Biomedical subjects

S J Kleene

Publications and source records attributed to S J Kleene.

At least 19 recordsLinked to original sources

PubMed2002

Single ionic channels of two Caenorhabditis elegans chemosensory neurons in native membrane.

The genome of Caenorhabditis elegans contains representatives of the channel families found in both vertebrate and invertebrate nervous systems. However, it lacks the ubiquitous Hodgkin-Huxley Na+ channel that is integral to long-distance signaling in other animals. Nematode neurons are presumed to communicate by electrotonic conduction and graded depolarizations. This fundamental difference in operating principle may require different channel populations to regulate transmission and transmitter release. We have sampled ionic channels from the somata of two chemosensory neurons (AWA and AWC) of C. elegans. A Ca2+-activated, outwardly rectifying channel has a conductance of 67 pS and a reversal potential indicating selectivity for K+. An inwardly rectifying channel is active at potentials more negative than -50 mV. The inward channel is notably flickery even in the absence of divalent cations; this prevented determination of its conductance and reversal potential. Both of these channels were inactive over a range of membrane potentials near the likely cell resting potential; this would account for the region of very high membrane resistance observed in whole-cell recordings. A very-large-conductance (> 100 pS), inwardly rectifying channel may account for channel-like fluctuations seen in whole-cell recordings.

Animals↗

PubMed2000

Spontaneous gating of olfactory cyclic-nucleotide-gated channels.

In vertebrates, cilia on the olfactory receptor neurons have a high density of cyclic-nucleotide-gated (CNG) channels. During transduction of odorous stimuli, cyclic AMP is formed. cAMP gates the CNG channels and this initiates the neuronal depolarization. Here it is shown that the ciliary CNG channels also open spontaneously. In the absence of odorants and second messengers, olfactory cilia have a small basal conductance to cations. Part of this conductance is similar to the cAMP-activated conductance in its sensitivity to channel inhibitors and divalent cations. The basal conductance may help to stabilize the neuronal membrane potential while limiting the sensitivity of odorant detection.

1-Methyl-3-isobutylxanthine↗

PubMed2000

Neuronal Ca2+ -activated Cl- channels--homing in on an elusive channel species.

Ca2+ -activated Cl- channels control electrical excitability in various peripheral and central populations of neurons. Ca2+ influx through voltage-gated or ligand-operated channels, as well as Ca2+ release from intracellular stores, have been shown to induce substantial Cl- conductances that determine the response to synaptic input, spike rate, and the receptor current of various kinds of neurons. In some neurons, Ca2+ -activated Cl- channels are localized in the dendritic membrane, and their contribution to signal processing depends on the local Cl- equilibrium potential which may differ considerably from those at the membranes of somata and axons. In olfactory sensory neurons, the channels are expressed in ciliary processes of dendritic endings where they serve to amplify the odor-induced receptor current. Recent biophysical studies of signal transduction in olfactory sensory neurons have yielded some insight into the functional properties of Ca2+ -activated Cl- channels expressed in the chemosensory membrane of these cells. Ion selectivity, channel conductance, and Ca2+ sensitivity have been investigated, and the role of the channels in the generation of receptor currents is well understood. However, further investigation of neuronal Ca2+ -activated Cl- channels will require information about the molecular structure of the channel protein, the regulation of channel activity by cellular signaling pathways, as well as the distribution of channels in different compartments of the neuron. To understand the physiological role of these channels it is also important to know the Cl- equilibrium potential in cells or in distinct cell compartments that express Ca2+ -activated Cl- channels. The state of knowledge about most of these aspects is considerably more advanced in non-neuronal cells, in particular in epithelia and smooth muscle. This review, therefore, collects results both from neuronal and from non-neuronal cells with the intent of facilitating research into Ca2+ -activated Cl- channels and their physiological functions in neurons.

Animals↗

PubMed1999

Both external and internal calcium reduce the sensitivity of the olfactory cyclic-nucleotide-gated channel to CAMP.

In vertebrate olfaction, odorous stimuli are first transduced into an electrical signal in the cilia of olfactory receptor neurons. Many odorants cause an increase in ciliary cAMP, which gates cationic channels in the ciliary membrane. The resulting influx of Ca2+ and Na+ produces a depolarizing receptor current. Modulation of the cyclic-nucleotide-gated (CNG) channels is one mechanism of adjusting olfactory sensitivity. Modulation of these channels by divalent cations was studied by patch-clamp recording from single cilia of frog olfactory receptor neurons. In accord with previous reports, it was found that cytoplasmic Ca2+ above 1 microM made the channels less sensitive to cAMP. The effect of cytoplasmic Ca2+ was eliminated by holding the cilium in a divalent-free cytoplasmic solution and was restored by adding calmodulin (CaM). An unexpected result was that external Ca2+ could also greatly reduce the sensitivity of the channels to cAMP. This reduction was seen when external Ca2+ exceeded 30 microM and was not affected by the divalent-free solution, by CaM, or by Ca2+ buffering. The effects of cytoplasmic and external Ca2+ were additive. Thus the effects of cytoplasmic and external Ca2+ are apparently mediated by different mechanisms. There was no effect of CaM on a Ca2+-activated Cl- current that also contributes to the receptor current. Increases in Ca2+ concentration on either side of the ciliary membrane may influence olfactory adaptation.

Amino Acid Sequence↗

PubMed1997

High-gain, low-noise amplification in olfactory transduction.

It is desirable that sensory systems use high-gain, low-noise amplification to convert weak stimuli into detectable signals. Here it is shown that a pair of receptor currents underlying vertebrate olfactory transduction constitutes such a scheme. The primary receptor current is an influx of Na+ and Ca2+ through cAMP-gated channels in the olfactory cilia. External divalent cations improve the signal-to-noise properties of this current, reducing the mean current and the current variance. As Ca2+ enters the cilium, it gates Cl- channels, activating a secondary depolarizing receptor current. This current amplifies the primary current, but introduces little additional noise. The system of two currents plus divalent cations in the mucus produces a large receptor current with very low noise.

Animals↗

PubMed1997

Noise analysis of ion channels in non-space-clamped cables: estimates of channel parameters in olfactory cilia.

Ion channels in the cilia of olfactory neurons are part of the transduction machinery of olfaction. Odorant stimuli have been shown to induce a biphasic current response, consisting of a cAMP-activated current and a Ca(2+)-activated Cl- current. We have developed a noise analysis method to study ion channels in leaky cables, such as the olfactory cilium, under non-space-clamp conditions. We performed steady-state noise analysis on ligand-induced currents in excised cilia, voltage-clamped at input and internally perfused with cAMP or Ca2+. The cAMP-activated channels analyzed by this method gave results similar to those of single-channel recordings (gamma = 8.3 pS). Single-channel currents have not yet been recorded for the Ca(2+)-activated Cl- channels. Using our noise analysis method, we estimate a unit conductance, gamma = 0.8 pS, for these channels. The density of channels was found to be approximately 70 channels/micron2 for both channel species.

Analysis of Variance↗

PubMed1996

Persistence of the olfactory receptor current in a wide variety of extracellular environments.

We measured the current activated by cytoplasmic adenosine 3':5'-cyclic monophosphate (cAMP) in olfactory cilia from the frog Rana pipiens. The odorant-induced current in frog olfactory receptor neurons was also measured for comparison. In both cases, recordings were performed near the neuronal resting potential in a variety of extracellular bath solutions. 2. In Ca(2+)-free baths, cAMP activated an inward current in excised olfactory cilia that was carried entirely by cations. As extracellular Ca2+ was increased, the cationic current decreased while a second current, carried by C1-, increased. Total cAMP-activated current decreased with increasing extracellular CA2+. When external Na+ but not Ca2+ was eliminated, only the C1- component of the current persisted. When external Na+ and Ca2+ were both removed, there was no cAMP-activated current. 3. In receptor neurons, the total odorant-induced receptor current varied in a similar way with the extracellular ionic environment. Under conditions favoring the anionic receptor current, the response amplitude decreased and the latency increased. 4. It is known that olfactory receptor currents persist in a wide variety of extracellular environments. This persistence can be sufficiently explained by the balance between cationic and anionic currents demonstrated here.

Animals↗

PubMed1995

Block by external calcium and magnesium of the cyclic-nucleotide-activated current in olfactory cilia.

Olfactory transduction occurs on the cilia of olfactory receptor neurons, which are in close proximity to the external environment. Transduction is mediated by cyclic AMP, which directly gates channels in the ciliary membrane. Previous evidence indicates that one environmental influence, the level of divalent cations in the mucus, may strongly influence olfactory transduction by blocking the cyclic-AMP-gated channels. In this report the effects of external calcium and magnesium on the ciliary macroscopic current activated by cytoplasmic cyclic AMP were measured. External calcium and magnesium each reduced the cyclic-AMP-activated current at both negative and positive potentials. At the neuronal resting potential (-50 mV), half-maximal inhibition of the current was produced by 250 microM calcium or 1.3 mM magnesium. Reduction in current by external calcium was strongly voltage-dependent, with larger effects at negative potentials. Reduction by magnesium was weaker and less voltage-dependent. Block of the cyclic-AMP-activated current by divalent cations in the mucus may be one element of a system that increases the signal-to-noise ratio for detection of odorants.

Animals↗

PubMed1994

Solving buffering problems with Mathematica software.

Determining ionic concentrations in buffered solutions usually reduces to solving a set of simultaneous polynomial equations. Mathematica software offers a convenient method for doing this. Using buffering of Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) as an example, we provide a Mathematica script to estimate the apparent association constant. A second example shows how to calculate free ion concentrations when two ligands (Ca2+ and Mg2+) compete for one chelator (EGTA). Finally, the concentrations of all species are determined in a complex mixture containing Ca2+, EGTA, and calmodulin, a protein with four Ca(2+)-binding sites. Modifying the examples presented should allow analysis of most practical buffering problems.

Algorithms↗

PubMed1994

Guanine nucleotides modulate steady-state inactivation of voltage-gated sodium channels in frog olfactory receptor neurons.

The voltage for half-inactivation (V1/2) of Na+ currents in frog olfactory receptor neurons (ORNs) under whole-cell voltage clamp showed a shift to more negative potentials with time. Inclusion of guanosine triphosphate (GTP) or its nonhydrolyzable analogue, guanosine-5'-O-3-thiotriphosphate (GTP-gamma-S), which activates G proteins, in the recording pipette, not only gave a more positive V1/2, but also reduced and delayed the negative shift observed in the absence of nucleotides. Guanosine-5'-O-2-thiodiphosphate (GDP-beta-S), a nonhydrolyzable analogue that prevents the binding of GTP to G proteins, did not affect the V1/2 significantly by itself but blocked the positive shift induced by GTP. Since the steady-state activation was not affected, our results indicate that a G protein or a G-protein-dependent process may be important in regulating the steady-state inactivation of Na+ channels in ORNs of the frog.

Animals↗

PubMed1994

Inhibition of olfactory cyclic nucleotide-activated current by calmodulin antagonists.

1. In amphibian olfactory receptor neurones, much of the depolarizing current in response to odours is carried by cationic channels that are directly gated by cyclic AMP. The effects of four calmodulin antagonists on the cyclic AMP-activated receptor current were studied in single olfactory cilia of the frog. 2. Two antagonists, W-7 and trifluoperazine, were potent and reversible inhibitors of the cyclic AMP-activated current. IC50 values were 5 microM for W-7 and 13 microM for trifluoperazine. A third antagonist, calmidazolium, irreversibly blocked the current. The fourth, mastoparan, had little effect. 3. Calmodulin was unable to reverse the effects of W-7 and trifluoperazine, suggesting that these inhibitors act directly on the cyclic AMP-gated channels. 4. Neither W-7 nor trifluoperazine inhibited a Ca(2+)-activated Cl- current which also contributes to the odorant response. These compounds thus allow the two components of the olfactory receptor current to be discriminated.

Animals↗

PubMed1994

An electrophysiological survey of frog olfactory cilia.

Individual olfactory receptor neurons vary widely in their responses to odorants. Olfactory stimulus reception occurs in the cilia of the receptor neurons. Thus, the variability among individual neurons could in part be due to differences among the olfactory cilia. We have quantified the known conductance properties of each of 117 frog olfactory cilia. From a strictly qualitative viewpoint, the cilia were very homogeneous. All but a few of them had a basal conductance in the absence of odorants and second messengers, conductances stimulated by cytoplasmic cyclic AMP and by Ca2+ and a conductance measured in the presence of ATP and stimulated by GTP gamma S. However, the magnitudes of the conductances varied widely among the cilia. Amplitudes of the cyclic-AMP- and Ca(2+)-activated ciliary currents correlated strongly with one another across the 117 cilia and 24 frogs studied, suggesting that expression of the underlying channels may be co-regulated. None of the conductance properties correlated strongly with ciliary length, a marker of cell maturity. Given cytoplasmic MgATP as substrate, ciliary adenylate cyclase apparently produced cyclic AMP, which in turn gated membrane channels and increased the ciliary conductance. In some cilia, MgATP alone caused a very large increase in conductance. In others, there was little effect unless GTP gamma S, which increases cyclase activity, was also added. No effect of cytoplasmic inositol trisphosphate on ciliary conductance was detectable.

Adenosine Triphosphate↗

PubMed1993

The cyclic nucleotide-activated conductance in olfactory cilia: effects of cytoplasmic Mg2+ and Ca2+.

Olfactory receptor neurons depolarize in response to odorants. This depolarization is mediated by an increase in intracellular cyclic AMP, which directly gates channels in the membranes of the neuronal cilia. Previous evidence suggests that a Ca2+ influx during the odorant response may ultimately play a role in terminating the response. One way Ca2+ inside the cell could terminate the odorant response would be to directly inhibit the cAMP-gated channels. In this report the effects of cytoplasmic Ca2+ and Mg2+ on the cAMP-activated current were measured in single olfactory cilia. Near the neuronal resting potential, cytoplasmic Ca2+ and Mg2+ only slightly reduced the cAMP-activated current. Even at high levels (1.0 mM Ca2+ or 5.0 mM Mg2+), the average inhibition was only around 20%. It is therefore unlikely that an influx of divalent cations terminates the odorant response by a direct effect on the cAMP-gated channels.

Ambystoma↗

PubMed1993

A simple intrapipette salt bridge.

A simple method is described for achieving a salt bridge within a patch pipette. The tip of the pipette is filled with a Cl(-)-free solution that bathes the membrane patch. Above this is a Cl(-)-containing solution that surrounds the recording Ag/AgCl wire electrode. Addition of 0.07% (w/v) agarose to the solution in the tip prevents bulk mixing of the 2 phases within the pipette. This configuration makes it possible to remove Cl- from the membrane surface but maintain it at the electrode surface. The presence of the Cl(-)-free phase in the bottom of the patch pipette does not reduce the electrical stability of the recording electrode.

Animals↗

PubMed1993

Origin of the chloride current in olfactory transduction.

In the cilia of amphibian olfactory receptor neurons, odorants elicit a receptor current that has two components: a cationic current through cAMP-gated channels and a Cl- current. Here, a cascade of ciliary currents that accounts for the total receptor current is demonstrated. In isolated olfactory cilia, cAMP sequentially activates two currents. The first is a primary cationic current through channels directly gated by cAMP. Part of this current is carried by Ca2+, which in turn activates a Cl- current. This secondary current is eliminated by the presence of Cl- channel inhibitors, replacement of Cl- with methanesulfonate-, removal of external Ca2+, or blockers of the cAMP-gated cationic channels. When cytoplasmic Ca2+ buffering is low, small cationic currents can activate Cl- currents that are 20 times larger.

Animals↗

PubMed1992

Basal conductance of frog olfactory cilia.

The conductance of isolated frog olfactory cilia in the absence of odorants and second messengers has been measured. Current flowing through the pipette-membrane seal rather than the ciliary membrane was subtracted. In normal physiological solutions, each cilium has a conductance averaging 92 pS at the neuronal resting potential. This basal conductance allows current to be carried by K+ or Na+ but not by Cl-. In some cases, single channels with a unit conductance of 153 pS were observed. The conductance of the ciliary membrane implies a length constant for electrotonic conduction of about 160 microns. Since the reversal potential of the basal conductance is near the neuronal resting potential, it should help to stabilize the ciliary potential at some cost to stimulus transduction efficiency.

Animals↗

PubMed1991

Transmembrane currents in frog olfactory cilia.

We have measured transmembrane currents in intact single cilia from frog olfactory receptor neurons. A single cilium on a neuron was sucked into a patch pipette, and a high-resistance seal was formed near the base of the cilium. Action potentials could be induced by applying suction or a voltage ramp to the ciliary membrane. A transient current was seen in some cells on stimulation with odorants. After excision from the cell, most of the cilia showed increased conductance in a bath containing cAMP, indicating that the cytoplasmic face of the ciliary membrane was accessible to the bath. The estimated resistance of a single cilium was surprisingly low.

Action Potentials↗

PubMed1991

Calcium-activated chloride conductance in frog olfactory cilia.

We have measured the effects of cytoplasmic Ca2+ on the conductance of single cilia excised from frog olfactory receptor neurons. When free cytoplasmic Ca2+ is buffered at 0.1 microM, ciliary conductance is low. As Ca2+ is increased, ciliary conductance increases. Maximal conductance averages sevenfold higher than that measured in the absence of Ca2+. We estimate that the K1/2 for Ca2+ activation is 5 microM; the dose-response curve indicates some positive cooperativity of Ca2+ binding. Activation by Ca2+ is rapid and fully reversible. Most of the Ca(2+)-activated current is carried by Cl- and persists in the absence of Na+ and K+. The Cl- channel inhibitor 3',5-dichlorodiphenylamine-2-carboxylate (300 microM) reduces the Ca(2+)-activated current by 90%. Odorants induce a Ca2+ influx in some olfactory receptor neurons, but the consequences of this influx for neuronal function are not well understood. Our findings allow us to predict that a Ca2+ influx would increase the permeability of the olfactory cilia to Cl-. How this would affect the neuronal potential is uncertain, since the equilibrium potential for Cl- in olfactory receptor neurons is unknown.

Animals↗