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. 2010 Aug 27;285(35):26806-26814.
doi: 10.1074/jbc.M110.109975. Epub 2010 Jun 29.

Contribution of the S5-pore-S6 domain to the gating characteristics of the cation channels TRPM2 and TRPM8

Frank J P Kühn  1 , Katja Witschas  2 , Cornelia Kühn  2 , Andreas Lückhoff  2
Affiliations

Contribution of the S5-pore-S6 domain to the gating characteristics of the cation channels TRPM2 and TRPM8

Frank J P Kühn et al. J Biol Chem. .

Abstract

The closely related cation channels TRPM2 and TRPM8 show completely different requirements for stimulation and are regulated by Ca(2+) in an opposite manner. TRPM8 is basically gated in a voltage-dependent process enhanced by cold temperatures and cooling compounds such as menthol and icilin. The putative S4 voltage sensor of TRPM8 is closely similar to that of TRPM2, which, however, is mostly devoid of voltage sensitivity. To gain insight into principal interactions of critical channel domains during the gating process, we created chimeras in which the entire S5-pore-S6 domains were reciprocally exchanged. The chimera M2-M8P (i.e. TRPM2 with the pore of TRPM8) responded to ADP-ribose and hydrogen peroxide and was regulated by extracellular and intracellular Ca(2+) as was wild-type TRPM2. Single-channel recordings revealed the characteristic pattern of TRPM2 with extremely long open times. Only at far-negative membrane potentials (-120 to -140 mV) did differences become apparent because currents were reduced by hyperpolarization in M2-M8P but not in TRPM2. The reciprocal chimera, M8-M2P, showed currents after stimulation with high concentrations of menthol and icilin, but these currents were only slightly larger than in controls. The transfer of the NUDT9 domain to the C terminus of TRPM8 produced a channel sensitive to cold, menthol, or icilin but insensitive to ADP-ribose or hydrogen peroxide. We conclude that the gating processes in TRPM2 and TRPM8 differ in their requirements for specific structures within the pore. Moreover, the regulation by extracellular and intracellular Ca(2+) and the single-channel properties in TRPM2 are not determined by the S5-pore-S6 region.

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Figures

FIGURE 1.
FIGURE 1.
Design of TRPM2/TRPM8 channel chimeras. The presumed basic structure of TRP channels, including the exchanged S5-pore-S6 domain (gray shading), is illustrated. The corresponding amino acid sequences of TRPM2 and TRPM8 are shown in single-letter code, and conserved residues are shown in boldface. The site within S6 where the exchange of a hydrophobic residue with a lysine reverses the charge selectivity of the pore in both TRPM2 and TRPM8 is marked with an asterisk (19).
FIGURE 2.
FIGURE 2.
Activation of M2-M8P by ADPR and regulation by extracellular Ca2+. A and B, whole-cell (w.c.) currents of cells expressing M2-M8P and wild-type TRPM2, respectively, elicited by infusion of 0.6 mm ADPR through the patch pipette. Each example represents four to five similar experiments. The Ca2+ concentration of the pipette solution was buffered with EGTA to below 10 nm. Horizontal black bars indicate time periods during which the standard bath solution (1.2 mm Ca2+) was exchanged with either a divalent cation-free solution (DVF) or a solution containing NMDG as the main extracellular cation. C and D, I-V relations of currents shown in A and B, respectively, obtained during voltage ramps from −90 to +60 mV from a holding potential of −60 mV.
FIGURE 3.
FIGURE 3.
Inhibition of wild-type TRPM2 and M2-M8P by divalent cation-free extracellular solution but in the presence of 1 μm intracellular Ca2+. A and B, whole-cell (w.c.) currents of cells expressing M2-M8P and wild-type TRPM2, respectively. Each example represents four to six similar experiments. Currents were elicited by infusion of ADPR (0.05 mm) and Ca2+ (1 μm) through the patch pipette. The holding potential was −30 mV to attenuate the large currents. Note the delayed response of current inhibition during Ca2+ depletion and the renewed current development during replenishment of extracellular Ca2+. The kinetics of inhibition were analyzed and are reported in text. Horizontal black bars indicate time periods during which the standard bath solution (1.2 mm Ca2+) was exchanged with either a divalent cation-free solution (DVF) or a solution containing NMDG as the main extracellular cation. C and D, I-V relations of currents shown in A and B, respectively, obtained during voltage ramps from −90 to +60 mV from a holding potential of −60 mV.
FIGURE 4.
FIGURE 4.
M2-M8P and wild-type TRPM2 are not stimulated by intracellular Ca2+ alone but by extracellular hydrogen peroxide. A and B, whole-cell (w.c.) currents of cells expressing wild-type TRPM2 and M2-M8P, respectively, recorded in the presence of high concentrations of Ca2+ in the patch pipette (horizontal gray bars, concentrations as indicated). Hydrogen peroxide (10 mm) was applied to the extracellular (bath) solution as marked by arrows. C, I-V relations of currents shown in B.
FIGURE 5.
FIGURE 5.
Comparison of the single-channel kinetics of TRPM2, TRPM8, and M2-M8P. A–C show representative traces from inside-out patches from cells transfected with wild-type TRPM8, wild-type TRPM2, or M2-M8P, respectively, along with an analysis of the open times. The number of openings was attributed to bins of widths shown on the abscissa. Mono- or biexponential fits of the histograms yielded the open times (τopen) indicated for each panel. The holding potential was −60 mV. Data were sampled from a total of 12 patches. c, closed; o, open.
FIGURE 6.
FIGURE 6.
Behavior of M2-M8P at far-negative potentials. A, current-voltage relation of M2-M8P and TRPM2 obtained with voltage ramps from −150 to +150 mV. B and C, increased closed times of M2-M8P at strong hyperpolarization. B shows characteristic traces from TRPM2 (upper) and M2-M8P (lower) during activation by ADPR (0.1 mm in 1 μm Ca2+). After the opening of several channels, the holding potential was changed from −60 to −120 mV. C shows the effects of hyperpolarization to −150 mV of ADPR-activated M2-M8P at two different time scales.
FIGURE 7.
FIGURE 7.
Closed times of wild-type TRPM2 and M2-M8P are different at far-negative holding potentials. The duration of closings was attributed to bins and counted. The distribution could be well fitted to a monoexponential function in the case of TRPM2 (left panels), yielding a single τ similar for each holding potential (HP; −60, −120, and −140 mV). In the case of M2-M8P (right panels), a fit to a biexponential function was required at the two more negative potentials, yielding a second τ, which contributed 24% at −120 mV and 70% at −140 mV. Data are from a total of 14 patches for each channel.
FIGURE 8.
FIGURE 8.
Whole-cell patch-clamp analysis of the M8M2-nud chimera. A, illustration of the channel structure of the M8M2-nud chimera, in which the NUDT9 domain of TRPM2 was C-terminally linked to TRPM8 (amino acids 1097–1104 of TRPM8 were replaced with amino acids 1168–1503 of TRPM2). B, whole-cell currents of a cell expressing M8M2-nud. The pipette solution contained 1 mm ADPR and 1 μm Ca2+. After infusion of ADPR and Ca2+ for >2 min did not induce currents, the cell was exposed to ice-cold bath solution for ∼10 s (as indicated by the horizontal black bar) and then stimulated with the menthol analog WS-12 (30 μm; arrow). The example shown is representative of five similar experiments. C, I-V relation of currents shown in B obtained during voltage ramps from −150 to +150 mV from a holding potential of −60 mV. RT, room temperature.
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