Steven J. Kleene

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.

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.

The Electrochemical Basis of Odor Transduction in Vertebrate Olfactory Cilia

Most vertebrate olfactory receptor neurons share a common G-protein-coupled pathway for transducing the binding of odorant into depolarization. The depolarization involves 2 currents: an influx of cations (including Ca2+) through cyclic nucleotide-gated channels and a secondary efflux of Cl- through Ca2+-gated Cl- channels. The relation between stimulus strength and receptor current shows positive cooperativity that is attributed to the channel properties. This cooperativity amplifies the responses to sufficiently strong stimuli but reduces sensitivity and dynamic range. The odor response is transient, and prolonged or repeated stimulation causes adaptation and desensitization. At least 10 mechanisms may contribute to termination of the response; several of these result from an increase in intraciliary Ca2+. It is not known to what extent regulation of ionic concentrations in the cilium depends on the dendrite and soma. Although many of the major mechanisms have been identified, odor transduction is not well understood at a quantitative level.

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.

Mechanisms of neuronal chloride accumulation in intact mouse olfactory epithelium

When olfactory receptor neurons respond to odours, a depolarizing Cl− efflux is a substantial part of the response. This requires that the resting neuron accumulate Cl− against an electrochemical gradient. In isolated olfactory receptor neurons, the Na+–K+–2Cl− cotransporter NKCC1 is essential for Cl− accumulation. However, in intact epithelium, a robust electrical olfactory response persists in mice lacking NKCC1. This response is largely due to a neuronal Cl− efflux. It thus appears that NKCC1 is an important part of a more complex system of Cl− accumulation. To identify the remaining transport proteins, we first screened by RT‐PCR for 21 Cl− transporters in mouse nasal tissue containing olfactory mucosa. For most of the Cl− transporters, the presence of mRNA was demonstrated. We also investigated the effects of pharmacological block or genetic ablation of Cl− transporters on the olfactory field potential, the electroolfactogram (EOG). Mice lacking the common Cl−/HCO3− exchanger AE2 had normal EOGs. Block of NKCC cotransport with bumetanide reduced the EOG in epithelia from wild‐type mice but had no effect in mice lacking NKCC1. Hydrochlorothiazide, a blocker of the Na+–Cl− cotransporter, had only a small effect. DIDS, a blocker of some KCC cotransporters and Cl−/HCO3− exchangers, reduced the EOG in epithelia from both wild‐type and NKCC1 knockout mice. A combination of bumetanide and DIDS decreased the response more than either drug alone. However, no combination of drugs completely abolished the Cl− component of the response. These results support the involvement of both NKCC1 and one or more DIDS‐sensitive transporters in Cl− accumulation in olfactory receptor neurons.

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.

Transmembrane Currents in Frog Olfactory Cilia

SummaryWe 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.

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 μm for W‐7 and 13 μm 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 Ca2+‐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.

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.

Dissociation of frog olfactory epithelium with N-ethylmaleimide

Treatment of frog olfactory epithelium with 8 mM N-ethylmaleimide for 2 min results in extensive dissociation of the epithelium. The resulting cell suspension contains single olfactory receptor neurons, sustentacular cells, respiratory epithelial cells, and cells of Bowmans glands. The cells in suspension exhibit the same morphologies seen in histological sections of intact epithelium.