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At least 361 records · Page 20Linked to original sources

PubMed2000

Role of the calcium-independent transient outward current I(to1) in shaping action potential morphology and duration.

The Kv4.3-encoded current (I:(Kv4.3)) has been identified as the major component of the voltage-dependent Ca(2+)-independent transient outward current (I:(to1)) in human and canine ventricular cells. Experimental evidence supports a correlation between I:(to1) density and prominence of the phase 1 notch; however, the role of I:(to1) in modulating action potential duration (APD) remains unclear. To help resolve this role, Markov state models of the human and canine Kv4.3- and Kv1.4-encoded currents at 35 degrees C are developed on the basis of experimental measurements. A model of canine I:(to1) is formulated as the combination of these Kv4.3 and Kv1.4 currents and is incorporated into an existing canine ventricular myocyte model. Simulations demonstrate strong coupling between L-type Ca(2+) current and I:(Kv4.3) and predict a bimodal relationship between I:(Kv4.3) density and APD whereby perturbations in I:(Kv4.3) density may produce either prolongation or shortening of APD, depending on baseline I:(to1) current level.

Action Potentials↗

PubMed2000

Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened.

Secreted signaling proteins of the Hedgehog family organize spatial pattern during animal development. Two integral membrane proteins have been identified with distinct roles in Hedgehog signaling. Patched functions in Hedgehog binding, and Smoothened functions in transducing the signal. Current models view Patched and Smoothened as a preformed receptor complex that is activated by Hedgehog binding. Here we present evidence that Patched destabilizes Smoothened in the absence of Hedgehog. Hedgehog binding causes removal of Patched from the cell surface. In contrast, Hedgehog causes phosphorylation, stabilization, and accumulation of Smoothened at the cell surface. Comparable effects can be produced by removing Patched from cells by RNA-mediated interference. These findings raise the possibility that Patched acts indirectly to regulate Smoothened activity.

Animals↗

PubMed1996

Impaired coupling of glucose signal to the exocytotic machinery in diabetic GK rats: a defect ameliorated by cAMP.

The GK rat is a spontaneous model of NIDDM. The insulin response to 16.7 mmol/l glucose was markedly impaired in both isolated perfused pancreas and isolated islets from GK rats compared with control Wistar rats. Depolarization with 30 mmol/l KCl in the presence of 3.3 mmol/l glucose and 250 micromol/l diazoxide induced similar insulin responses in perfused pancreases of GK and control rats. In contrast, the glucose-stimulated insulin release was also severely impaired in GK pancreases in the depolarized state. Forskolin (1 micromol/l) markedly enhanced insulin release at 3.3 mmol/l glucose in GK but not control pancreases (54 +/- 15 vs. 3 +/- 1 pmol/10 min, P < 0.001). Dibutyryl cAMP (1 mmol/l) exerted effects similar to forskolin on insulin release in the perfused pancreas. In depolarized pancreases of GK but not control rats, forskolin also induced a marked insulin response at 3.3 mmol/l glucose (163 +/- 48 vs. 16 +/- 1 pmol/20 min, P < 0.03). Similarly, in studies on isolated islets from GK rats cultured in 5.5 or 16.7 mmol/l glucose for 48 h, forskolin (5 pmol/l) restored insulin release in response to 16.7 mmol/l glucose but had no effect on islet glucose utilization at 3.3 or 16.7 mmol/l glucose. Forskolin markedly stimulated insulin release at 3.3 mmol/l glucose in GK but not control rat islets cultured for 48 h in 5.5 mmol/l glucose, whereas 20 mmol/l arginine had an almost identical effect in both islet varieties. However, in islets cultured in 16.7 mmol/l glucose, forskolin stimulated insulin release similarly both in control and GK islets at 3.3 mmol/l glucose. In conclusion, this study suggests that the insulinotropic effects of glucose are coupled to a direct regulation of the exocytotic machinery in the pancreatic beta-cell. This pathway is markedly impaired in GK rats, contributing to defective insulin response to glucose. In this model, cAMP generation restores the insulin response to 16.7 mmol/l glucose and exerts a marked insulin release even at 3.3 mmol/l glucose.

Animals↗

PubMed2005

Temporal autocorrelation can enhance the persistence and abundance of metapopulations comprised of coupled sinks.

In spatially heterogeneous landscapes, some habitats may be persistent sources, providing immigrants to sustain populations in unfavorable sink habitats (where extinction is inevitable without immigration). Recent theoretical and empirical studies of source-sink systems demonstrate that temporally variable local growth rates in sinks can substantially increase average abundance of a persisting population, provided that the variation is positively autocorrelated--in effect, temporal variation inflates average abundance. Here we extend these results to a metapopulation in which all habitat patches are sinks. Using numerical studies of a population with discrete generations (buttressed by analytic results), we show that temporal variation and moderate dispersal can jointly permit indefinite persistence of the metapopulation and that positive autocorrelation both lowers the magnitude of variation required for persistence and increases the average abundance of persisting metapopulations. These effects are weakened--but not destroyed--if variation in local growth rates is spatially synchronized and dispersal is localized. We show that the inflationary effect is robust to a number of extensions of the basic model, including demographic stochasticity and density dependence. Because ecological and environmental processes contributing to temporally variable growth rates in natural populations are typically autocorrelated, these observations may have important implications for species persistence.

Ecosystem↗

PubMed2006

Ion interaction at the pore of Lc-type Ca2+ channel is sufficient to mediate depolarization-induced exocytosis.

The coupling of voltage-gated Ca2+ channel (VGCC) to exocytotic proteins suggests a regulatory function for the channel in depolarization-evoked exocytosis. To explore this possibility we have examined catecholamine secretion in PC12 and chromaffin cells. We found that replacing Ca2+ with La3+ or other lanthanide ions supported exocytosis in divalent ion-free solution. Cd2+, nifedipine, or verapamil inhibited depolarization-evoked secretion in La3+, indicating specific binding of La3+ at the pore of L-type VGCC, probably at the poly-glutamate (EEEE) locus. Lanthanide efficacy was stringently dependent on ionic radius with La3+>Ce3+>Pr3+, consistent with a size-selective binding interface of trivalent cations at the channel pore. La3+ inward currents were not detected and the highly sensitive La3+/fura-2 imaging assay (approximately 1 pm) detected no La3+ entry, cytosolic La3+ build-up, or alterations in cytosolic Ca2. These results provide strong evidence that occupancy of the pore of the channel by an impermeable cation leads to a conformational change that is transmitted to the exocytotic machinery upstream of intracellular cation build-up (intracellular Ca2+ concentration). Our model allows for a tight temporal and spatial coupling between the excitatory stimulation event and vesicle fusion. It challenges the conventional view that intracellular Ca2+ ion build-up via VGCC permeation is required to trigger secretion and establishes the VGCC as a plausible Ca2+ sensor protein in the process of neuroendocrine secretion.

Adrenal Medulla↗

PubMed2004

Crystal structure of a platelet-agglutinating factor isolated from the venom of Taiwan habu (Trimeresurus mucrosquamatus).

Platelet glycoprotein Ib (GPIb)-binding proteins (GPIb-BPs) from snake venoms are usually C-type lectins, which target specific sites of GPIbalpha and elicit distinct effects on platelets. In the present paper, we report a tetrameric platelet-agglutinating factor (molecular mass 121.1 kDa), termed mucrocetin, purified from the venom of Taiwan habu (Trimeresurus mucrosquamatus ). Mucrocetin is a GPIbalpha agonist with a binding site distinct from that of flavocetin-A (a snake venom GPIbalpha antagonist) on GPIbalpha, in spite of the high sequence identity (94.6%) between the two venom lectins. The crystal structure of mucrocetin was solved and refined to 2.8 A (1 A=0.1 nm) resolution, which shows an interesting crystal packing of six-layer cylinders of doughnut-shaped molecules. The four alphabeta heterodimers are arranged in an unusual square-shaped ring stabilized by four interdimer 'head-to-tail' disulphide bridges. Detailed structural comparison between mucrocetin and flavocetin-A suggests that their disparate platelet effects are probably attributable to different charge distributions on the putative concave binding surface. A unique positively charged patch on the binding surface of mucrocetin, formed by Lys102, Lys108, Lys109 and Arg123 in the alpha-subunit coupled with Lys22, Lys102, Lys116 and Arg117 in the beta-subunit, appears to be the primary determinant of its platelet-agglutinating activity. Conceivably, this interesting venom factor may provide a useful tool to study platelet agglutination by binding to the GPIb-IX-V complex.

Amino Acid Sequence↗

PubMed1989

ATP-sensitive K+ channels in a plasma membrane H+-ATPase mutant of the yeast Saccharomyces cerevisiae.

A mutant in the plasma membrane H+-ATPase gene of the yeast Saccharomyces cerevisiae with a reduced H+-ATPase activity, when examined at the single-channel level with the patch-clamp technique, was found to exhibit K+ channels activated by intracellular application of ATP. In the parent strain, the same channel, identified by its conductance and selectivity, is not activated by ATP. This activity in the mutant is blocked by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide. ADP and the ATP analog adenosine 5'-[gamma-[35S]thio]triphosphate do not activate the channel. These findings suggest a tight physical coupling between the plasma membrane ATPase and the K+ channel.

Adenosine Triphosphate↗

PubMed1995

Redistribution of myocardial blood flow with topical nitroglycerin in patients with coronary artery disease.

BACKGROUND: Unlike nonselective coronary vasodilators, nitroglycerin (GTN) is said to exert its primary vasodilatory effect on epicardial conductance vessels. Thus, in experimental models of coronary occlusion GTN appears to preferentially direct blood flow to poststenotic zones of ischemia. This phenomenon has, to date, not been tested in humans. Using positron emission tomography we examined the effect of transdermal GTN on global and regional myocardial perfusion in patients with angiographically proven coronary artery disease. METHODS AND RESULTS: Myocardial perfusion with [13N]ammonia was estimated from dynamic time-activity curves at baseline and 3 hours following application of either a 0.4 mg/h GTN skin patch (n = 10) or a placebo patch (n = 10) in a double-blind parallel design. From resliced cross-sectional images, regional flow, expressed as [13N]ammonia retention, was estimated from 216 myocardial sectors. Ischemia was defined as a significant reduction (> 2 SDs from average counts/pixel in maximally perfused zones) in [13N]ammonia retention within 10 contiguous myocardial sectors coupled with an increase or no change in counts derived from [18F]fluorodeoxyglucose. There was no change in global myocardial blood flow as expressed by [13N]ammonia retention following either placebo (0.61 +/- 0.14 to 0.62 +/- 0.12 min-1) or GTN (0.75 +/- 0.22 to 0.74 +/- 0.19 min-1). Conversely, there was a significant increase in the proportion of blood flow to the ischemic zones with GTN (73.9 +/- 12.6% to 94.9 +/- 17.8%; P < .05). No change in the distribution of blood flow to either ischemic or nonischemic zones was observed with placebo. A slight but insignificant decrease in [13N]ammonia retention in nonischemic zones was observed with GTN (1.01 +/- 0.31 to 0.93 +/- 0.26 min-1). CONCLUSIONS: This study suggests that under resting conditions topical GTN alters myocardial perfusion by preferentially increasing flow to areas of reduced perfusion with little or no change in global myocardial perfusion in patients whose angina is responsive to GTN.

Administration, Cutaneous↗

PubMed1995

Enhancement by GABA of the association rate of picrotoxin and tert-butylbicyclophosphorothionate to the rat cloned alpha 1 beta 2 gamma 2 GABAA receptor subtype.

1. We examined how gamma-aminobutyric acid (GABA) influences interaction of picrotoxin and tert-butylbicyclophosphorothionate (TBPS) with recombinant rat alpha 1 beta 2 gamma 2 GABAA receptors stably expressed in human embryonic kidney cells (HEK293), as monitored with changes in Cl- currents measured by the whole-cell patch clamp technique. 2. During application of GABA (5 microM) for 15 s, picrotoxin and TBPS dose-dependently accelerated the decay of inward GABA-induced currents (a holding potential of -60 mV under a symmetrical Cl- gradient). The drugs, upon preincubation with the receptors, also reduced the initial current amplitude in a preincubation time and concentration-dependent manner. This indicates their interaction with both GABA-bound and resting receptors. 3. The half maximal inhibitory concentration for picrotoxin and TBPS at the beginning of a 15 s GABA (5 microM) pulse was several times greater than that obtained at the end of the pulse. GABA thus appears to enhance picrotoxin and TBPS potency, but only at concentrations leading to occupancy of both high and low affinity GABA sites, i.e., 5 microM. Preincubation of the receptors with the drugs in the presence of GABA at 200 nM, which leads to occupancy of only high affinity GABA sites in the alpha 1 beta 2 gamma 2 subtype, produced no appreciable change in potency of picrotoxin or TBPS. This indicates that they preferentially interact with multiliganded, but not monoliganded receptors, unlike U-93631, a novel ligand to the picrotoxin site, which has higher affinity to both mono- and multiliganded receptors than resting receptors. 4. The time-dependent decay and preincubation time-dependent reduction of initial amplitude of GABA-induced Cl- currents followed monoexponential time courses, and time constants thus obtained displayed a linear relationship with drug concentration. Analysis of the data using a kinetic model with a single drug site showed that GABA (5 microM) enhanced the association rate for picrotoxin and TBPS nearly 100 fold, but their dissociation rate only 10 fold. The dissociation rate obtained from current recovery from picrotoxin or TBPS block yielded nearly identical values to the above analysis.5. We conclude that picrotoxin and TBPS interact with both resting and GABA-bound receptors, but their affinity for the latter is about 10 times greater than that for the former, largely due to a markedly increased association rate to the multiliganded receptors (but not monoliganded ones). This and our earlier study with U-93631 improves our understanding of functional coupling between GABA and picrotoxin sites, which appears to be useful in characterizing the mode of interaction for various picrotoxin site ligands.

Animals↗

PubMed1986

Effect of thiamine deficiency and its reversal on cerebral blood flow in the rat. Observations on the phenomena of hyperperfusion, "no reflow," and delayed hypoperfusion.

Local CBF (LCBF) was determined in the same rat model and at the same intervals of thiamine deficiency and reversal as in previous studies of local cerebral glucose utilization (LCGU) and pH (LCpH). The results showed that prior to the appearance of the clinical sequelae of thiamine deficiency (opisthotonus, which usually occurs on day 18 of deficiency) cerebral structures such as the mammillary body, vestibular nucleus, inferior colliculus, and thalamus showed significant hyperperfusion, reaching greater than 200% of control values. At opisthotonus, there was a general decline in LCBF, but, in addition, the larger of these structures developed inhomogeneous perfusion with patches of hyperperfusion adjacent to others of low flow. Seven days of thiamine replenishment at opisthotonus resulted in delayed hypoperfusion notably in the mammillary body, inferior colliculus, and thalamic nuclei. Superimposition of the LCBF, LCGU, and LCpH data reveals that structures known to be vulnerable to the development of histological lesions in this model showed an early phase of hyperperfusion uncoupled from declining LCGU and normal LCpH. Then, following a significant but only focal rise in LCGU between days 11 and 14 of deficiency, hyperperfusion persisted while the pH was dropping and LCGU was rapidly declining. The phase of patchy perfusion occurred only in the histologically vulnerable structures when LCGU was very low and acidosis was at its peak, suggesting that it may have resulted from these opposing influences on LCBF. Following replenishment with thiamine, the vulnerable structures showed delayed hypoperfusion coupled to LCGU.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1998

Verapamil block of the delayed rectifier K current in chick embryo dorsal root ganglion neurons.

We have used the patch-clamp method in the whole-cell configuration to investigate the mechanism of block of the delayed rectifier K current (IDRK) by verapamil in embryonic chick dorsal root ganglion (DRG) neurons. Verapamil induced a dose-dependent decay of the current, without altering its activation kinetics. This observation, together with the good description of IDRK time course at various blocker concentrations with the computer simulation of a three-state chain model (closed left and right arrow open left and right arrow open-blocked), indicates that verapamil acts as a state-dependent, open-channel blocker. To account for the double-exponential time course of recovery from block, this minimal kinetics scheme was expanded to include a closed-blocked state resulting from channel closure (at hyperpolarized voltages) with verapamil still bound to it. The apparent block and unblock rate constants assessed from verapamil-induced current decay in the presence of external Na were 0.95 +/- 0.05 ms-1mM-1 and 0.0037 +/- 0.0016 ms-1, respectively. When external Na was replaced by K, only the unblock rate constant changed, to 0.02 +/- 0.009 ms-1. Under these ionic conditions it was also observed that the recovery from block was modified from the double-exponential time course in the presence of external Na (tau1 = 160 ms; tau2 = 1600 ms), to a faster single-exponential recovery (tau = 100 ms). We tested the voltage dependence of block by applying stimulation protocols aimed at eliminating bias easily introduced by the shift of the gating equilibrium and by the coupling of channel activation and block. Under these experimental conditions the resulting block rate constant was not measurably voltage dependent.

Animals↗

PubMed2006

Ryanodine receptor-transmitter release site coupling increases quantal size in a synapse-specific manner.

The mechanisms by which presynaptic neurones differentially regulate synaptic transmission with multiple postsynaptic targets in the brain are not fully understood. Using intracellular sharp electrode and whole-cell voltage-clamp recordings of soma-soma synapses between identified Lymnaea neurones, we provide direct evidence that quantal size is regulated presynaptically through the coupling of multiple release sites. This coupling effectively multiplies quantal size, thereby providing significant influence over parameters of synaptic transmission that are influenced by quantal size, such as the variance in transmitter release at stationary release probabilities. Variation in the degree of coupling is dependent on the identity of the postsynaptic cell, even though the variation in quantal size is of presynaptic origin. We have therefore demonstrated the presence of a novel mechanism by which presynaptic neurones may differentially regulate quantal size at select synaptic connections, in turn providing them with a means of regulating synaptic transmission with multiple postsynaptic cells.

Animals↗

PubMed1986

GABAA and GABAB receptors on porcine pars intermedia cells in primary culture: functional role in modulating peptide release.

A primary culture of porcine pars intermedia cells with particularly high yields has been developed. The cells, grown in monolayers, secrete the pro-opiomelanocortin-derived peptide alpha-melanocyte-stimulating hormone over several weeks. The patch-clamp technique has been used to demonstrate the presence of gamma-aminobutyrateA (GABAA) receptors on the cells. GABA or the selective GABAA receptor agonist isoguvacine produced a depolarizing increase in chloride conductance that desensitized rapidly. The response was antagonized by bicuculline and by the aminopyridazine derivative of GABA (SR 95103), a novel GABAA receptor antagonist. The effects of specific agonists for each receptor were tested on peptide release from cells maintained in a perfusion system. Isoguvacine (10 microM) potentiated Ba2+-evoked release of alpha-melanocyte-stimulating hormone, whereas (-)-baclofen (50 microM) decreased both basal and stimulated hormone release. This negative effect on peptide secretion was reproduced when GABA (50 microM) was perfused in the presence of bicuculline (10 microM) to block GABAA receptor activation. The possible mechanisms underlying these GABAA and GABAB effects on stimulus-secretion coupling in this neuroendocrine model are discussed.

Animals↗

PubMed2005

Actions of emigrated neutrophils on Na(+) and K(+) currents in rat ventricular myocytes.

Interactions between neutrophils and the ventricular myocardium can contribute to tissue injury, contractile dysfunction and generation of arrhythmias in acute cardiac inflammation. Many of the molecular events responsible for neutrophil adhesion to ventricular myocytes are well defined; in contrast, the resulting electrophysiological effects and changes in excitation-contraction coupling have not been studied in detail. In the present experiments, rat ventricular myocytes were superfused with either circulating or emigrated neutrophils and whole-cell currents and action potential waveforms were recorded using the nystatin-perforated patch method. Almost immediately after adhering to ventricular myocytes, emigrated neutrophils caused a depolarization of the resting membrane potential and a marked prolongation of myocyte action potential. Voltage clamp experiments demonstrated that following neutrophil adhesion, there was (i) a slowing of the inactivation of a TTX-sensitive Na(+) current, and (ii) a decrease in an inwardly rectifying K(+) current. One cytotoxic effect of neutrophils appears to be initiated by enhanced Na(+) entry into the myocytes. Thus, manoeuvres that precluded activation of Na(+) channels, for example holding the membrane potential at -80 mV, significantly increased the time to cell death or prevented contracture entirely. A mathematical model for the action potential of rat ventricular myocytes has been modified and then utilized to integrate these findings. These simulations demonstrate the marked effects of (50-fold) slowing of the inactivation of 2-4% of the available Na(+) channels on action potential duration and the corresponding intracellular Ca(2+) transient. In ongoing studies using this combination of approaches, are providing significant new insights into some of the fundamental processes that modulate myocyte damage in acute inflammation.

Action Potentials↗

PubMed1994

A- and C-type rat nodose sensory neurons: model interpretations of dynamic discharge characteristics.

1. Neurons of the nodose ganglia provide the sole connection between many types of visceral sensory inputs and the central nervous system. Electrophysiological studies of isolated nodose neurons provide a practical means of measuring individual cell membrane currents and assessing their putative contributions to the overall response properties of the neuron and its terminations. Here, we present a comprehensive mathematical model of an isolated nodose sensory neuron that is based upon numerical fits to quantitative voltage- and current-clamp data recorded in our laboratory. Model development was accomplished using an iterative process of electrophysiological recordings, nonlinear parameter estimation, and computer simulation. This work is part of an integrative effort aimed at identifying and characterizing the fundamental ionic mechanisms participating in the afferent neuronal limb of the baroreceptor reflex. 2. The neuronal model consists of two parts: a Hodgkin-Huxley-type membrane model coupled to a lumped fluid compartment model that describes Ca2+ ion concentration dynamics within the intracellular and external perineuronal media. Calcium buffering via a calmodulin-type buffer is provided within the intracellular compartment. 3. The complete model accurately reproduces whole-cell voltage-clamp recordings of the major ion channel currents observed in enzymatically dispersed nodose sensory neurons. Specifically, two Na+ currents exhibiting fast (INaf) and slow tetrodotoxin (TTX)-insensitive (INas) kinetics; low- and high-threshold Ca2+ currents exhibiting transient (ICa,t) and long-lasting (ICa,n) dynamics, respectively; and outward K+ currents consisting of a delayed-rectifier current (IK), a transient outward current (I(t)) and a Ca(2+)-activated K+ current (IK,Ca). 4. Whole-cell current-clamp recordings of somatic action-potential dynamics were performed on enzymatically dispersed nodose neurons using the perforated patch-clamp technique. Stimulus protocols consisted of both short (< or = 2.0 ms) and long (> or = 200 ms) duration current pulses over a wide range of membrane holding potentials. These studies clearly revealed two populations of nodose neurons, often termed A- and C-type cells, which exhibit markedly different action-potential signatures and stimulus response properties. 5. Using a single set of equations, the model accurately reproduces the electrical behavior of both A- and C-type nodose neurons in response to a wide variety of stimulus conditions and membrane holding potentials. The structure of the model, as well as the majority of its parameters are the same for both A- and C-type implementations.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2006

Neuromodulation of Na+ channel slow inactivation via cAMP-dependent protein kinase and protein kinase C.

Neurotransmitters modulate sodium channel availability through activation of G protein-coupled receptors, cAMP-dependent protein kinase (PKA), and protein kinase C (PKC). Voltage-dependent slow inactivation also controls sodium channel availability, synaptic integration, and neuronal firing. Here we show by analysis of sodium channel mutants that neuromodulation via PKA and PKC enhances intrinsic slow inactivation of sodium channels, making them unavailable for activation. Mutations in the S6 segment in domain III (N1466A,D) either enhance or block slow inactivation, implicating S6 segments in the molecular pathway for slow inactivation. Modulation of N1466A channels by PKC or PKA is increased, whereas modulation of N1466D is nearly completely blocked. These results demonstrate that neuromodulation by PKA and PKC is caused by their enhancement of intrinsic slow inactivation gating. Modulation of slow inactivation by neurotransmitters acting through G protein-coupled receptors, PKA, and PKC is a flexible mechanism of cellular plasticity controlling the firing behavior of central neurons.

Amino Acid Sequence↗

PubMed1998

Three residues predicted by molecular modeling to interact with the purine moiety alter ligand binding and channel gating in cyclic nucleotide-gated channels.

Cytoplasmic cAMP and cGMP are soluble cellular messengers that directly activate cyclic nucleotide-gated (CNG) channels. These channels mediate sensory transduction in photoreceptors and olfactory neurons. The closely related CNG channels in these cell types have different nucleotide activation profiles, and we have investigated the molecular basis of their nucleotide selectivity properties. Previously, we predicted that the purine moiety of the nucleotide interacts with residues F533, K596, and D604 (bovine rod alpha CNG channel subunit sequences) of the nucleotide binding domain. In this study, we replaced these three residues with the corresponding residues of the bovine olfactory CNG channel. Mutations at each position altered the nucleotide activation of the rod CNG channels. In a mutant where K596 was replaced with arginine, cAMP-activated currents were enhanced 8-12-fold, suggesting that residue 596 influences channel gating. Thermodynamic cycle analysis of the data showed that (1) the residues are energetically coupled and (2) energetic coupling exists between the potentiating effects of Ni2+ and the replacement of F533 with tyrosine. These data suggest that changes in one of the residues alter the purine contacts with the other residues and that F533 communicates with the C-linker region of the channel involved in Ni2+ potentiation.

Amino Acid Substitution↗

PubMed2003

Inhibition of Caenorhabditis elegans social feeding by FMRFamide-related peptide activation of NPR-1.

Social and solitary feeding in natural Caenorhabditis elegans isolates are associated with two alleles of the orphan G-protein-coupled receptor (GPCR) NPR-1: social feeders contain NPR-1 215F, whereas solitary feeders contain NPR-1 215V. Here we identify FMRFamide-related neuropeptides (FaRPs) encoded by the flp-18 and flp-21 genes as NPR-1 ligands and show that these peptides can differentially activate the NPR-1 215F and NPR-1 215V receptors. Multicopy overexpression of flp-21 transformed wild social animals into solitary feeders. Conversely, a flp-21 deletion partially phenocopied the npr-1(null) phenotype, which is consistent with NPR-1 activation by FLP-21 in vivo but also implicates other ligands for NPR-1. Phylogenetic studies indicate that the dominant npr-1 215V allele likely arose from an ancestral npr-1 215F gene in C. elegans. Our data suggest a model in which solitary feeding evolved in an ancestral social strain of C. elegans by a gain-of-function mutation that modified the response of NPR-1 to FLP-18 and FLP-21 ligands.

Action Potentials↗