Constructs, Cell Culture, and Transfection—The construct
encoding human TRPM2 (8 (link)) with a
C-terminal EE epitope (29 ) was
used. Mutations were introduced using QuikChange system (Stratagene) and
confirmed by sequencing. The constructs encoding subunit concatemers with a
C-terminal EE epitope were made as follows. Firstly, the sequence (nucleotides
1–700) encoding part of the TRPM2 N terminus (TRPM2N) was amplified by
PCR using Pfu and forward primer
5′-TCTCTAGAATGGAGCCCTCAGCCCTGAGG-3′ (XbaI
sequence underlined and TRPM2 sequence in italic) and reverse primer
5′-TCAGTACAGGTAGAGCAAGGTGTCC-3′. The resultant PCR product
containing XbaI site was inserted into pCR2.1 following the manufacturer's
instructions (Invitrogen) to generate TRPM2N-pCR2.1. Secondly, the vector
sequence between EcoRI and XbaI and the TRPM2 sequence between XbaI and SacI
were separately excised from TRPM2N-pCR2.1 and ligated with the sequence
between SacI and EcoRI from TRPM2-EE-pcDNA3.1 to generate TRPM2-EE-pCR2.1.
Finally, the TRPM2-EE sequence between XbaI and HindIII was excised from
TRPM2-EE-pCR2.1 to replace the sequence between XbaI and PmeI in
TRPM2-Myc-pcDNA3.1 to produce the constructs encoding concatenated subunits
(see Fig. 4A).
Maintenance of human embryonic kidney cells (HEK293) and transient
transfection with plasmids were described previously
(29 ).
Biotin Labeling and Western Blotting Analysis—Experiments
were performed as described previously
(29 ,
30 (link)). Proteins were resolved on
SDS-PAGE gels and detected using primary rabbit anti-EE antibody (1:2000
dilution; Bethyl Laboratories) and secondary goat horseradish
peroxidase-conjugated anti-rabbit IgG antibody (1:2000 dilution; Santa Cruz
Biotechnology).
Electrophysiological Recording—Whole-cell recordings were
made using an Axopatch 200B amplifier at room temperature 24–48 h after
transfection as described previously
(29 ,
30 (link)). The data were filtered at
2 kHz and sampled at 10 kHz. Cells were held at –40 mV and voltage ramps
with a 1-s duration from –120 mV to 80 mV were applied every 5 s. The
currents at –80 mV denoted in the figures by circles (see Figs.
1, B and C,
3, A–D, and
4, C and D)
were obtained from the current responses to voltage ramps. In some
experiments, cells were held constantly at –40 mV or 40 mV, and the
currents were plotted as continuous lines (see
Fig. 3E).
Intracellular solution contained (in mm): 147 NaCl, 0.05 EGTA, 1
MgCl2, 10 HEPES, 1 Na2ATP, and 1 ADPR.
Table 1 lists the compositions
of extracellular solutions used. Flufenamic acid (FFA) (0.5 mm)
(31 (link)) or
N-(p-amylcinnamoyl) anthranilic acid (20 μm)
(32 (link)) was applied at the end of
each recording via a RSC-160 system (Biologic Science Instruments) to confirm
TRPM2 channel-mediated currents.

Compositions of extracellular recording solutions (in
mm)

Solutions
NaClKClMgCl2CaCl2HEPESGlucose
Standard
 147
 2
 1
 2
 10
 13
147 NaCl + 2 CaCl2 + 1 MgCl2 147
 0
 1
 2
 10
 13
147 NaCl (no CaCl2)
 147
 0
 0
 0
 10
 24
147 NaCl + 0.1 CaCl2 147
 0
 0
 0.1
 10
 24
147 NaCl + 0.3 CaCl2 147
 0
 0
 0.3
 10
 22
147 NaCl + 1 CaCl2 147
 0
 0
 1
 10
 21
147 NaCl + 2 CaCl2 147
 0
 0
 2
 10
 17
147 NaCl + 3 CaCl2 147
 0
 0
 3
 10
 11
147 NaCl + 10 CaCl2 147
 0
 0
 10
 10
 0
147 NaCl + 2 MgCl2 147
 0
 2
 0
 10
 20
110 CaCl2 0
 0
 0
 110
 10
 24
110 MgCl2 0
 0
 110
 0
 10
 24
The reversal or zero-current potentials (Er) were
determined from current responses to the aforementioned voltage ramps. After
cell-attached configuration was established in standard extracellular
solution, application of voltage ramps started and continued throughout
experiments, during which whole-cell configuration was achieved at least 2 min
after the external solution was replaced with the indicated extracellular
solutions. The flufenamic acid/anthranilic acid-insensitive current components
were negligible (e.g. Fig.
1B), and no subtraction from the total currents was made.
The reversal potentials were corrected for liquid junction potentials as we
described previously (33 (link)). Ion
activities were used, converted from ion concentrations using the following
coefficients: γNa = 0.75, γCa = 0.28, and
γMg = 0.34. The relative permeability
PX/PNa (X = calcium or
magnesium) were derived using the Goldman-HodgkinKatz equation
(8 (link),
23 (link)):
PX/PNa = [Na]i exp(ErF/RT)(1 + exp
(ErF/RT))/4[X]o,
where F, R, and T are Faraday constant, gas constant, and
absolute temperature.
Data Analysis—All the data, where appropriate, are presented
as mean ± S.E. The calcium inhibition was estimated by fitting to the
Hill equation: I/I (%) = (100–C)/[1+
([calcium]/IC50)n], where I is the
sustained current as a percentage of the peak current (Ip),
C is the Ca2+-insensitive current component,
IC50 is the concentration producing half-maximal inhibition, and
n is the Hill coefficient. Curve fitting was carried using Origin
(OriginLab, Northampton, MA). Comparisons were made using the Student's
t test between two groups or analysis of variance (post hoc Tukey)
between multiple groups with significance at the level of p <
0.05.