Sensory stimuli alter the membrane conductance of receptor cells. Noise analysis studies in several types of receptors suggest that ionic channels mediate this change in conductance, but no direct evidence for such channels has been obtained. We have now investigated the mechanism of ion permeation underlying the light-induced conductance in the ventral photo-receptors of Limulus polyphemus by means of the patch-clamp technique and have resolved single-channel currents that are activated by light.

Olfactory sensory neurons respond to odorants increasing Ca(2+) concentrations in their chemosensory cilia. Calcium enters the cilia through cAMP-gated channels, activating Ca(2+)-dependent chloride or potassium channels. Calcium also has a fundamental role in odour adaptation, regulating cAMP turnover rate and the affinity of the cyclic nucleotide-gated channels for cAMP. It has been shown that a Na(+)/Ca(2+) exchanger (NCX) extrudes Ca(2+) from the cilia. Here we confirm previous evidence that olfactory cilia also express plasma membrane Ca(2+)-ATPase (PMCA), and show the first evidence supporting a role in Ca(2+) removal. Both transporters were detected by immunoblot of purified olfactory cilia membranes. The pump was also revealed by immunocytochemistry and immunohistochemistry. Inside-out cilia membrane vesicles transported Ca(2+) in an ATP-dependent fashion. PMCA activity was potentiated by luminal Ca(2+) (K(0.5) = 670 nm) and enhanced by calmodulin (CaM; K(0.5) = 31 nm). Both carboxyeosin (CE) and calmidazolium reduced Ca(2+) transport, as expected for a CaM-modulated PMCA. The relaxation time constant (tau) of the Ca(2+)-dependent Cl(-) current (272 +/- 78 ms), indicative of luminal Ca(2+) decline, was increased by CE (2181 +/- 437 ms), by omitting ATP (666 +/- 49 ms) and by raising pH (725 +/- 65 ms), suggesting a role of the pump on Ca(2+) clearance. Replacement of external Na(+) by Li(+) had a similar effect (tau = 442 +/- 8 ms), confirming the NCX involvement in Ca(2+) extrusion. The evidence suggests that both Ca(2+) transporters contribute to re-establish resting Ca(2+) levels in the cilia following olfactory responses.

A B S T R A C T Removing the glial cells that encaseLimulus ventral photoreceptors allows direct observation of the cell surface. Light microscopy of denuded photoreceptors reveals a subdivision of the cell body into lobes. Often one lobe, but sometimes several, is relatively clear and translucent (the R lobes). The lobe adjacent to the axon (the A lobe) has a textured appearance. Scanning electron microscopy shows that microvilli cover the surface ot R lobes and are absent from the surface of A lobes. When a dim spot of light is incident on the R lobe, the probability of evoking a single photon response is two to three orders of magnitude higher than when the same spot is incident on the A lobe. We conclude that the sensitivity of the cell to light is principally a function of the R lobe.

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The mechanisms that power the physiological events occurring in cilia, flagella, and microvilli are of fundamental importance for the functions of these important and ubicuous organelles. The olfactory epithelium is mostly populated by ciliated olfactory sensory neurons (OSNs) and surrounding sustentacular cells (SCs) with apical microvilli. The only OSN dendrite extends to the surface forming a knob projecting several chemosensory cilia of ϳ50 ϫ 0.2 m, devoid of inner membranes embedded in a mucus layer. Upon odorant binding, odor receptors couple to G-protein activating adenylyl cyclase, producing cAMP. cAMP opens cyclic nucleotide-gated channels allowing a Ca 2ϩ influx that opens Ca 2ϩ -activated Cl Ϫ channels, generating the receptor potential. Many enzymes are activated in chemotransduction to hydrolyze ATP. The knob contains approximately two mitochondria; assuming that the cilia ATP is 1 mM and diffuses along it at ϳ10 m in 500 ms, ATP from the knob mitochondria may not fulfill the demands of transduction over the full length of the cilium, which suggests an additional ATP source. We measured millimolar glucose in rat mucus; we detected glucose transporter GLUT3 in rat and toad (Caudiverbera caudiverbera) OSN cilia, SC microvilli, and glycolytic enzymes in rat cilia. We also found that the cilia and knob can incorporate and accumulate 2-deoxyglucose (glucose analog),butnotwhenblockingGLUT.Glucoseremovalandtheinhibitionofglycolysisoroxidativephospholylationimpairedtheodorresponse. This evidence strongly suggests that glycolysis in the cilia and knob oxidative phosphorylation together fuel chemotransduction.

The cornea is extensively innervated by trigeminal ganglion cold thermoreceptor neurons expressing TRPM8 (transient receptor potential cation channel subfamily M member 8). These neurons respond to cooling, hyperosmolarity and wetness of the corneal surface. Surgical injury of corneal nerve fibers alters tear production and often causes dry eye sensation. The contribution of TRPM8-expressing corneal cold-sensitive neurons (CCSNs) to these symptoms is unclear. Using extracellular recording of CCSNs nerve terminals combined with in vivo confocal tracking of reinnervation, Ca 2ϩ imaging and patch-clamp recordings of fluorescent retrogradely labeled corneal neurons in culture, we analyzed the functional modifications of CCSNs induced by peripheral axonal damage in male mice. After injury, the percentage of CCSNs, the cold-and menthol-evoked intracellular [Ca 2ϩ ] rises and the TRPM8 current density in CCSNs were larger than in sham animals, with no differences in the brake K ϩ current I KD. Active and passive membrane properties of CCSNs from both groups were alike and corresponded mainly to those of canonical low-and high-threshold cold thermoreceptor neurons. Ongoing firing activity and menthol sensitivity were higher in CCSN terminals of injured mice, an observation accounted for by mathematical modeling. These functional changes developed in parallel with a partial reinnervation of the cornea by TRPM8(ϩ) fibers and with an increase in basal tearing in injured animals compared with sham mice. Our results unveil key TRPM8-dependent functional changes in CCSNs in response to injury, suggesting that increased tearing rate and ocular dryness sensation derived from deep surgical ablation of corneal nerves are due to enhanced functional expression of TRPM8 channels in these injured trigeminal primary sensory neurons.

The identity of the second messenger that directly activates the light-dependent conductance in invertebrate photoreceptors remains unclear; the available evidence provides some support for cGMP and Ca2'. To resolve this issue we have applied these second messengers to membrane patches excised from the light-sensitive lobe of Limulus ventral photoreceptors. Our results show that these patches contain channels that can be opened by cGMP, but not by Ca2+. These cGMP-activated channels closely resemble the channels activated by light in cell-attached patches. This evidence suggests that cGMP is the messenger that opens the light-dependent channel in invertebrate photoreceptors.In photoreceptors, large numbers (102-103) of membrane channels are affected by the absorption of a single photon (1, 2). This amplification is produced by a second messenger cascade (3). In vertebrate rods and cones (4-8) the final step of this cascade involves the direct control of ion channels by cGMP. Efforts to establish which second messenger controls the light-dependent channels in invertebrate photoreceptors have given conflicting results (9). Injection of inositol trisphosphate (IP3) (10, 11) or Ca2+ (12) into Limulus ventral photoreceptors leads to the activation of the light-dependent conductance. This evidence, together with biochemical evidence that the inositol phospholipid pathway (11,(13)(14)(15) METHODS To apply the patch clamp technique to Limulus ventral photoreceptors, it is first necessary to remove the glial cells and connective tissue that surround the photoreceptor cell that is to be studied. This was done mechanically by using a suction pipette while observing the cell through a x 100 compound microscope, as described (17). Gigaohm seals were made on the light-sensitive microvillar membrane ofthe photoreceptor (18) by using 1-to 2-Mfl electrodes filled with artificial seawater (ASW) and coated with Sylgard (Dow). After obtaining the seals, patches were excised in the insideout configuration into an "internal" solution (described below) resembling the ionic composition ofthe cytoplasm. As described in Results, it was important to prevent exposure of the excised patch to ASW. In our initial work, we achieved this by exposing the region of the cell-attached patch to internal solution delivered from a separate pipette. We found, however, that this procedure was cumbersome and that obtaining stable excised patches was rare. We then switched to a different strategy that gave a higher yield of stable excised patches. In this procedure we perfused the entire bath with internal solution before making the seals (depolarizing the membrane voltage to near zero). The patch could therefore be excised directly into the bath without exposure to ASW. To apply different solutions to the excised patch, the pipette tip was placed near the mouth of one of a series of parallel glass capillaries through which different solutions Abbreviations: IP3, inositol trisphosphate; ASW, artificial seawater. 7938The publication costs of this a...

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