Ectopic expression of cone-specific G-protein-coupled receptor kinase GRK7 in zebrafish rods leads to lower photosensitivity and altered responses.

To investigate the roles of G-protein receptor kinases (GRKs) in the light responses of vertebrate photoreceptors, we generated transgenic zebrafish lines, the rods of which express either cone GRK (GRK7) or rod GRK (GRK1) in addition to the endogenous GRK1, and we then measured the electrophysiological characteristics of single-cell responses and the behavioural responses of intact animals. Our study establishes the zebrafish expression system as a convenient platform for the investigation of specific components of the phototransduction cascade. The addition of GRK1 led to minor changes in rod responses.

Electrical coupling in sustentacular cells of the mouse olfactory epithelium.

Sustentacular cells (SCs) line the apical surface of the olfactory epithelium (OE) and provide trophic, metabolic, and mechanical support for olfactory receptor neurons. Morphological studies have suggested that SCs possess gap junctions, although physiological evidence for gap junctional communication in mammalian SCs is lacking. In the present study we investigated whether coupling exists between SCs situated in tissue slices of OE from neonatal (P0-P4) mice.

Ionic conductances in sustentacular cells of the mouse olfactory epithelium.

The electrical properties of sustentacular cells (SCs) in the olfactory epithelium (OE) were investigated in tissue slices taken from neonatal mice (P0-P4). Conventional whole-cell recordings were obtained from SCs and also from olfactory receptor neurones (ORNs) in situ. SCs had a larger apparent cell capacitance (C(cell)) (18.

Suppression of a slow post-spike afterhyperpolarization by calcineurin inhibitors.

A subset of myenteric neurons in the intestine (AH neurons) generate prolonged (>5 s) post-spike afterhyperpolarizations (slow AHPs) that are insensitive to apamin and tetraethylammonium. Generation of slow AHPs depends critically on Ca(2+) entry and intracellular release of Ca(2+) from stores, which then leads to the activation of a K(+) conductance that underlies the slow AHP (g(sAHP)). Slow AHPs are inhibited by stimulation of the cAMP/protein kinase A (PKA) pathway, suggesting that phosphorylation of the K(+)-channels that mediate the g(sAHP) (K(sAHP)-channels) is responsible for suppression of slow AHPs and possibly for the repolarization phase of slow AHPs.

SK channels and the varieties of slow after-hyperpolarizations in neurons.

Action potentials and associated Ca2+ influx can be followed by slow after-hyperpolarizations (sAHPs) caused by a voltage-insensitive, Ca2+-dependent K+ current. Slow AHPs are a widespread phenomenon in mammalian (including human) neurons and are present in both peripheral and central nervous systems. Although, the molecular identity of ion channels responsible for common membrane potential mechanisms has been largely determined, the nature of the channels that underlie the sAHPs in neurons, both in the brain and in the periphery, remains unresolved.

Altered excitability of intestinal neurons in primary culture caused by acute oxidative stress.

Neurons were isolated from the intestine of guinea pigs and grown in primary culture for < or =15 days. Using conventional whole cell recording techniques, we demonstrated that the majority of neurons express a prolonged poststimulus afterhyperpolarization (slow AHP). These neurons also had large-amplitude (approximately 100 mV), broad-duration (approximately 2 ms) action potentials and generated a hyperpolarization activated inward current (Ih).

PKA-mediated inhibition of a novel K+ channel underlies the slow after-hyperpolarization in enteric AH neurons.

Postspike after-hyperpolarizations (AHPs) control the excitability of neurons and are important in shaping firing patterns. The duration of some of these events extends to tens of seconds and they can render neurons inexcitable for much of their time course. While consensus is strong that the medium duration (< 1 s AHPs are mediated by the opening of small conductance Ca2+-activated K+ channels, the K+ channels mediating slow AHPs (> 5 s in a subset of enteric (AH) neurons) have an intermediate unit conductance (IKCa).

Controlling the excitability of IPANs: a possible route to therapeutics.

Recent studies have shown that intrinsic primary afferent neurons (IPANs) express a much larger range of ionic currents than non-sensory neurons of the enteric nervous system. These ionic currents can be modulated by neurotransmitters that are synaptically released onto the soma (unlike cranial and spinal sensory neurons). The membrane receptors and ionic channels that are involved in the sensory transduction processes of IPANS are beginning to be defined.

Action potential afterdepolarization mediated by a Ca2+-activated cation conductance in myenteric AH neurons.

We investigated the nature of afterdepolarizing potentials in AH neurons from the guinea-pig duodenum using whole-cell patch-clamp recordings in intact myenteric ganglia. Afterdepolarizing potentials were minimally activated following action-potential firing under normal conditions, but after application of charybdotoxin (40 nM) or tetraethyl ammonium (TEA; 10-20 mM) to the bathing solution, prominent afterdepolarizing potentials followed action potentials. The whole-cell current underlying afterdepolarizing potentials (I(ADP)) in the presence of TEA (10-20 mM) reversed at -38 mV and was not voltage-dependent.

Regulation of K+ channels underlying the slow afterhyperpolarization in enteric afterhyperpolarization-generating myenteric neurons: role of calcium and phosphorylation.

1. Myenteric afterhyperpolarization-generating myenteric (AH) neurons serve as intrinsic primary afferent neurons of the enteric nervous system and generate prolonged or slow afterhyperpolarizing potentials (slow AHP). The slow AHP is generated by an increase in a Ca2+-activated K+ conductance (gK-Ca) and is inhibited by enteric neurotransmitters leading to increased excitability.

Sensitization of enteric reflexes in the rat colon in vitro.

We have investigated sensitization of reflexes in the isolated rat colon in order to develop a model that might prove useful for investigating how the sensitivity of enteric reflexes can be altered by prior stimulation. Records were taken of circular muscle tension, 7-10 mm oral and anal to radial distension exerted by a hook passed through the wall of the colon. A test stimulus of 1.

TEA- and apamin-resistant K(Ca) channels in guinea-pig myenteric neurons: slow AHP channels.

The patch-clamp technique was used to record from intact ganglia of the guinea-pig duodenum in order to characterize the K(+) channels that underlie the slow afterhyperpolarization (slow AHP) of myenteric neurons. Cell-attached patch recordings from slow AHP-generating (AH) neurons revealed an increased open probability (P(o)) of TEA-resistant K(+) channels following action potentials. The P(o) increased from < 0.

Afterhyperpolarization current in myenteric neurons of the guinea pig duodenum.

Whole cell patch and cell-attached recordings were obtained from neurons in intact ganglia of the myenteric plexus of the guinea pig duodenum. Two classes of neuron were identified electrophysiologically: phasically firing AH neurons that had a pronounced slow afterhyperpolarization (AHP) and tonically firing S neurons that lacked a slow AHP. We investigated the properties of the slow AHP and the underlying current (I(AHP)) to address the roles of Ca(2+) entry and Ca(2+) release in the AHP and the characteristics of the K(+) channels that are activated.

Potassium channels in gastrointestinal smooth muscle.

1. Electromechanical coupling in smooth muscle serves to coordinate the contractile activity of the syncytium. Electrical activity of smooth muscle of the gut is generated by ionic conductances that regulate and in turn are regulated by the membrane potential of smooth muscle cells.

Functional innervation of the biliary sphincter of the guinea-pig revealed by anti-autonomic drugs.

1. The roles of excitatory and inhibitory intrinsic motor nerves on contractions reflexly evoked by wall distension were investigated in the isolated sphincter of Oddi of the guinea-pig (SO-GP). 2.

Diverse ionic currents and electrical activity of cultured myenteric neurons from the guinea pig proximal colon.

The aim of this study was to perform a patch-clamp analysis of myenteric neurons from the guinea pig proximal colon. Neurons were enzymatically dispersed, cultured for 2-7 days, and recorded from using whole cell patch clamp. The majority of cells fired phasically, whereas about one-quarter of the neurons fired in a tonic manner.

Recording ionic events from cultured, DiI-labelled myenteric neurons in the guinea-pig proximal colon.

To date investigations of enteric neurons by patch clamping/calcium imaging have been limited by studying unidentified heterogeneous populations of neurons. In DiI-labelled colonic myenteric neurons, the feasibility of recording ionic events was determined by applying DiI either to the mucosa or the circular muscle, dispersing neurons after 48 h organotypic culture, and patch-clamping/calcium imaging labeled neurons after 3-7 days in culture. Myenteric neurons with diffuse DiI fluorescence were typically smooth and agranular.

Voltage-dependent large conductance channels in visceral smooth muscle.

The ionic conductances that underlie the resting membrane potential of visceral smooth muscle are not fully understood. Using the patch-clamp technique in the whole-cell configuration, single large conductance channels (LCCs) with unitary conductances of up to 400 pS were recorded in isolated smooth muscle cells of the opossum esophagus. These channels were active at physiological potentials (-100 to -40 mV) and opened with increasing frequency as the membrane potential was hyperpolarized.

Inhibition of voltage-activated K+ currents in smooth muscle cells of the guinea pig proximal colon by noradrenergic agonists.

1. The effects of noradrenaline and isoprenaline on the Ca2+i-insensitive, voltage-activated K+ current in smooth muscle cells from the circular muscle layer of the guinea pig proximal colon were investigated by using standard whole-cell patch-clamp techniques at room temperature (22-24 degrees C). 2.

An intermediate conductance K+ channel in the cell membrane of mouse intestinal smooth muscle.

Single channel currents were recorded from cell-attached and inside-out patches in smooth muscle cells of the mouse ileum in order to identify TEA-sensitive Ca2+-dependent K+ channels. Cells were bathed in high-K+ (150 mM) solution with [Ca2+] buffered to 80-150 nM with EGTA and patch pipettes were filled with low-K+ (2.5 mM) physiological solution.

Molecular identification of a component of delayed rectifier current in gastrointestinal smooth muscles.

Kv2.2, homologous to the shab family of Drosophila voltage-gated K+ channels, was isolated from human and canine colonic circular smooth muscle-derived mRNA. Northern hybridization analysis performed on RNA prepared from tissues and RT-PCR performed on RNA isolated from dispersed and selected smooth muscle cells demonstrate that Kv2.

Nitric oxide suppresses a Ca(2+)-stimulated Cl- current in smooth muscle cells of opossum esophagus.

Nitric oxide (NO) hyperpolarizes visceral smooth muscles. Using the patch-clamp technique, we investigated the possibility that NO-mediated hyperpolarization in the circular muscle of opossum esophagus results from the suppression of a Ca(2+)-stimulated Cl- current. Smooth muscle cells were dissociated from the circular layer and bathed in high-K+ Ca(2+)-EGTA-buffered solution.

Activation of small conductance Ca(2+)-dependent K+ channels by purinergic agonists in smooth muscle cells of the mouse ileum.

1. Whole-cell and single-channel K+ currents were recorded at room temperature (22-24 degrees C), from smooth muscle cells enzymatically dispersed from the mouse ileum, using variations of the patch-clamp technique. 2.

Cloning and expression of the large-conductance Ca(2+)-activated K+ channel from colonic smooth muscle.

We have cloned cDNAs encoding the alpha- and beta-subunits of a large-conductance Ca(2+)-activated K+ channel (BK channel) from canine colonic smooth muscle (cslo-alpha and cslo-beta). Nucleotide sequence homology of cslo-alpha with mslo and dslo suggests that it is the canine homologue of these genes. The carboxy-terminal end of the protein is the most diverse between species, and we have also found alternative exons in cslo-alpha in this region.

Suppression of two cloned smooth muscle-derived delayed rectifier potassium channels by cholinergic agonists and phorbol esters.

Functional coupling between muscarinic (m3) receptors and two voltage-gated K+ (Kv) channels (Kv1.2 and Kv1.5) cloned originally from canine colonic smooth muscle was studied using the Xenopus oocytes expression system and a mammalian cell line (COS cells).