Calcium-dependent modulation of the agonist affinity of the mammalian olfactory cyclic nucleotide-gated channel by calmodulin and a novel endogenous factor.
The calcium-dependent modulation of the affinity of the cyclic nucleotide-gated (CNG) channels for adenosine 3',5'-cyclic monophosphate (cAMP) was studied in enzymatically dissociated rat olfactory receptor neurons, by recording macroscopic cAMP-activated currents from inside-out patches excised from their dendritic knobs. Upon intracellular addition of 0.2 mM Ca2+ (0.2 Ca) the concentration of cAMP required for the activation of half-maximal current (EC50) was reversibly increased from 3 microM to about 30 microM. This Ca2+-induced affinity shift was insensitive to the calmodulin antagonist, mastoparan, was abolished irreversibly by a 2-min exposure to 3 mm Mg2+ + 2 mm EGTA (Mg + EGTA), and was not restored by the application of calmodulin (CAM). Addition of CAM plus 0.2 mM Ca2+ (0.2 Ca + CAM), further reversibly shifted the cAMP affinity from 30 microM to about 200 microM. This affinity shift was not affected by Mg + EGTA exposure, but was reversed by mastoparan. Thus, the former Ca2+-only effect must be mediated by an unknown endogenous factor, distinct from CAM. Removal of this factor also increased the affinity of the channel for CAM. The affinity shift induced by Ca2+-only was maintained in the presence of the nonhydrolyzable cAMP analogue, 8-bromo-cAMP and the phosphatase inhibitor, microcystin-LR, ruling out modulation by phosphodiesterases or phosphatases. Our results indicate that the olfactory CNG channels are modulated by an as yet unidentified factor distinct from CAM.
Balasubramanian
S
S
School of Physiology and Pharmacology, University of New South Wales, Sydney 2052, Australia.
Lynch
J W
JW
Barry
P H
PH
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D000242:Cyclic AMP / Q000502:physiology*; D054815:Cyclic Nucleotide-Gated Cation Channels; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D036341:Intercellular Signaling Peptides and Proteins; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000187:drug effects* / Q000502:physiology; D008322:Mammals / Q000502:physiology; D008387:Marine Toxins; D052998:Microcystins; D009812:Odorants; D018034:Olfactory Receptor Neurons / Q000187:drug effects* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010455:Peptides; D010456:Peptides, Cyclic / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D051381:Rats; D017208:Rats, Wistar; D014862:Wasp Venoms / Q000494:pharmacology
D002147:Calmodulin; D054815:Cyclic Nucleotide-Gated Cation Channels; D004791:Enzyme Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D007473:Ion Channels; D008387:Marine Toxins; D052998:Microcystins; D010455:Peptides; D010456:Peptides, Cyclic; D014862:Wasp Venoms; C089414:olfactory cyclic-nucleotide-gated channel 2; D015124:8-Bromo Cyclic Adenosine Monophosphate; C037500:mastoparan; D000242:Cyclic AMP; D010744:Phosphoric Monoester Hydrolases; D000262:Adenylyl Cyclases; C057862:cyanoginosin LR; D002118:Calcium