doi: 10.

1038/nature06861

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Supplementary Figures

GFP-Control

GFP-OR83b

GFP-OR22a

Rhodamine

Rhodamine

Rhodamine

Merge

Merge

Merge

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Supplementary Figure 1. Visualization of OR proteins in HEK293 cells. Confocal micrographs of HEK293 cells transfected with GFP control plasmid, GFP-OR83b N-terminal fusion protein, and GFP-OR22a N-terminal fusion protein. Top row: GFP fluorescence; 2nd row: Rhodamine fluorescence imaging the rhodamine-labelled plasma membrane. 3rd row: Merge of GFP (green) and rhodamine (red) signals. 4th row: Magnified areas with paths used for quantitative image analysis. Bottom row: Fluorescence intensity along the paths indicated in the magnified panels above. Membrane insertion of the OR proteins is demonstrated by colocalising the GFP signal with red fluorescence of the rhodamine-labelled plasma membrane.

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a
Control 2 nM Etb 100 M Etb

b
250
200

Ca2+-free

200 ]i (nM) [Ca

2+

150 100 50 0
on tro l nM Et 10 b 0 M Et b C on 10 tro 10 0 0 M l M Et Et b b + C a 2+ 2 C

Control Ca2+-free

100 M Etb Ca2+-free

100 M Etb + Ca2+

100

Supplementary Figure 2. Coexpressed OR22a and OR83b mediate ethyl butyrate-stimulated Ca2+ influx. a, Images of the free intracellular calcium concentration ([Ca2+]i in nM) in HEK293 cells expressing OR22a and OR83b; top: before (Control) and one minute after application of ethyl butyrate (Etb) in a saline containing 1 mM Ca2+; bottom: Etb-induced elevation of [Ca2+]i in a Ca2+free bath solution is only detectable when Ca2+ is coapplied with the odorant Etb (right). Scale bar, 10 m. b, [Ca2+]i measured in the regions indicated in a.

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hKCNQ4 + OR22a + OR83b

+40 mV 200 pA 200 ms Control 100 pM Etb 100 nM Etb

-90 mV

b
hKCNQ4 ORC 1.0 0.5 0.0 -6 -8 Control -10 log [Etb] (M) -4 1.0 0.5 0.0

I/IControl

Supplementary Figure 3. Testing for odour signal transduction via the G protein Gq. a, Odour stimulation by Etb of OR22a and OR83b coexpressed in HEK293 cells with human KCNQ4, a voltage-gated potassium channel that reports Gq protein activation by an alteration of its activity, does not affect the KCNQ4 current activated by a voltage step to +40 mV. Linearly voltagedependent currents were subtracted using a P/n protocol. b, While increasing Etb concentrations produce an increasing olfactory receptor current (ORC, black squares, data normalized to maximum response, the fitted curve is described by EC50 = 256 pM and Hill coefficient = 0.41) the KCNQ current at +40 mV (blue circles) remains unaffected (data presented as means and s.e.m.; n = 6).

I/Imax

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hHCN2 + OR22a + OR83b

b
-40 mV -140 mV

-40 mV

1.00 0.75

c
Control V (mV) Etb

15 10 5 0

100 nM Etb

I/Imax

0.50 0.25

***

200 pA 500 ms

0.00 -140 800

**

-100 -60 V (mV)

-140 mV

at -140 mV (ms)

-40 mV

OR22a OR22a OR83b +OR83b 600

500 400 300 200 Con OR22aOR22aOR83b +OR83b

(ms)

600

Control Etb

200 pA 500 ms

**
400 Control Etb -140 -120 -100 V (mV) -80

Supplementary Figure 4. Testing for odour signal transduction via the G protein Gs. a, OR22a, OR83b and the human HCN2 channel, which is activated by hyperpolarization and modulated by cAMP, were coexpressed in HEK293 cells. The current family (top) was activated under control conditions according to the given pulse protocol. Odour stimulation (2 min 100 nM Etb) accelerated the current time course (bottom). b, Normalized maximal currents (top) and activation time constants (bottom) as a function of test potential of hHCN2 channels coexpressed with OR22a before and after stimulation with 100 nM Etb. n = 7; **, P < 0.01; ***, P < 0.0001; ANOVA test. c, Effect of 100 nM Etb on the depolarizing shift of the HCN2 current half-maximal activation potential (V, top) and activation time constant at 140 mV (bottom) in cells coexpressing the indicated ORs. n = 7; *, P < 0.05; **, P < 0.01; Students t test. Error bars represent s.e.m.

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a
+80 mV 0 mV -80 mV Control 8-br-cAMP Control 8-br-cGMP 500 pA 50 ms PA

b
OR47a+OR83b 1000 I (pA)

500

PA

8-br- 8-brcAMP cGMP

Supplementary Figure 5. Cyclic nucleotides activate currents through the receptor complex OR47a/OR83b. a, Superimposed current responses in cells expressing OR47a and OR83b to the indicated pulse protocol (top) obtained in the absence (Control) and in the presence of 100 M pentyl acetate (PA, middle) or 100 M 8-bromo-cAMP and 8-bromo-cGMP (bottom), respectively. b, Maximal inward currents at 100 mV activated by 100 M PA, 8-bromo-cAMP and 8bromo-cGMP (n = 8). Error bars represent s.e.m.

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Supplementary Tables

Supplementary Table 1. Parameters characterizing currents activated by ethyl butyrate application (100 nM, 1 s) in HEK293 cells expressing OR22a (C-terminal and N-terminal GFP fusion protein) and OR83b. Currents were measured in the whole-cell configuration at -60 mV in the presence of ATP and GTP (+) and in the presence of ADP and GDP (). Ii, ionotropic current; Im, metabotropic current; Decay time is the time at which the current response terminated. The latency of Ii was < 0.5 s.
OR 22a C N C N ATP GTP + + Ii Peak size (pA) 100 23 98 23 133 51 132 38 Ii Peak time (s) 1.10 0.03 1.16 0.06 1.13 0.02 0.90 0.17 Ii Decay time (s) 10.5 0.7 10.2 0.9 10.8 0.6 10.4 0.9 Im Latency (s) 10 2 11 3 18 8 24 5 Im Peak size (pA) 291 54 311 51 77 7 51 6 Im Peak time (s) 58 4 62 9 43 9 44 6 Im Decay time (s) 79 5 80 8 68 8 63 9 n

16 8 6 11

Supplementary Table 2. Parameters characterizing currents activated by ethyl butyrate application (100 nM, 1 s) in HEK293 cells expressing OR22a (C-terminal GFP fusion protein) and OR83b. Currents were measured in the outside-out configuration at -60 mV in the presence of ATP and GTP (+) and in the presence of ADP and GDP (). Ii, ionotropic current; Im, metabotropic current; Decay time is the time at which the current response terminated. The latency of Ii was < 0.5 s.

OR 22a C C

ATP GTP +

Ii Peak size (pA) 1.3 0.3 1.6 0.3

Ii Peak time (s) 1.13 0.11 1.38 0.23

Ii Decay time (s) 5.2 1.1 4.5 1.2

Im Latency (s) 10 2 11 3

Im Peak size (pA) 1.7 0.4 0.5 0.2

Im Peak time (s) 33 4 36 8

Im Decay time (s) 45 7 58 9

10 6

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