Calcium Entry through Cyclic Nucleotide-Gated Channels in Individual Cilia of Olfactory Receptor Cells: Spatiotemporal Dynamics

Abstract

Transient elevations of intracellular Ca 2ϩ play an important role in regulating the sensitivity of olfactory transduction, but such elevations have not been demonstrated in the olfactory cilia, which are the site of primary odor transduction. To begin to understand Ca 2ϩ signaling in olfactory cilia, we used highresolution imaging techniques to study the Ca 2ϩ transients that occur in salamander olfactory receptor neurons (ORNs) as a result of cyclic nucleotide-gated (CNG) channel activation. To visualize ciliary Ca 2ϩ signals, we loaded ORNs with the Ca 2ϩ indicator dye Fluo-3 AM and measured fluorescence with a laser scanning confocal microscope. Application of the phosphodiesterase inhibitor IBMX increased fluorescence in the cilia and other neuronal compartments; the ciliary signal occurred first and was more transient. This signal could be abolished by lowering external Ca 2ϩ or by applying LY83583, a potent blocker of CNG channels, indicating that Ca 2ϩ entry through CNG channels was the primary source of fluorescence increases. Direct activation of CNG channels with low levels of 8-Br-cGMP (1 M) led to tonic Ca 2ϩ signals that were restricted locally to the cilia and the dendritic knob. Elevated external K ϩ , which depolarizes cell membranes, increased fluorescence signals in the cell body and dendrite but failed to increase ciliary Ca 2ϩ fluorescence. The results demonstrate the existence and spatiotemporal properties of Ca 2ϩ transients in individual olfactory cilia and implicate CNG channels as a major pathway for Ca 2ϩ entry into ORN cilia during odor transduction.

Cyclic Nucleotide-gated Channels Mediate Membrane Depolarization following Activation of Store-operated Calcium Entry in Endothelial Cells

“…Indeed, homomeric CNG channels are selectively permeable to Ca 2ϩ (53). Fourth, pretreatment with the CNG channel inhibitor LY83,583 (49,50) nearly abolished the observed current. These data collectively support the idea that CNG channels contribute to the non-selective cation permeability in endothelial cells.…”

“…We next examined whether the cationic current was inhibited by LY83,583, a potent CNG channel blocker (49,50), by using physiological salt solutions. PAECs were incubated for 15 min in the presence of 40 M extracellular LY83,583 prior to establishing a seal.…”

Molecular components of signal amplification in olfactory sensory cilia

“…A few μM cAMP is, however, sufficient to activate the cAMP-gated transduction channels. These channels respond to 1-10 μM cAMP, conduct Ca 2+ and monovalent cations into the ciliary lumen (52,53), and generate an initial receptor potential that amounts to <10% of the total receptor potential, as can be gleaned from electroolfactographic measurements in mice. The cAMP-dependent Ca 2+ influx serves as a trigger that sets off a much larger Cl − efflux.…”

Response properties of isolated mouse olfactory receptor cells

“…During the odour response, Ca 2+ , which enters the cell through cyclic nucleotide-gated channels (Frings et al 1995;Leinders-Zufall et al 1997), activates an excitatory Ca 2+ -activated Cl _ conductance thereby increasing the magnitude of the total receptor current (Kleene & Gesteland, 1991;Kurahashi & Yau, 1993;Lowe & Gold, 1993;Zhainazarov & Ache, 1995). Furthermore, Ca 2+ also has profound negative feedback effects on various stages of olfactory signal transduction including the cyclic nucleotidegated channel, adenylyl cyclase and phosphodiesterase (Borisy et al 1992;Kramer & Siegelbaum, 1992;Chen & Yau, 1994;Yan et al 1995;Wei et al 1996Wei et al , 1998Kurahashi & Menini, 1997;Leinders-Zufall et al 1999).…”