JGP study (Takeuchi and Kurahashi. 2023. J. Gen. Physiol.https://doi.org/10.1085/jgp.202213165) reveals that segregation of signals within sensory cilia allows Ca2+ to play opposing roles in olfactory signal transduction.
Our sense of smell begins in the sensory cilia that protrude from the surface of olfactory receptor cells (ORCs), where the chemical signal of odorant molecules is converted into an electrical signal that can be transmitted to the brain. The binding of molecules to odorant receptors in the ciliary membrane triggers production of the second messenger cAMP, which, in turn, binds and activates CNG ion channels, leading to an influx of Ca2+ ions into the cilium (1). In this issue of JGP, Takeuchi and Kurahashi successfully image Ca2+ dynamics within the long, thin cilia of ORCs, allowing them to explain how Ca2+ plays contrasting roles in subsequent signal transduction events (2).
After entering through CNG channels, Ca2+ amplifies the olfactory signal by activating Cl− channels to fully depolarize the ORC (3). However, in a feedback process known as olfactory adaptation, Ca2+, via the Ca2+-binding protein calmodulin, suppresses the activity of CNG channels to reduce the signals generated by lingering smells (4, 5). “At first glance, one might think that these two processes cancel each other out, but the actual cellular response is not like that,” says Hiroko Takeuchi, an associate professor at Osaka University. “We wanted to investigate how Ca2+ plays both excitatory and inhibitory roles within a submicron area of the cell.”
The thin structure of ORC sensory cilia has made it difficult to analyze Ca2+’s divergent effects on olfactory signaling. But Takeuchi, together with Professor Takashi Kurahashi, devised a method to simultaneously measure Ca2+ dynamics and membrane currents in the same cilium (2). Isolated ORCs are patch-clamped in a whole-cell configuration and the recording pipette is used to introduce a fluorescent Ca2+-sensitive dye and caged cAMP to the ciliary cytoplasm. UV photolysis with a laser scanning microscope can then be used to uncage the cAMP and activate CNG channels in a small region of the cilium, triggering Ca2+ influx and the generation of membrane currents.
When the UV stimulus ceased, intraciliary Ca2+ levels returned to baseline in <2 s, showing a similar time course to the decline in membrane current (including Ca2+-activated Cl− components). But this immediate return to basal Ca2+ levels was surprising, because Ca2+-dependent olfactory adaptation is known to persist for >10 s (4). Indeed, when Takeuchi and Kurahashi subjected cilia to two pulses of UV stimulation, the second pulse elicited a smaller current response (indicating adaptation) even though Ca2+ levels returned to baseline in the intervening period.
One explanation for this segregation of Ca2+-dependent signal boosting and Ca2+-mediated adaptation could be that the presence of the Ca2+ buffer EGTA in the recording pipette alters the dynamics of ciliary Ca2+ in some unexpected way. “However, removing EGTA did not change the result, which indicates that the same segregation phenomenon happens in native cilia,” Takeuchi says.
Takeuchi explains that, after the initial influx of Ca2+ through CNG channels, the ability of Ca2+-activated Cl− channels to boost the signal is most likely regulated by free Ca2+ in the ciliary cytoplasm. But, when cytoplasmic Ca2+ levels return to baseline, some Ca2+ remains bound to calmodulin, which mediates olfactory adaptation by binding to CNG channels.
Having established a method to simultaneously measure intraciliary Ca2+ and membrane current, the researchers now hope to gain a more quantitative understanding of Ca2+ dynamics in sensory cilia and how it influences this crucial first stage in our sense of smell.