The N-Terminal Domain of the IP3 Receptor Gates Store-Operated hTrp3 Channels  Kirill Kiselyov, Gregory A Mignery, Michael X Zhu, Shmuel Muallem  Molecular Cell  Volume 4, Issue 3, Pages 423-429 (September 1999) DOI: 10.1016/S1097-2765(00)80344-5

Figure 2 The Effect of IP3R Constructs on Spontaneous Activity of hTrp3 Channels (A) Ba2+ influx was measured in unstimulated control cells and cells expressing hTrp3 and ΔTMIP3R. (B) Summary of the effect of different IP3R constructs on basal PM permeability of control and hTrp3-expressing cells determined from the average amplitude of the Ba2+ influx into unstimulated cells. Note that ΔTMIP3R and BDIP3R but not FLIP3R or TMIP3R increased the PM permeability of unstimulated cells. Asterisks next to the bars denote a statistically significant difference (P < 0.05) relative to control cells (*) and relative to cells expressing only hTrp3 (**). (C) Single-channel recordings of the spontaneous hTrp3 activity in unstimulated cells and in membrane patches excised from them. The cells were transfected with cDNA coding for hTrp3 and FLIP3R or ΔTMIP3R, as indicated. Membrane potential was +60 mV. (D) Averaged open probability of spontaneously active hTrp3 channels in intact cells and excised PM patches. All cells were transiently transfected with hTrp3 and with or without the indicated IP3R constructs. The number of experiments is given in the columns. The model describes possible interactions between hTrp3 and IP3R constructs. Molecular Cell 1999 4, 423-429DOI: (10.1016/S1097-2765(00)80344-5)

Figure 1 Expression of IP3R Constructs and Their Interaction with hTrp3 in HEK 293 Cells (A) The structural domains of IP3R constructs used in the present work. FLIP3R (FL), full-length IP3R; ΔTMIP3R (ΔTM), full-length IP3R with the transmembrane region deleted; TMIP3R (TM), the transmembrane region of the IP3R; BDIP3R (BD), the IP3-binding domain of the IP3R; ΔBDIP3R (ΔBD), full-length IP3R with the IP3-binding domain deleted. (B) Time course of Tg-induced Ca2+ release from internal stores of cells expressing hTrp3 and native IP3R and cells expressing hTrp3 and recombinant full-length type 1 IP3R. (C) The initial rates of Tg-induced Ca2+ release in cells expressing hTrp3 and the indicated IP3R constructs were measured as in (B). The results are the average of 5–15 experiments. Asterisks (*) above the bars indicate results significantly different from control (293+hTrp3) (P < 0.05). (D–G) Coimmunoprecipitation of hTrp3 and IP3R constructs. Cells stably expressing HA-tagged hTrp3 were transfected with FLIP3R, ΔBDIP3R, or BDIP3R. Proteins were extracted, and hTrp3 was immunoprecipitated (IP) with anti-HA antibodies (Ab) as described in Experimental Procedures. Immunoprecipitated proteins were detected with antibodies specific for HA (D). Extracts from cells expressing hTrp3 were used as standard (Stnd). Extracts from control HEK 293 cells were used as a control for the IP. The anti-HA Ab immunoprecipitated similar amounts of hTrp3 from all cells. In (E), immunoprecipitated proteins were detected with Ab specific for the C terminus of type 1 IP3R. Brain microsomes were used as standard. The weak signal observed with cells expressing hTrp3 alone is due to the native type 1 IP3R expressed in these cells (Kiselyov et al. 1998). Strong signal was obtained from cells expressing FLIP3R. Weak or no signal was obtained with cells expressing ΔBDIP3R (recognized by these antibodies, but does not bind to hTrp3) and BDIP3R (which is not recognized by these antibodies). Binding of BDIP3R was detected with Ab recognizing the N-terminal domain of IP3R (F) and with Ab encoding a proton pump tag, which was fused to this domain (G). For the standard of FLIP3R (F), brain microsomes were used, and for the standard of BDIP3R, extracts from HEK 293 cells transfected with the BDIP3R construct were used. Extracts from untransfected cells expressing hTrp3 were used for control of the IP in both panels. Molecular Cell 1999 4, 423-429DOI: (10.1016/S1097-2765(00)80344-5)

Figure 3 The Effect of IP3R Constructs on hTrp3 Activity in Stimulated Cells (A) Single-channel recordings of spontaneous and UTP-activated hTrp3 channel in HEK 293 cells transfected with cDNA coding for hTrp3 alone or hTrp3 together with FLIP3R or BDIP3R, as indicated. (B) Summary of results. hTrp3 open probability (NPo) was measured before and after UTP stimulation and was calculated as the ratios of NPo(UTP)/NPo(control), which were then averaged. The number of experiments is given in the columns. (C) Top panel, time course of UTP-induced Ba2+ influx in control HEK 293 cells, cells expressing hTrp3, or cells expressing hTrp3 and FLIP3R. UTP (100 μM) was applied in Ca2+-free solution 300–400 s before the addition of 2 mM Ba2+ to the bath solution. Middle panel, averaged amplitudes of UTP-induced Ba2+ influx in control cells and cells expressing hTrp3 and the indicated IP3R constructs. Bottom panel, the absolute values of UTP-induced Ba2+ influx were obtained by subtracting the average amplitude of the spontaneous Ba2+ influx in Figure 2 from the amplitude of UTP-induced Ba2+ influx in the middle panel. Asterisks next to the bars denote statistically significant difference (P < 0.05) relative to control cells (*) and relative to cells expressing only hTrp3 (**). Note that FLIP3R but not ΔTMIP3R increased PM permeability in stimulated cells. (D) The same analysis was performed on hTrp3-mediated Ba2+ influx induced by 2 μM Tg. Note activation of hTrp3 by FLIP3R but not BDIP3R in response to store depletion. Molecular Cell 1999 4, 423-429DOI: (10.1016/S1097-2765(00)80344-5)

Figure 4 Reconstitution of hTrp3 Gating by the IP3R Constructs in Excised Patches (A) A typical experiment with the cytoplasmic ΔTMIP3R. Application of 4 μM IP3 to the patch caused hTrp3 activation (expressed as channel open probability, NPo). After extensive washes of the patch surface, the ability of IP3 to activate hTrp3 was lost. However, it was restored by exposing the patch to ΔTMIP3R and IP3. The experiment was performed at +60 mV. (B) Segments of current from the experiment in (A) in periods 1 and 2. (C and D) Reconstitution of hTrp3 sensitivity to IP3 by BDIP3R. Note that in the absence of IP3, BDIP3R was ineffective in activating hTrp3. (E) Current traces from an experiment with a cytoplasmic extract prepared from control cells. (F) The ability and efficiency of different IP3R constructs to reactivate hTrp3 channels is compared. The numbers denote successful attempts/total number of attempts. Molecular Cell 1999 4, 423-429DOI: (10.1016/S1097-2765(00)80344-5)