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Deciphering the function of the CNGB1b subunit in olfactory CNG channels.
Nache V
,
Wongsamitkul N
,
Kusch J
,
Zimmer T
,
Schwede F
,
Benndorf K
.
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Olfactory cyclic nucleotide-gated (CNG) ion channels are key players in the signal transduction cascade of olfactory sensory neurons. The second messengers cAMP and cGMP directly activate these channels, generating a depolarizing receptor potential. Olfactory CNG channels are composed of two CNGA2 subunits and two modulatory subunits, CNGA4, and CNGB1b. So far the exact role of the modulatory subunits for channel activation is not fully understood. By measuring ligand binding and channel activation simultaneously, we show that in functional heterotetrameric channels not only the CNGA2 subunits and the CNGA4 subunit but also the CNGB1b subunit binds cyclic nucleotides and, moreover, also alone translates this signal to open the pore. In addition, we show that the CNGB1b subunit is the most sensitive subunit in a heterotetrameric channel to cyclic nucleotides and that it accelerates deactivation to a similar extent as does the CNGA4 subunit. In conclusion, the CNGB1b subunit participates in ligand-gated activation of olfactory CNG channels and, particularly, contributes to rapid termination of odorant signal in an olfactory sensory neuron.
Figure 1. The CNGB1b subunit does not bind cyclic nucleotides when coexpressed with disabled CNGA2 subunits.(a) Micrographs of a pipette tip with a patch in situ containing multiple CNGA2:A4:B1b channels either in the absence or presence of 5 μM fcGMP. (b) Binding of fcGMP to CNGA2, CNGA4 and CNGB1b measured with 10 μM fcGMP. When coexpressed with disabled CNGA2 (CNGA2TM), CNGB1b does not bind fcGMP. This result obtained in one of our previous studies13 was strengthened with four additional measurements. The fluorescence intensity for CNGA2:A4:B1b channels13 is displayed to compare it with that of CNGA2TM:B1b channels. (c) fcAMP and fHcAMP do not detectably bind to CNGB1b. The nonspecific fluorescence, measured in patches from water-injected oocytes, has been subtracted. The cartoons symbolize the four subunits (squares), their binding site (big circle) and the respective ligand (small circle). In all the cartoons, the R538E mutation in the binding domain is indicated by a red cross and the T539M mutation by a blue cross.
Figure 2. In a CNGA2:A4 background the CNGB1b subunit binds both fcGMP and fHcAMP and activates the channel.(a) Binding of fcGMP to the CNGB1b subunit evokes channel activation. Binding of fcGMP to CNGB1b was observed when coexpressed with either CNGA2RE:A4RE or CNGA2TM:A4RE (upper diagram). When disabling the binding domain of CNGB1b, the fluorescence intensity is significantly reduced. Correspondingly, binding to CNGB1b in CNGA2RE:A4RE:B1b and CNGA2TM:A4RE:B1b channels evoked moderate channel activation which did not appear when the CNGB1b subunit was disabled (lower diagram). The bar graph shows the current amplitude at 10 μM fcGMP, I, corresponding to the binding experiments in the upper diagram, normalized to the current amplitude, Imax, recorded at 4 mM cGMP. (b) Binding of fHcAMP to CNGB1b was observed when coexpressed with both CNGA2RE and CNGA4RE. When disabling the binding domain of CNGB1b, the fluorescence intensity was nearly abolished. Correspondingly, CNGA2RE:A4RE:B1b channels were activated by the ligand only when the CNGB1b binding site was available.
Figure 3. Contribution of the CNGB1b and CNGA4 subunit to channel activation.Concentration-activation relationships for heterotetrameric CNG channels containing different disabled subunits and cGMP. The voltage was +100 mV. The data points represent means of 7–12 measurements each. The continuous curves (red) are the best fit with equation (2). The fits suggest the presence of a component with high apparent affinity (dashed gray lines) and a component with low apparent affinity. (a) CNGA2RE:A4RE:B1b. EC50,l = 177.8 μM cGMP, Hl = 1.2, EC50,h = 3.6 μM cGMP, Hh = 1.0 (fixed), a = 0.07. (b) CNGA2RE:A4:B1bRE. EC50,l = 364.2 μM cGMP, Hl = 1.1, EC50,h = 8.86 μM cGMP, Hh = 1.0 (fixed), a = 0.24. (c) CNGA2RE:A4:B1b. EC50,l = 733.2 μM cGMP, Hl = 1.2, EC50,h = 5.7 μM cGMP, Hh = 1.05, a = 0.89. The fit parameters are summarized in Table 1.
Figure 4. Effects of the CNGB1b and CNGA4 subunit on the kinetics of channel activation and deactivation.Superimposition of activation (a) and deactivation (b) time courses following a concentration jump to 10 μM fcGMP and back to zero for CNGA2 (green), CNGA2:A4:B1b (black), CNGA2RE:A4RE:B1b (blue), and CNGA2RE:A4:B1bRE (orange) channels. The traces are averages obtained from 5–7 traces obtained from different patches. All traces were normalized with respect to the initial and late current (I/Imax). The voltage was set to +10 mV. The respective activation and deactivation time courses of the individual measurements were fitted with monoexponential functions apart from the activation of CNGA2RE:A4RE:B1b channels that required a biexponential function (Equation S1, Supplementary Information). The time constants are illustrated by bar graphs below the traces. For small differences a t-test with respect to CNGA2:A4:B1b channels was performed. An asterisk indicates a significant difference (p < 0.05).
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