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The neuropeptide head activator induces activation and translocation of the growth-factor-regulated Ca²⁺-permeable channel GRC

Katrin Boels^1,*, Günter Glassmeier^2,*, Doris Herrmann¹, I. Björn Riedel¹, Wolfgang Hampe¹, Itaru Kojima³, Jürgen R. Schwarz² and H. Chica Schaller^1,

¹ Zentrum für Molekulare Neurobiologie, Universität Hamburg, Martinistraße 52, 20246 Hamburg, Germany
² Institut für Physiologie, Universität Hamburg, Martinistraße 52, 20246 Hamburg, Germany
³ Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan
^* These authors contributed equally to this work

View larger version (68K): [in a new window]   Fig. 1. PCR amplification of putative GRC- and VR1-channel cDNAs from the HA-responsive cell lines NT2, NH15-CA2 (Nb), and BON. No PCR products were visible after 30 cycles, but reamplification showed bands of the expected sizes (681 bp for mouse, 690 bp for human GRC).

View larger version (63K): [in a new window]   Fig. 2. HA-induced translocation of GRC in NH15-CA2 cells. NH15-CA2 cells were treated for the times indicated with 2 nM monomerized HA, permeabilized by fixation in ice-cold 1% acetic acid in ethanol, and immunostained with the antiserum against GRC.

View larger version (68K): [in a new window]   Fig. 3. Stimulation of GRC cell-surface expression by HA and inhibition of translocation by the Ca2+-channel blocker SK&F 96365. BON cells were incubated for 0, 10 and 20 minutes with 2 nM HA, then for 20 minutes at 37°C with the antiserum against GRC (top panel). GRC translocation was blocked if NH15-CA2 cells were pretreated for 20 minutes with SK&F 96365 (10 µM), before HA (2 nM) was added (bottom panel).

View larger version (44K): [in a new window]   Fig. 4. Expression of GRC and GRC-FLAG in COS-7 and of GRC in CHO cells. COS-7 and CHO cells were transiently transfected by electroporation with the indicated pcDNA3 constructs. GRC in the membrane fraction was made visible by immunostaining with antibodies against GRC or FLAG.

View larger version (54K): [in a new window]   Fig. 5. HA-induced translocation of GRC in COS-7 and CHO cells transfected (Tf) with GRC or GRC-FLAG. Two days after transfection, cells were treated for 30 minutes with 2 nM HA. After fixation they were immunostained as indicated with the antibodies (Ab) against GRC or FLAG.

View larger version (28K): [in a new window]   Fig. 6. HA-induced increase of membrane currents in COS-7 (A-E) and in CHO cells (F,G) transfected with GRC-pcDNA3. (A) 400 ms potential steps were applied from -120 mV to 60 mV in increments of 20 mV from a holding potential of -60 mV. Membrane currents recorded in external NaCl solution are shown of a mock-injected COS-7 cell not expressing GRC and of a cell expressing GRC, both after a treatment for 20 minutes with HA. (B) The amplitudes of the membrane currents as determined in A at the end of the pulses were plotted versus the pulse potential. Data points were connected by straight lines (mock, ; GRC, ). (C) Activation of GRC by HA was reduced after application of 10 µM ruthenium red (RR) in an external CsCl solution. Membrane currents were recorded with a 250 ms voltage ramp command from -100 mV to 60 mV from a holding potential of -60 mV. (D) HA-activated GRC was Ca2+ permeable, if the measurement was performed in an external CaCl2 solution, and the currents were blockable by RR. (E) The membrane current of a cell expressing GRC, but not of a mock-injected COS-7 cell, increased after HA application as measured in the perforated-patch configuration using standard external NaCl solution. Membrane currents activated by HA in cells expressing GRC were blocked by application of 1 mM La3+ or 10 µM RR. Dashed lines denote zero current, arrows indicate the time of HA or blocker application (F,G). In CHO cells heterologously expressed GRC was not activated by HA (F), but by serum (G). Membrane currents were elicited as described in (C). In an external CaCl2 solution a CHO cell pretreated with HA for 20 minutes did not exhibit increased membrane currents (F). Pretreatment with 10% fetal calf serum activated GRC-mediated membrane currents, which could be reduced by 10 µM RR (G).

View larger version (77K): [in a new window]   Fig. 7. Binding of HA to GRC-transfected COS-7 cells, but not to GRC itself. (A) Mock- and GRC-transfected COS-7 cells were incubated with 125I-HA bipeptide (1.3x106 cpm/ml) for 30 minutes at 37°C. After fixation with 4% formaldehyde in 7% acetic acid and 7% glycerol, slides were covered with dipping film and developed for autoradiography 8 days later. Shown on the left are phase-contrast and on the right bright field micrographs. (B) 125I-HA bipeptide was crosslinked to GRC-FLAG or mock-transfected COS-7 cells by EDC and sulfo-NHS. Membrane fractions were solubilized, and subjected to pull-down with anti-FLAG agarose. GRC was recovered quantitatively in the immunoprecipitate, but contained no radioactive HA. (HA-AR, HA autoradiography).

View larger version (71K): [in a new window]   Fig. 8. Second messengers involved in HA signaling to GRC. GRC-transfected COS-7 cells were treated with various inhibitors for 20 minutes at 37°C, before HA was added. Blockage of Ca2+ channels by SK&F 93625 (SKF), of inhibitory G proteins by pertussis toxin (PTX), of Ca2+/calmodulin-dependent kinases by KN-93 (KN), and of PI(3)-kinase by wortmannin (WORT) inhibited the HA-induced translocation of GRC, whereas roscovitine (ROS), which blocks cyclin-dependent kinases, had no effect.

View larger version (17K): [in a new window]   Fig. 9. Inhibition of the HA signaling cascade leading to increased membrane currents. Membrane current densities of mock- and GRC-injected COS-7 cells were recorded at a membrane potential of -80 mV in standard external NaCl solution. The cells were pretreated with the inhibitors as described in Fig. 8, except for PTX, which was preincubated for 30 minutes to 8 hours, and for SK&F 93625, which was added after induction of GRC currents by HA. Each symbol represents one measured cell.

View larger version (24K): [in a new window]   Fig. 10. Model for HA signaling to GRC. After binding HA the HA receptor SorLA together with a postulated second receptor component (Rec) activates a pertussis-toxin sensitive heterotrimeric G protein (1), which interacts via PI3-K (2) with surface GRC (3) to induce Ca2+ influx (4). The increase in Ca2+ leads to activation of CaMK (5), which possibly together with PI3-K (?), triggers shuttling of more GRC molecules to the plasma membrane (6).