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SUPPLEMENTARY MATERIALS Responsiveness of COS-1 cells to different compounds 0.0 20 15 10 5 Relative response, % 0.5 20 M Glutamate 1.0 40 M ACh 1 M ATP + 100 M suramin B 20 M Serotonin Normalized ATP response A 20 M GABA I. 0 10-8 10-7 10-6 10-5 10-4 10-3 10-2 ATP, M Figure 1. (A) Magnitude of Ca2+ transients in the COS-1 cytoplasm versus concentration of bath ATP. Every COS-1 cell (n = 18) was stimulated first with 10 M ATP and then with the nucleotide at several concentrations (n=3-5). For comparison, a Ca2+ response (R) at a given ATP concentration ([ATP]) were normalized to the magnitude of the control response (R10) elicited 10 M ATP. The continuous line corresponds to the equation: R/R10 = Amax[ATP]/(1 + [ATP]/K0.5) with Amax = 1.16 and K0.5 = 2.1 M. (B) Relative responsiveness of COS-1 cells to different compounds (n=3-6). Every COS-1 cell was stimulated by 1 M ATP as a control prior to application of other stimuli, and responses to the indicated compounds were normalized to the magnitude of the control response. II. ATP release on serial depolarization of a taste cell with spike-like pulses. Figure 2. Response of the ATP-sensor elicited by serial depolarization of a taste cell with 7 ms pulses to 10 mV (n=200) (upper insert). A change in Fluo-4 fluorescence became visible after application of 170 pulses. 1 III. Fraction of gustducin-positive taste cells. By introducing the population fraction for A type cells, fA = (number of type A cells)/(total number of taste bud cells), and that for cells of the other types, the fraction of type A cells in the GFP-negative (non-green) cell population can be written as: F = (1-x)fA/((1-x)fA+fB+fC+fN) where x is the fraction of GFP-positive cells in the A subcategory. Taking F = 12/43 = 0.28 (Fig.1E) and fA – fN equal to 0.52, 0.31, 0.13, and 0.04, respectively (Romanov & Kolesnikov, 2006), the above equation yields x = 0.64. Thus, in a mouse CV taste bud, nearly two of every three cells of the type A are gustducin-positive. IV. Linear T7-based aRNA amplification and PCR analysis. The whole-cell patch-clamp technique was used to identify enzymatically dissociated taste cells. First-strand cDNA was synthesized directly from cell lysates harvested via patch pipettes. RNaseOUT RNase inhibitor (Invitrogen) was included in the patch pipette. After recording, the cytoplasmic contents were drawn into the recording pipette by suction and expelled into a PCR tube containing 100 ng of oligo(dT)15-T7 (5’AAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGCGCTTTTTTTTTTTTTTT3’) primer in water. A sample of 10 pooled cells was subjected to heat denaturation at 70°C for 5 min followed by snap cooling on ice. Reverse transcription was carried out in a 10 µl volume containing 2 µl first-strand reaction buffer, 0.5 µl 0.1 M DTT, 0.5 µl 10 mM dNTPs, 20 U RNaseOUT (Invitrogen), 100 U SuperScript III reverse transcriptase (Invitrogen) at 50ºC for 30 min. Second-strand cDNA was synthesized with 0.3 µl RNase H (Invitrogen) and 10 U DNA polymerase I (Promega). The reaction was carried out at 12ºC for 30 min and then at 22ºC for 1 h. cDNA was blunt-ended by treatment with 20 U T4 DNA polymerase (Promega) for 10 min at 37ºC followed by inactivation at 80°C for 3 min. The antisense RNA (aRNA) amplification by in vitro transcription was carried out in 100 µl reaction mixture for 3 h with the use of T7 RNA polymerase (Promega) following the manufacturer’s instructions. After RNA amplification, the DNA template was removed by incubation of the reaction with 4 U DNaseI (Sigma) for 15 min. Amplified aRNA was purified using RNeasy MinElute Kit (Qiagen) and reverse-transcribed with 200 U SuperScript III (Invitrogen) and 200 ng random hexanucleotides in a 20 µl reaction at 50°C for 1 h. The experiment was performed in triplicate. Control in vitro transcription reaction contained no template but 100 ng oligo(dT)15-T7 primer. Reaction condition for PCR were 95ºC for 2 min, followed by 40 cycles at 95ºC for 40s, 56ºC for 40s (or 60º for PLCβ2), and 72ºC for 50 s with the use of PCR Master Mix (Promega). Primers for Cx 26 were 285-306 2 (CAAGAATGTATGCTACGACCAC) sense and 484-501 (CAGGGACCCTTCGATACG) antisense oligonucleotides (GenBank M81445), yielding a 217 bp product; primers for Cx 30.3 were 371-390 (TCACCTGTCCTTCCCTGTTA) sense and 640-657 (TCGGGCGATGTAACAGTC) antisense oligonucleotides (GenBank M91443), yielding a 287 bp product; primers for Cx 31.1 were 124-144 (AACTGGAGTGTTTTTGAGGGA) sense and 842-862 (AGATGAGGTCGCATGAGAGGA) antisense oligonucleotides (GenBank M91236), yielding a 739 bp product; primers for Cx 32 were 217-240 (TCCTCTTTCATCTGTAACACCCTC) sense and 796-820 (TGTTGATCTCATTCTGCTTGTATTC) antisense oligonucleotides (GenBank M63802), yielding a 604 bp product; primers for Cx 33 were 1980-1998 (GCCTATTCTCTTATACCTG) sense and 2370-2389 (CCAAGACAAAAGACACCACT) antisense oligonucleotides (GenBank AY465183), yielding a 410 bp product; primers for Cx 36 were 336-353 (ATCTTGGAGAGGCTGCTG) sense and 1867-1886 (ATCTTCTCGTTTGCTCCCTC) antisense oligonucleotides (GenBank AF016190), yielding a 411 bp product; primers for Cx 43 were 377-401 (CTATGTGATGAGAAAGGAAGAGAAG) sense and 882-901 (GAGCAGCCATTGAAGTAAGC) antisense oligonucleotides (GenBank X61576), yielding a 525 bp product; primers for Cx 45 were 530-550 (TCACTGTGCTGATTGTCTTTC) sense and 1013-1031 (AGAAAGCCCACCTCAAACA) antisense oligonucleotides (GenBank X63100), yielding a 502 base product; primers for Cx 47 were 641-660 (GTGGCTCAGCTGGTGGTTAG) sense and 1285-1302 (TCTCTGCTGCCTGTACTG) antisense oligonucleotides (GenBank AJ276435), yielding a 662 bp product; primers for pannexin 1 were 763-784 (TTCTTCCCCTACATCCTACTGC) sense and 1478-1498 (TGTTCTCCAGCACCTTCAGAC) antisense oligonucleotides (GenBank BC049074 ), yielding a 736 bp product; primers for TRPM5 were 2411-2430 (TCACAGATGAGGACACGCAC) sense and 2841-2864 (AGCAGAGGGTGAAGAGAACAGTTC) antisense oligonucleotides (GenBank AY280364), yielding a 435 bp product; primers for phospholipase C beta 2 were 521544 (CCCCTTACCATGCCAGCTCTCTTC) sense and 833-853 (CACCACGCCCTTCAGTTCCCA) antisense oligonucleotides (GenBank BC038048), yielding a 333 bp product. V. Immunohostochemistry Paraffin embedded sections (4m thick) from mouse circumvallate papillae were incubated overnight at 4°C with the indicated primary antibodies. Primary antibodies were diluted in blocking solution (2% goat serum, 0.1% Triton X-100 in PBS) as follows: 1:500 for rabbit antiTRPM5 (Damak et al., 2005); 1:500 for chicken anti-Panx1 (Diatheva); 1:500 for rabbit anti3 PLC2 (Santa Cruz). For double immunostaining, sections were incubated with anti-Panx1 along with either anti-TRPM5 or anti- PLC2. Sections were then washed three times for ten minutes each in PBS at room temperature, then incubated for two hours at room temp with the indicated secondary antibodies. Secondary antibodies were diluted in blocking solution as follows: 1:500 for alexa fluor 594 goat anti-rabbit IgG (Molecular probes); 1:500 for fluorescein conjugated goat anti chicken IgY antibody (Aves). Following incubation with the secondary antibody, sections were washed three times for ten minutes each in PBS, then mounted. Images were taken using confocal microscopy. Imunostaining controls consisted of (a) omitting the primary antiserum and (b) omitting the secondary antibody. No staining was seen under these control conditions. 4