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Channel Isoforms (channel + isoform)
Selected AbstractsBiochemical properties of V91G calmodulin: A calmodulin point mutation that deregulates muscle contraction in DrosophilaPROTEIN SCIENCE, Issue 12 2004Bo Wang Abstract A mutation (Cam7) to the single endogenous calmodulin gene of Drosophila generates a mutant protein with valine 91 changed to glycine (V91G D-CaM). This mutation produces a unique pupal lethal phenotype distinct from that of a null mutation. Genetic studies indicate that the phenotype reflects deregulation of calcium fluxes within the larval muscles, leading to hypercontraction followed by muscle failure. We investigated the biochemical properties of V91G D-CaM. The effects of the mutation on free CaM are minor: Calcium binding, and overall secondary and tertiary structure are indistinguishable from those of wild type. A slight destabilization of the C-terminal domain is detectable in the calcium-free (apo-) form, and the calcium-bound (holo-) form has a somewhat lower surface hydrophobicity. These findings reinforce the indications from the in vivo work that interaction with a specific CaM target(s) underlies the mutant defects. In particular, defective regulation of ryanodine receptor (RyR) channels was indicated by genetic interaction analysis. Studies described here establish that the putative CaM binding region of the Drosophila RyR (D-RyR) binds wild-type D-CaM comparably to the equivalent CaM-RyR interactions seen for the mammalian skeletal muscle RyR channel isoform (RYR1). The V91G mutation weakens the interaction of both apo- and holo-D-CaM with this binding region, and decreases the enhancement of the calcium-binding affinity of CaM that is detectable in the presence of the RyR target peptide. The predicted functional consequences of these changes are consonant with the in vivo phenotype, and indicate that D-RyR is one, if not the major, target affected by the V91G mutation in CaM. [source] Expression pattern of neuronal and skeletal muscle voltage-gated Na+ channels in the developing mouse heartTHE JOURNAL OF PHYSIOLOGY, Issue 3 2005Volker Haufe In the mammalian heart, a variety of voltage-gated Na+ channel transcripts and proteins have been detected. However, little quantitative information is available on the abundance of each transcript during development, or the contribution of TTX-sensitive Na+ channels to the cardiac sodium current (INa). Using competitive and real-time RT-PCR we investigated the transcription of six Na+ channels (Nav1.1,Nav1.6) and the ,1 subunit during mouse heart development. Nav1.5 was predominantly expressed in the adult heart, whereas the splice variant Nav1.5a was the major Na+ channel isoform in embryonic hearts. The TTX-resistant Na+ channel transcripts (Nav1.5 and Nav1.5a) increased 1.7-fold during postnatal development. Transcripts encoding TTX-sensitive Na+ channels (Nav1.1,Nav1.4) and the ,1 subunit gradually increased up to fourfold from postnatal day (P)1 to P126, while the Nav1.6 transcript level remained low and constant over the same period. In adults, TTX-sensitive channel mRNA accounted for 30,40% of the channel pool in whole-heart preparations (Nav1.3 > Nav1.4 > Nav1.2 , Nav1.1 , Nav1.6), and 16% in mRNA from isolated cardiomyocytes (Nav1.4 > Nav1.3 > Nav1.2 > Nav1.1 > Nav1.6). Confocal immunofluorescence on ventricular myocytes suggested that Nav1.1 and Nav1.2 were localized at the intercalated disks and in the t tubules. Nav1.3 labelling predominantly produced a diffuse but strong intracellular signal. Nav1.6 fluorescence was detected only along the Z lines. Electrophysiological recordings showed that TTX-sensitive and TTX-resistant Na+ channels, respectively, accounted for 8% and 92% of the INa in adult ventricular cardiomyocytes. Our data suggest that neuronal and skeletal muscle Na+ channels contribute to the action potential of cardiomyocytes in the adult mammalian heart. [source] Regulated expression of HCN channels and cAMP levels shape the properties of the h current in developing rat hippocampusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2006Rainer Surges Abstract The hyperpolarization-activated current (Ih) contributes to intrinsic properties and network responses of neurons. Its biophysical properties depend on the expression profiles of the underlying hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels and the presence of cyclic AMP (cAMP) that potently and differentially modulates Ih conducted by HCN1, HCN2 and/or HCN4. Here, we studied the properties of Ih in hippocampal CA1 pyramidal cells, the developmental evolution of the HCN-subunit isoforms that contribute to this current, and their interplay with age-dependent free cAMP concentrations, using electrophysiological, molecular and biochemical methods. Ih amplitude increased progressively during the first four postnatal weeks, consistent with the observed overall increased expression of HCN channels. Activation kinetics of the current accelerated during this period, consonant with the quantitative reduction of mRNA and protein expression of the slow-kinetics HCN4 isoform and increased levels of HCN1. The sensitivity of Ih to cAMP, and the contribution of the slow component to the overall Ih, decreased with age. These are likely a result of the developmentally regulated transition of the complement of HCN channel isoforms from cAMP sensitive to relatively cAMP insensitive. Thus, although hippocampal cAMP concentrations increased over twofold during the developmental period studied, the coordinated changes in expression of three HCN channel isoforms resulted in reduced effects of this signalling molecule on neuronal h currents. [source] Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissuesFEBS JOURNAL, Issue 6 2001Sven Moosmang Hyperpolarization-activated cation currents (Ih) have been identified in cardiac pacemaker cells and a variety of central and peripheral neurons. Four members of a gene family encoding hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCN1,4) have been cloned recently. Native Ih currents recorded from different cell types exhibit distinct activation kinetics. To determine if this diversity of Ih currents may be caused by differential expression of HCN channel isoforms, we investigated the cellular distribution of the transcripts of HCN1,4 in the murine sinoatrial node, retina and dorsal root ganglion (DRG) by in situ hybridization. In the sinoatrial node, the most prominently expressed HCN channel is HCN4, whereas HCN2 and HCN1 are detected there at moderate and low levels, respectively. Retinal photoreceptors express high levels of HCN1, whereas HCN2, 3 and 4 were not found in these cells. In DRG neurons, the dominant HCN transcript is HCN1, followed by HCN2. We next determined the functional properties of recombinant HCN1,4 channels expressed in HEK293 cells. All four channel types gave rise to Ih currents but displayed marked differences in their activation kinetics. Our results suggest that the heterogeneity of native Ih currents is generated, at least in part, by the tissue-specific expression of HCN channel genes. [source] Complementation of Physiological and Behavioral Defects by a Slowpoke Ca2+ -Activated K+ Channel TransgeneJOURNAL OF NEUROCHEMISTRY, Issue 3 2000Robert Brenner Abstract: The Drosophila slowpoke gene encodes a large conductance calcium-activated potassium channel used in neurons, muscle, and some epithelial cells. Tissue-specific transcriptional promoters and alternative mRNA splicing generate a large array of transcripts. These distinct transcripts are thought to tailor the properties of the channel to the requirements of the cell. Presumably, a single splice variant cannot satisfy the specific needs of all cell types. To test this, we examined whether a single slowpoke splice variant was capable of complementing all slowpoke behavioral phenotypes. Null mutations in slowpoke cause animals to be semiflightless and to manifest an inducible "sticky-feet" phenotype. The well-characterized slowpoke transcriptional control region was used to direct the expression of a single slowpoke splice variant (cDNA H13) in transgenic flies. The endogenous gene in these flies had been inactivated by the slo4 mutation. Action-potential recordings and voltage-clamp recordings demonstrated the production of functional channels from the transgene. The transgene completely complemented the flight defect, but not the sticky-feet phenotype. We conclude that distinct slowpoke channel isoforms, produced by alternative splicing, are not interchangeable and are required for proper function of different cell types. [source] The Role of K+ Channels in Determining Pulmonary Vascular Tone, Oxygen Sensing, Cell Proliferation, and Apoptosis: Implications in Hypoxic Pulmonary Vasoconstriction and Pulmonary Arterial HypertensionMICROCIRCULATION, Issue 8 2006ROHIT MOUDGIL ABSTRACT Potassium channels are tetrameric, membrane-spanning proteins that selectively conduct K+ at near diffusion-limited rates. Their remarkable ionic selectivity results from a highly-conserved K+ recognition sequence in the pore. The classical function of K+ channels is regulation of membrane potential (EM) and thence vascular tone. In pulmonary artery smooth muscle cells (PASMC), tonic K+ egress, driven by a 145/5 mM intracellular/extracellular concentration gradient, contributes to a EM of about ,60 mV. It has been recently discovered that K+ channels also participate in vascular remodeling by regulating cell proliferation and apoptosis. PASMC express voltage-gated (Kv), inward rectifier (Kir), calcium-sensitive (KCa), and two-pore (K2P) channels. Certain K+ channels are subject to rapid redox regulation by reactive oxygen species (ROS) derived from the PASMC's oxygen-sensor (mitochondria and/or NADPH oxidase). Acute hypoxic inhibition of ROS production inhibits Kv1.5, which depolarizes EM, opens voltage-sensitive, L-type calcium channels, elevates cytosolic calcium, and initiates hypoxic pulmonary vasoconstriction (HPV). Hypoxia-inhibited K+ currents are not seen in systemic arterial SMCs. Kv expression is also transcriptionally regulated by HIF-1, and NFAT. Loss of PASMC Kv1.5 and Kv2.1 contributes to the pathogenesis of pulmonary arterial hypertension (PAH) by causing a sustained depolarization, which increases intracellular calcium and K+, thereby stimulating cell proliferation and inhibiting apoptosis, respectively. Restoring Kv expression (via Kv1.5 gene therapy, dichloroacetate, or anti-survivin therapy) reduces experimental PAH. Electrophysiological diversity exists within the pulmonary circulation. Resistance PASMC have a homogeneous Kv current (including an oxygen-sensitive component), whereas conduit PASMC current is a Kv/KCa mosaic. This reflects regional differences in expression of channel isoforms, heterotetramers, splice variants, and regulatory subunits as well as mitochondrial diversity. In conclusion, K+ channels regulate pulmonary vascular tone and remodeling and constitute potential therapeutic targets in the regression of PAH. [source] Functional expression of the hyperpolarization-activated, non-selective cation current If in immortalized HL-1 cardiomyocytesTHE JOURNAL OF PHYSIOLOGY, Issue 1 2002Laura Sartiani HL-1 cells are adult mouse atrial myocytes induced to proliferate indefinitely by SV40 large T antigen. These cells beat spontaneously when confluent and express several adult cardiac cell markers including the outward delayed rectifier K+ channel. Here, we examined the presence of a hyperpolarization-activated If current in HL-1 cells using the whole-cell patch-clamp technique on isolated cells enzymatically dissociated from the culture at confluence. Cell membrane capacitance (Cm) ranged from 5 to 53 pF. If was detected in about 30 % of the cells and its occurrence was independent of the stage of the culture. If maximal slope conductance was 89.7 ± 0.4 pS pF,1 (n= 10). If current in HL-1 cells showed typical characteristics of native cardiac If current: activation threshold between ,50 and ,60 mV, half-maximal activation potential of ,83.1 ± 0.7 mV (n= 50), reversal potential at ,20.8 ± 1.5 mV (n= 10), time-dependent activation by hyperpolarization and blockade by 4 mm Cs+. In half of the cells tested, activation of adenylyl cyclase by the forskolin analogue L858051 (20 ,m) induced both a ,6 mV positive shift of the half-activation potential and a ,37 % increase in the fully activated If current. RT-PCR analysis of the hyperpolarization-activated, cyclic nucleotide-gated channels (HCN) expressed in HL-1 cells demonstrated major contributions of HCN1 and HCN2 channel isoforms to If current. Cytosolic Ca2+ oscillations in spontaneously beating HL-1 cells were measured in Fluo-3 AM-loaded cells using a fast-scanning confocal microscope. The oscillation frequency ranged from 1.3 to 5 Hz and the spontaneous activity was stopped in the presence of 4 mm Cs+. Action potentials from HL-1 cells had a triangular shape, with an overshoot at +15 mV and a maximal diastolic potential of ,69 mV, i.e. more negative than the threshold potential for If activation. In conclusion, HL-1 cells display a hyperpolarization-activated If current which might contribute to the spontaneous contractile activity of these cells. [source] | |||||||||||||