Evidence that OMP regulates NCX1 through interaction with CaM and Bex.

<p>A, NCX1 and OMP are highly expressed and co-localized in cell bodies, dendrites, dendritic knobs (long arrows) and cilia (short arrows) of mature OSNs of WT mice. Scale bar, 5 µm. B, Sensorgrams for interaction of XIP peptide immobilized on a CM5 sensorchip and CaM (10, 25, 50,100, 250 nM) using a BIAcore 3000 biosensor. Running buffer used during recording contained 0.1 mM Ca²⁺. Data were fitted with a 1∶1 Langmuir binding model using BIAevaluation software, giving K_d = 20 nM. C, Sensorgram for interaction of immobilized Bex1 (50–75) peptide and CaM (31.25, 62.5, 125, 250, 500 nM) on a CM5 chip in presence of 0.1 mM Ca²⁺. D, Because of the rapid on/off kinetics of the interaction between CaM and Bex1 peptide, saturation curves of equilibrium response versus CaM concentration were analyzed using a nonlinear 1∶1 binding isotherm model, giving K_d = 280 nM.</p

NCX is critical for reducing elevated intracellular Ca²⁺ levels in OSN knobs.

<p>A, Comparison of the kinetic properties of the Ca²⁺ transients (normalized responses) in OSN knobs evoked by a 1-s pulse of KCl (80 mM) before (control) and after thapsigargin (200 nM) treatment. B, Ca²⁺ response (ΔF/F) of a single knob stimulated with a 5-s pulse of caffeine (10 mM) followed by a 1-s pulse of KCl (80 mM), confirming that thapsigargin (200 nM) pretreatment depleted Ca²⁺ from intracellular stores. C, Comparison of the kinetic properties of the Ca²⁺ transients (normalized responses) in OSN knobs evoked by a 1-s pulse of IBMX (100 µM) before (control) and after treatment with the PMCA inhibitor carboxyeosin (10 µM). D, Comparison of the kinetic properties of IBMX-induced Ca²⁺ transients (normalized responses) in OSN knobs before (control) and after treatment with the NCX inhibitor 3,4-dichlorobenzamil hydrochloride (DCB; 10 µM). E, Bar graphs showing collected decay time constants from control (gray) and treated (black) OSN knobs. For thapsigargin (Tg), control: τ = 21.1±1.9 s (<i>n</i> = 13, <i>N</i> = 4), Tg: τ = 19.0±1.9 s (<i>n</i> = 14, <i>N</i> = 4). For carboxyeosin (CE), control: τ = 8.5±1.9 s (<i>n</i> = 25, <i>N</i> = 8), CE: τ = 13.3±1.4 s (<i>n</i> = 16, <i>N</i> = 4). For 3,4-dichlorobenzamil hydrochloride (DCB), control: τ = 11.0±1.4 s (<i>n</i> = 12, <i>N</i> = 4), DCB: τ = 35.1±4.4 s (<i>n</i> = 12, <i>N</i> = 4). NS, not significant; *<i>p</i><0.05; **<i>p</i><0.0001. The Ca²⁺ transients in A,C, and D were rescaled to give the same peak amplitude.</p

Recovery of elevated intracellular Ca²⁺ levels is compromised in OMP^−/− mice.

<p>A–C, Comparison of fluorescence intensity changes (ΔF/F) of Ca²⁺ responses in WT and OMP^−/− knobs to a 1-s pulse of 100 µM IBMX (A, B) or 80 mM KCl (C) using 1 mM external Ca²⁺. Recovery time course of the signals was fitted with single exponential functions (dashed lines). The decay time constants (τ) of the fitted curves are indicated. These results demonstrate an apparent defect in the kinetics of removal of elevated Ca²⁺_i from the dendritic knobs of the OSNs of OMP^−/− mice. D, Ca²⁺ response of a single WT knob stimulated with a 1-s pulse of IBMX (100 µM) followed by a 5-s pulse of caffeine (10 mM), both in low external Ca²⁺ solution (0.6 µM), confirming that the caffeine-induced Ca²⁺ transient depends on an intracellular source. E, Comparison of Ca²⁺ transients in WT and OMP^−/− knobs evoked by a 5-s pulse of caffeine (10 mM) in low extracellular Ca²⁺ solution (0.6 µM). Recovery time course of the signals was fitted with single exponential functions (dashed lines). The decay time constants (τ) of the fitted curves are indicated. F, Bar graphs showing collected results from OSN knobs of WT and OMP^−/− mice. For stimulation with IBMX, WT: τ = 8.5±1.3 s (<i>n</i> = 25, <i>N</i> = 8), OMP^−/−: τ = 21.3±2.2 s (<i>n</i> = 13, <i>N</i> = 4). For stimulation with KCl, WT: τ = 20.1±1.7 s (<i>n</i> = 13, <i>N</i> = 4), OMP^−/−: τ = 28.7±3.2 s (<i>n</i> = 13, <i>N</i> = 4). For stimulation with caffeine in 1 mM Ca²⁺_o, WT: τ = 13.9±1.6 s (<i>n</i> = 15, <i>N</i> = 5), OMP^−/−: τ = 26.8±1.9 s (<i>n</i> = 15, <i>N</i> = 4), **<i>p</i><0.0001 and in low external Ca²⁺ (0.6 µM), WT: τ = 13.1±1.8 s (<i>n</i> = 19, <i>N</i> = 6), OMP^−/−: τ = 27.3±1.9 s (<i>n</i> = 15, <i>N</i> = 5), *<i>p</i><0.001; **<i>p</i><0.0001.</p

OMP facilitates NCX activity.

<p>A, The activity of NCX was probed in reverse mode by monitoring the rise of Ca²⁺_i in response to a stepwise reduction of Na⁺_o (by substituting Na⁺ by Li⁺). Ca²⁺ responses are reversibly inhibited by the NCX inhibitor KB-R7943 (10 µM) as well as by dichlorobenzamil (DCB, 10 µM). This confirms that the rise in Ca²⁺ induced by low Na⁺_o is a result of Ca²⁺ entry through NCX. B, Bar histogram of the Ca²⁺ responses obtained after treatment with pharmacological agents that modulate NCX activity. Concentrations are given below each bar (in µM). Number of knobs tested are indicated in parentheses above each bar. C, Averaged Ca²⁺ responses due to reverse mode activity of NCX from WT (gray) and OMP^−/− knobs (black) show a 2.5-fold increase (p<0.0001) in time-to-peak in OMP^−/− (<i>n</i> = 27; <i>N</i> = 5) <i>vs</i>. WT mice (<i>n</i> = 26; <i>N</i> = 4). D, Averaged decay time courses of the same signal in WT (gray) and OMP^−/− knobs (black) reveal an approximately 2-fold increase in the recovery rate in OMP^−/− mice (p<0.03). Data were normalized to the value obtained immediately at the end of the 20-s low Na⁺ stimulus.</p