Electrophysiological and molecular dissection of two very similar currents encoded by different proteins

1. Separation of Siamese twins Orai1-encoded store-operated channel and TRPC1-dependent IP 3 receptor- operated channel Vladislav Zarayskiy, Ph.D. Ion Channel and Calcium Signaling Unit Boston University, Boston, MA

2. Ca 2+ influx pathways IP 3 IP 3 -sensitive Ca 2+ store Store-operated IP 3 receptor ? ? ? SERCA ? Ryanodine- sensitive store Ca 2+ Voltage-gated Ligand-gated Mechano-gated Other G PLC IP 3 Ca 2+ Physiological responses

3. Methods BAPTA 145 Glu, 165 CsOH 3 MgCl 2, 10 HEPES 130 NaCl, 3 CsCl, 2 CaCl 2 1 MgCl 2 , 10 HEPES

4. CIF activates I CRAC CIF(-) CIF(+) CIF(-) CIF(+) RBL-2H3 mast cells Csutora et al., 2006 -2 -1 0 100 200 300 DES I (pA/pF) time (s) 0.1 BAPTA 20 Ca 2+ 130 Na + 165 Cs + -2 -1 -80 -40 0 40 80 V (mV) I (pA/pF)

5. iPLA 2 is crucial for CIF activation of I CRAC Csutora et al., 2006 0.1 BAPTA 20 Ca 2+ 130 Na + 165 Cs + a/s Control iPLA 2 ß I (pA/pF) -2 -1 0 100 200 300 DES time (s) -2 -1 -80 -40 0 40 80 V (mV) I (pA/pF) a/s Control a/s Control a/s Control iPLA 2 ß

6. iPLA 2 is crucial for I CRAC development Csutora et al., 2006 10 BAPTA 20 Ca 2+ 130 Na + 165 Cs + I (pA/pF) time (s) + rec- iPLA 2 ß -2 -1 0 100 200 300 + rec- iPLA 2 ß -2 -1 80 40 -40 -80 I (pA/pF) V (mV) 0 a/s a/s DES * 0 25 µM 25 µM 0 20 40 60 80 100 Current (%) ( S ) -BEL ( R ) -BEL Control -2 -1 -100 -50 50 V (mV) I (pA/pF) 0 0 ( S ) -BEL ( R ) -BEL

7. depleted Ca 2+ stores CIF Ca 2+ Store-Operated iPLA 2 CaM R G PLC SERCA IP 3 Ca 2+ IP 3 R physiological responses SOC (CRAC) ER/SR LysoPL Bolotina, Science STKE, July 2004 Ca 2+ influx pathways Ca 2+ IP 3 R IP 3 ROC IP 3 Receptor-Operated

8. Differences in the mechanism of activation : Store depletion versus IP 3 and the role of iPLA 2 β Zarayskiy et al., 2007 I CRAC 20 Ca 2+ 130 Na + 165 Cs + I IP3ROC IP 3 BAPTA 20 Ca 2+ 130 Na + 165 Cs + Control iPLA 2 a/s -2 -1 1 -80 -40 40 V (mV) I (pA/pF) -2 -1 0 0.01 0.1 1 10 100 IP 3 concentration (8) (3) (3) (4) (4) (5) µM I (pA/pF) a/s iPLA 2 I (pA/pF) -2 -1 0 100 200 Time (s) DES Control a/s iPLA 2 Control iPLA 2 a/s V (mV) I (pA/pF) -2 -1 1 -80 -40 40 -2 -1 0 100 200 Time (s) I (pA/pF) DES Control a/s iPLA 2 BAPTA

9. I CRAC and I IP3ROC : Biophysical and molecular discrimination Zarayskiy et al., 2007 Inwardly rectifying ~ Linear Inwardly rectifying Inwardly rectifying Current-voltage relationship (I/V) (Na + ,Ca 2+ ) out / (Cs + ) in (Na + ,Ca 2+ ) out / (Na + ,Cs + ) in Yes Yes Inhibition by DES Yes Yes Inhibition by 2-APB Inhibition No effect TRPC1/IP 3 R peptide Inhibition No effect Calyculin A Present Absent iPLA 2 β-deficient cells Absent Present TRPC1-deficient cells Present Present STIM1-deficient cells Present Absent Orai1-deficient cells P Ca2+ : P Na+ <0.4 P Ca2+ : P Na+ >1000 Selectivity >+50 mV +5 mV > +50 mV >+50 mV Reversal potential: (Na + ,Ca 2+ ) out / (Cs + ) in (Na + ,Ca 2+ ) out / (Na + ,Cs + ) in Yes No Requirement of IP 3 No Yes Activation by Ca 2+ store depletion I IP3ROC I CRAC

10. Zarayskiy et al., 2007 I CRAC I IP3ROC Differences in cation selectivity : Ca 2+ -selective I CRAC and cation-selective I IP3ROC. 20 Ca 2+ 165 Cs + 130 Na + 130 NMDG + 130 Cs + a/s iPLA 2 130 NMDG + 130 Cs + 0 50 100 Control NMDG + Cs + Current, (%) ( 6 ) ( 6 ) (5) 0 50 100 Current, (%) Control NMDG + Cs + ( 4 ) ( 4 ) ( 3 ) BAPTA IP 3 BAPTA 20 Ca 2+ 165 Cs + 130 Na +

11. Zarayskiy et al., 2007 I CRAC I IP3ROC Intracellular Cs + as a reason for inward rectification of I IP3ROC . 165 Cs + 20 Ca 2+ 130 Na + 130 Na + 130 Na + V (mV) I (pA/pF) -1 1 -80 -40 40 [Na + ] i [Cs + ] i [Na + ] i [Cs + ] i V( mV) I (pA/pF) -1 1 -80 -40 0 40 a/s iPLA 2 [Na + ] i [Cs + ] i BAPTA IP 3 BAPTA 20 Ca 2+ 130 Na + 165 Cs + 165 Cs +

12. I CRAC and I IP3ROC : Biophysical and molecular discrimination Zarayskiy et al., 2007 Inwardly rectifying ~ Linear Inwardly rectifying Inwardly rectifying Current-voltage relationship (I/V) (Na + ,Ca 2+ ) out / (Cs + ) in (Na + ,Ca 2+ ) out / (Na + ,Cs + ) in Yes Yes Inhibition by DES Yes Yes Inhibition by 2-APB Inhibition No effect TRPC1/IP 3 R peptide Inhibition No effect Calyculin A Present Absent iPLA 2 β-deficient cells Absent Present TRPC1-deficient cells Present Present STIM1-deficient cells Present Absent Orai1-deficient cells P Ca2+ : P Na+ <0.4 P Ca2+ : P Na+ >1000 Selectivity >+50 mV +5 mV > +50 mV >+50 mV Reversal potential: (Na + ,Ca 2+ ) out / (Cs + ) in (Na + ,Ca 2+ ) out / (Na + ,Cs + ) in Yes No Requirement of IP 3 No Yes Activation by Ca 2+ store depletion I IP3ROC I CRAC

13. I CRAC I IP3ROC Zarayskiy et al., 2007 Differences in molecular identity Orai1-encoded I CRAC and TRPC1-dependent I IP3ROC TRPC1 a/s Orai1 siRNA V (mV) I (pA/pF) -1 1 -80 -40 40 V (mV) I (pA/pF) -1 1 -80 -40 40 TRPC1 a/s Control Orai1 siRNA BAPTA IP 3 BAPTA

14. I CRAC I IP3ROC Zarayskiy et al., 2007 Differences in molecular identity Orai1-encoded I CRAC and TRPC1-dependent I IP3ROC Orai1 siRNA V (mV) I (pA/pF) -1 1 -80 -40 40 TRPC1 a/s V (mV) I (pA/pF) -1 1 -80 -40 40 IP 3 BAPTA Control CRAC+IP 3 ROC -1 1 -80 -40 40 80 V (mV) I (pA/pF)

15. I CRAC I IP3ROC Zarayskiy et al., 2007 Differences in molecular identity Orai1-encoded I CRAC and TRPC1-dependent I IP3ROC Orai1 siRNA V (mV) I (pA/pF) -1 1 -80 -40 40 TRPC1 a/s V (mV) I (pA/pF) -1 1 -80 -40 40 V (mV) I (pA/pF) -1 1 -80 -40 40 TRPC1 a/s Control Orai1 siRNA IP 3 BAPTA

16. I CRAC and I IP3ROC : Biophysical and molecular discrimination Zarayskiy et al., 2007 Inwardly rectifying ~ Linear Inwardly rectifying Inwardly rectifying Current-voltage relationship (I/V) (Na + ,Ca 2+ ) out / (Cs + ) in (Na + ,Ca 2+ ) out / (Na + ,Cs + ) in Yes Yes Inhibition by DES Yes Yes Inhibition by 2-APB Inhibition No effect TRPC1/IP 3 R peptide Inhibition No effect Calyculin A Present Absent iPLA 2 β-deficient cells Absent Present TRPC1-deficient cells Present Present STIM1-deficient cells Present Absent Orai1-deficient cells P Ca2+ : P Na+ <0.4 P Ca2+ : P Na+ >1000 Selectivity >+50 mV +5 mV > +50 mV >+50 mV Reversal potential: (Na + ,Ca 2+ ) out / (Cs + ) in (Na + ,Ca 2+ ) out / (Na + ,Cs + ) in Yes No Requirement of IP 3 No Yes Activation by Ca 2+ store depletion I IP3ROC I CRAC

17. I CRAC I IP3ROC Zarayskiy et al., 2007 I IP3ROC but not I CRAC depends on conformational coupling with IP 3 R : differential effects of TRPC1 / IP 3 R binding peptide TRPC1 a/s Peptide (+) (-) V (mV) I (pA/pF) -1 1 -80 -40 40 Orai1 siRNA Peptide (+) (-) V (mV) I (pA/pF) -1 1 -80 -40 0 40 BAPTA IP 3 BAPTA

18. I CRAC and I IP3ROC : Biophysical and molecular discrimination Zarayskiy et al., 2007 Inwardly rectifying ~ Linear Inwardly rectifying Inwardly rectifying Current-voltage relationship (I/V) (Na + ,Ca 2+ ) out / (Cs + ) in (Na + ,Ca 2+ ) out / (Na + ,Cs + ) in Yes Yes Inhibition by DES Yes Yes Inhibition by 2-APB Inhibition No effect TRPC1/IP 3 R peptide Inhibition No effect Calyculin A Present Absent iPLA 2 β-deficient cells Absent Present TRPC1-deficient cells Present Present STIM1-deficient cells Present Absent Orai1-deficient cells P Ca2+ : P Na+ <0.4 P Ca2+ : P Na+ >1000 Selectivity >+50 mV +5 mV > +50 mV >+50 mV Reversal potential: (Na + ,Ca 2+ ) out / (Cs + ) in (Na + ,Ca 2+ ) out / (Na + ,Cs + ) in Yes No Requirement of IP 3 No Yes Activation by Ca 2+ store depletion I IP3ROC I CRAC

19. I CRAC and I IP3ROC : Biophysical and molecular discrimination Zarayskiy et al., 2007 Inwardly rectifying ~ Linear Inwardly rectifying Inwardly rectifying Current-voltage relationship (I/V) (Na + ,Ca 2+ ) out / (Cs + ) in (Na + ,Ca 2+ ) out / (Na + ,Cs + ) in Yes Yes Inhibition by DES Yes Yes Inhibition by 2-APB Inhibition No effect TRPC1/IP 3 R peptide Inhibition No effect Calyculin A Present Absent iPLA 2 β-deficient cells Absent Present TRPC1-deficient cells Present Present STIM1-deficient cells Present Absent Orai1-deficient cells P Ca2+ : P Na+ <0.4 P Ca2+ : P Na+ >1000 Selectivity >+50 mV +5 mV > +50 mV >+50 mV Reversal potential: (Na + ,Ca 2+ ) out / (Cs + ) in (Na + ,Ca 2+ ) out / (Na + ,Cs + ) in Yes No Requirement of IP 3 No Yes Activation by Ca 2+ store depletion I IP3ROC I CRAC

20. depleted Ca 2+ stores CIF Ca 2+ iPLA 2 CaM R G PLC SERCA Ca 2+ Na + IP 3 IP 3 Receptor-Operated Ca 2+ IP 3 R IP 3 R physiological responses IP 3 ROC SOC ER/SR LysoPL Ca 2+ influx pathways Orai1 TRPC1 (+other TRPCs?) Store-Operated

21. I IP3ROC depends on Ca 2+ buffering Zarayskiy et al., 2007 0.5 1 1.5 20 40 60 80 100 I (pA/pF) sec 0 +80 mV 10 µM IP 3 + 1 mM BAPTA (4) (4) IP 3 BAPTA

22. BAPTA + OAG OAG activates additional current besides I CRAC 20 Ca 2+ 130 Na + 165 Cs + 2 1 -4 -2 0 2 Current (pA/pF)  ( 2-1) -2 -1 0 1 2 0 100 200 2 1 Current (normalized) Time (sec) +80mV -80mV BAPTA

23. Calyculin A inhibits I IP3ROC but not I CRAC Zarayskiy et al., 2007 V (mV) CalyA a/s iPLA 2 Control I (pA/pF) V (mV) I (pA/pF) Control CalyA -1 1 -80 -40 40 -1 1 -80 -40 40 I CRAC I IP3ROC BAPTA IP 3 BAPTA

24. I IP3ROC can be inhibited from inside and outside Zarayskiy et al., 2007 I (pA/pF) -2 -1 1 2 -80 -40 40 80 V (mV) IP 3 + BAPTA -2 -1 0 1 2 Control 1 µM La 3+ 500 µg/ml Heparin iPLA 2 a/s iPLA 2 a/s Control La 3 + Heparin IP 3 BAPTA

26. depleted Ca 2+ stores CIF Ca 2+ iPLA 2 CaM R G PLC SERCA Ca 2+ Na + IP 3 IP 3 Receptor-Operated Ca 2+ IP 3 R IP 3 R physiological responses IP 3 ROC SOC ER/SR LysoPL Ca 2+ influx pathways Orai1 TRPC1 (+other TRPCs?) Store-Operated