Species-Specific Regulation of TRPM2 by PI(4,5)P2 via the Membrane Interfacial Cavity

Abstract

The human apoptosis channel TRPM2 is stimulated by intracellular ADR-ribose and calcium. Recent studies show pronounced species-specific activation mechanisms. Our aim was to analyse the functional effect of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂), commonly referred to as PIP₂, on different TRPM2 orthologues. Moreover, we wished to identify the interaction site between TRPM2 and PIP₂. We demonstrate a crucial role of PIP₂, in the activation of TRPM2 orthologues of man, zebrafish, and sea anemone. Utilizing inside-out patch clamp recordings of HEK-293 cells transfected with TRPM2, differential effects of PIP₂ that were dependent on the species variant became apparent. While depletion of PIP₂ via polylysine uniformly caused complete inactivation of TRPM2, restoration of channel activity by artificial PIP₂ differed widely. Human TRPM2 was the least sensitive species variant, making it the most susceptible one for regulation by changes in intramembranous PIP₂ content. Furthermore, mutations of highly conserved positively charged amino acid residues in the membrane interfacial cavity reduced the PIP₂ sensitivity in all three TRPM2 orthologues to varying degrees. We conclude that the membrane interfacial cavity acts as a uniform PIP₂ binding site of TRPM2, facilitating channel activation in the presence of ADPR and Ca²⁺ in a species-specific manner.

Keywords: PIP2; TRPM2; patch clamp; phosphoinositides; phospholipid.

Figure 1

PIP₂ is a necessary cofactor for TRPM2 activation. Inside-out patch clamp recordings of HEK-293 cells expressing TRPM2 at −60 mV and 1 µM Ca²⁺ unless stated otherwise. Currents were activated by cytosolic exposure to ADPR (black bar). (A) Representative current trace of hsTRPM2 activated by cytosolic exposure to 300 µM ADPR (black bar) and 25 µM PIP₂ (green bar) and blocked by 15 µg/mL polylysine (PL, orange bar). (B) Summary of the effects of ADPR, PIP_2, and polylysine on hsTRPM2 currents. Statistical analysis reveals a significant current increase via PIP₂ after the initial ADPR induced current plateaued. The current was abolished upon exposure to polylysine and unable to recover via a second PIP₂ application. Control (Ctrl) represents standard bath solution in absence of ADPR. (C,D) Representative current trace and statistics of the PIP₂ effect following elevated intracellular Ca²⁺ concentrations (100 µM) for hsTRPM2. Application of 100 µM Ca²⁺ causes a strong current increase in hsTRPM2 which is only slightly further increased upon external PIP₂ addition. Current traces of drTRPM2 (E) and nvTRPM2 (G) exposed to 100 µM ADPR, 25 µM PIP₂ and 15 µg/mL polylysine. Statistical analysis shows no significant difference for drTRPM2 (F) and nvTRPM2 (H) between the current observed during exposure to ADPR and the additional application of PIP₂. (F) Fractional reactivation of drTRPM2 currents by second PIP₂ application after the current was abolished via polylysine while nvTRPM2 (H) fully recovered. Data are presented as mean ± SEM analysed via one-way ANOVA with Dunn’s multiple comparison test; n = 3–8; * p < 0.05, ** p < 0.01, ns = not significant.

Figure 2

Influence of high calcium and PIP₂ derivatives on TRPM2 current restoration. Recordings of HEK-293 cells expressing TRPM2 at −60 mV in the inside-out patch clamp configuration. Currents were activated by cytosolic exposure to ADPR (black bar) and PIP₂ (25 µM) (green bar). Representative current trace and statistics of hsTRPM2 (A,B) and drTRPM2 (C,D) at high Ca²⁺ concentrations (100 µM). High Ca²⁺ in presence of PIP₂ and ADPR (blue bar) causes current restoration after the current was inhibited via 15 µg/mL polylysine (PL, orange bar). Representative recordings and summary of current statistics of the effect of PI derivatives on hsTRPM2 (E,F) and drTRPM2 (G,H) currents using 1 µM cytosolic Ca²⁺. Statistical analysis was performed via paired Student’s t-test and data presented as mean ± SEM; n = 4–7; * p < 0.05.

Figure 3

PIP₂ sensitivity of TRPM2. PIP₂ dose-response recordings of inside-out patches of HEK-293 cells expressing hsTRPM2 (A), drTRPM2 (C), and nvTRPM2 (D). ADPR (black bar) was used to initiate TRPM2 currents followed by increasing concentrations of PIP₂ (green bars). Polylysine (PL, orange bar) was utilized to scavenge PIP₂. Summary of the PIP₂ dose-response recordings calculated as a percentage of the maximum current obtained at 100 µM PIP₂ (hsTRPM2 (B), drTRPM2, and nvTRPM2 (E)). Data were analysed from five to six independent experiments.

Figure 4

Sequence alignment of TRPM2 and TRPM8. Sequence alignment of the membrane interfacial cavity (formed by: Pre-S1 domain and MHR4 from the adjacent subunit (green), junction between S4 and S5 (purple) and TRP domain (orange)) of hs-, dr- and nvTRPM2 and hsTRPM8. Target positions (yellow) within MHR4-PreS1 labelled mutant I (hsW743, drW718, and nvW705), S4–S5 linker labelled mutant II (hsK918, drR934, and nvR969) and TRP domain separated in mutant III (hsR1067, drR1082, nvR1104) and mutant IV (hsR1077, drR1092, nvR1114).

Figure 5

PIP₂ sensitivity of hsTRPM2 membrane interfacial cavity mutant channels. (A) Current amplitudes of wild-type and mutant hsTRPM2 channels in response to 300 µM ADPR. Representative current trace of K918A (II) (B) and R1077A (IV) (C) exposed to increasing PIP₂ (green bar) concentrations in the presence of 300 µM ADPR (black bar). 15 µg/mL polylysine (PL, orange bar) was used to block PIP₂ mediated currents. (D) Data summary of PIP₂ dose-response experiments showing a clear right shift of mutants K918A (II) and R1077A (IV) compared with the wild-type. Data presented as mean ± SEM; n = 5.

Figure 6

Biotinylation assay of TRPM2 expressed in HEK-293 cells. Representative surface and total expression of hs-, dr- and nvTRPM2 WT and point mutations via biotinylation. β-actin was used as the loading control.

Figure 7

PIP₂ sensitivity of drTRPM2 membrane interfacial cavity mutant channels. (A) ADPR (100 µM, black bar) induced currents of wild-type and mutant drTRPM2 channels. Current trace of R934A (II) (B) and R1092A (IV) (C) perfused with increasing concentrations of PIP₂ (green bars). Polylysine (15 µg/mL, PL, orange bar) was used to scavenge PIP₂ since even in the absence of external PIP₂ the channels are already saturated with natural membrane PIP₂. (D) Summary of the presented data showing a right shift in PIP₂ sensitivity of mutants R934A (II) and R1092A (IV). Data represented as mean ± SEM; n = 5–6.

Figure 8

PIP₂ sensitivity of nvTRPM2 membrane interfacial cavity mutant channels. (A) Current amplitudes induced by ADPR (100 µM, black bar) of wild-type and mutant nvTRPM2 channels. PIP₂ (green bars) dose-response recordings of nvTRPM2 R969A (II) (B), R1104A (III) (C) and R1114A (IV) (D) in presence of 100 µM ADPR (black bar). Since maximum currents were already observed in absence of external PIP₂, natural PIP₂ was scavenged via polylysine (15 µg/mL, PL, orange bar) and subsequently restored via increasing PIP₂ concentrations. (E) Data summary of PIP₂ sensitivity of mutants R969A (II), R1104A (III), and R1114A (IV) showing dominant right shift compared with wild-type nvTRPM2. Data represented as mean ± SEM; n = 5–6.