKSs), and supported a convergent model for NCS chromophore biosynthesis from the deoxy aminosugar, naphthoic acid, and enediyne core building blocks. These findings shed light into deoxysugar biosynthesis, further support the iterative type I PKS paradigm for enediyne core biosynthesis, and unveil a mechanism for microbial polycyclic aromatic polyketide biosynthesis by an iterative type I PKS.

Human KCNQ4 mutations known as DFNA2 cause non-syndromic, autosomal-dominant, progressive high-frequency hearing loss in which the cellular and molecular basis is unclear. We provide immunofluorescence data showing that Kcnq4 expression in the adult cochlea has both longitudinal (base to apex) and radial (inner to outer hair cells) gradients. The most intense labeling is in outer hair cells at the apex and in inner hair cells as well as spiral ganglion neurons at the base. Spatiotemporal expression studies show increasing intensity of KCNQ4 protein labeling from postnatal day 21 (P21) to P120 mice that is most apparent in inner hair cells of the middle turn. We have identified four alternative splice variants of Kcnq4 in mice. The alternative use of exons 9 -11 produces three transcript variants (v1-v3), whereas the fourth variant (v4) skips all three exons; all variants have the same amino acid sequence at the C termini. Both reverse transcription-PCR and quantitative PCR analyses demonstrate that these variants have differential expression patterns along the length of the mouse organ of Corti and spiral ganglion neurons. Our expression data suggest that the primary defect leading to highfrequency loss in DFNA2 patients may be attributable to high levels of the dysfunctional Kcnq4_v3 variant in the spiral ganglion and inner hair cells in the basal hook region. Progressive hearing loss associated with aging may result from an increasing mutational load expansion toward the apex in inner hair cells and spiral ganglion neurons.

Multiple Ca2+ channels confer diverse functions to hair cells of the auditory and vestibular organs in the mammalian inner ear. We used gene-targeting technology to generate a 1D Ca 2+ channel-deficient mice to determine the physiological role of these Ca 2+ channels in hearing and balance. Analyses of auditory-evoked brainstem recordings confirmed that a 1D )/) mice were deaf and revealed that heterozygous (a 1D +/) ) mice have increased hearing thresholds. However, hearing deficits in a 1D +/) mice were manifested mainly by the increase in threshold of low-frequency sounds. In contrast to impaired hearing, a 1D )/) mice have balance performances equivalent to their wild-type littermates. Light and electron microscope analyses of the inner ear revealed outer hair cell loss at the apical cochlea, but no apparent abnormality at the basal cochlea and the vestibule. We determined the mechanisms underlying the auditory function defects and the normal vestibular functions by examining the Ba 2+ currents in cochlear inner and outer hair cells versus utricular hair cells in a 1D +/) mice. Whereas the whole-cell Ba 2+ currents in inner hair cells consist mainly of the nimodipine-sensitive current (85%), the utricular hair cells express only 50% of this channel subtype. Thus, differential expression of a 1D channels in the cochlear and utricular hair cells confers the phenotype of the a 1D null mutant mice. Because vestibular and cochlear hair cells share common features and null deletion of several genes have yielded both deafness and imbalance in mice, a 1D null mutant mice may serve as a model to disentangle vestibular from auditory-specific functions.

The structural phenotype of neural connections in the auditory brainstem is sculpted by spontaneous and stimulus-induced neural activities during development. However, functional and molecular mechanisms of spontaneous action potentials (SAPs) in the developing cochlea are unknown. Additionally, it is unclear how regenerating hair cells establish their neural ranking in the constellation of neurons in the brainstem. We have demonstrated that a transient Ca 2؉ current produced by the Cav3.1 channel is expressed early in development to initiate spontaneous Ca 2؉ spikes. Ca 2ϩ currents ͉ cochlea ͉ hearing ͉ spontaneous activity

Amomum villosum is an important medicinal and edible plant with several pharmacologically active volatile oils. However, identifying A. villosum from A. villosum var. xanthioides and A. longiligulare which exhibit similar morphological characteristics to A. villosum, is difficult. The main goal of this study, therefore, is to mine genetic resources and improve molecular methods that could be used to distinguish these species. A total of eight complete chloroplasts (cp) genomes of these Amomum species which were collected from the main producing areas in China were determined to be 163,608–164,069 bp in size. All genomes displayed a typical quadripartite structure with a pair of inverted repeat (IR) regions (29,820–29,959 bp) that separated a large single copy (LSC) region (88,680–88,857 bp) from a small single copy (SSC) region (15,288–15,369 bp). Each genome encodes 113 different genes with 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. More than 150 SSRs were identified in the entire cp genomes of these three species. The Sanger sequencing results based on 32 Amomum samples indicated that five highly divergent regions screened from cp genomes could not be used to distinguish Amomum species. Phylogenetic analysis showed that the cp genomes could not only accurately identify Amomum species, but also provide a solid foundation for the establishment of phylogenetic relationships of Amomum species. The availability of cp genome resources and the comparative analysis is beneficial for species authentication and phylogenetic analysis in Amomum.

Zingiber officinale, commonly known as ginger, is an important plant of the family Zingiberaceae and is widely used as an herbal medicine and condiment. The lack of chloroplast genomic information hinders molecular research and phylogenetic analysis on ginger. We introduced the complete chloroplast genome of Z. officinale and identified its phylogenetic position in Zingiberaceae. The chloroplast genome of Z. officinale is 162,621 bp with a four-part circular structure and 36.1% GC content. All 113 unique genes were annotated. A total of 78 simple sequence repeats (SSRs) and 42 long repeat sequences, which are potential areas for species authentication, were found. Comparative analysis revealed some highly variable regions, including rps16-trnQ-UUG, atpH-atpI, trnT-UGU-trnL-UAA, ycf1, and psaC-ndhE. Moreover, the small single-copy (SSC) region was the most variable region in all four shared regions, indicating that it may be undergoing rapid nucleotide substitution in the family Zingiberaceae. Phylogenetic analysis based on all available chloroplasts of Zingiberales in the National Center for Biotechnology Information indicated that Zingiber is a sister branch to Kaempferia species. The availability of the Z. officinale chloroplast genome provided invaluable data for species-level authentication and phylogenetic analysis and can thus benefit further investigations on species in the family Zingiberaceae.