Calcium permeability of transient receptor potential canonical (TRPC) 4 channels measured by TRPC4-GCaMP6s

Abstract

Conflicting evidence has been obtained regarding whether transient receptor potential cation channels (TRPC) are store-operated channels (SOCs) or receptor-operated channels (ROCs). Moreover, the Ca/Na permeability ratio differs depending on whether the current-voltage (I-V) curve has a doubly rectifying shape or inward rectifying shape. To investigate the calcium permeability of TRPC4 channels, we attached GCaMP6s to TRPC4 and simultaneously measured the current and calcium signals. A TRPC4 specific activator, (-)-englerin A, induced both current and calcium fluorescence with the similar time course. Muscarinic receptor stimulator, carbachol, also induced both current and calcium fluorescence with the similar time course. By forming heteromers with TRPC4, TRPC1 significantly reduced the inward current with outward rectifying I-V curve, which also caused the decrease of calcium fluorescence intensity. These results suggest that GCaMP6s attached to TRPC4 can detect slight calcium changes near TRPC4 channels. Consequently, TRPC4-GCaMP6s can be a useful tool for testing the calcium permeability of TRPC4 channels.

Keywords: Calcium; GCaMP6s; Receptor-operated channels; TRPC1/4 heteromer; TRPC4.

Fig. 1. GCaMP6s is suitable for measuring small calcium changes.

(A) Image recordings of GECIs measuring calcium concentration changes (GCaMP6s, R-GECO, YC 6.1 FRET). (B, C) Fluorescence (measured as change in GCaMP6s and R-GECO fluorescence, ΔF, over maximum fluorescence F_max; ΔF/F_max) evoked by low calcium (0 mM) and IM (10 µM) in GCaMP6s-transfected (B) or R-GECO-transfected (C) HEK293 cells. The area enclosed by the dashed box includes a magnified form. Each image was captured every 2 sec. (D) FRET efficiency as determined by fluorescence changes in YC 6.1-transfected cells. The conditions are the same as in B~C.

Fig. 2. Simultaneous recordings of the TRPC4 current and channel specific calcium.

(A) Simultaneous recording of calcium influx (grey) and inward current (black) induced by EA (100 nM) in cells transfected with mTRPC4β and GCaMP6s. The maximum calcium response was induced by IM. Calcium is expressed as change in GCaMP6s fluorescence, ΔF, over basal fluorescence F₀; ΔF/F₀. (B) mTRPC4β-GCaMP6s fusion strategy. GCaMP6s was fused to the C-terminus of mTRPC4β. (C) Full current trace of mTRPC4β-GCaMP6s stimulated by EA. (D) TRPC4-specific calcium influx by mTRPC4β-GCaMP6s. Calcium influx was triggered by EA, thereby stimulating TRPC4 channels. (E) Data from cells transfected with mTRPC4β-GCaMP6s. The conditions are the same as in A. (F) Comparable peak times of current and calcium; blue boxes and bars show means±SEMs.

Fig. 3. Calcium increases through TRPC4 mediated by its physiological signaling pathway.

(A) CCh (100 µM) stimulation in mTRPC4β-GCaMP6s and M2 coexpressing cells. TG (1 µM) was added before CCh to block the ability of cells to pump calcium into the ER. (B) I-V curve indicated by the arrow in A. (C) Rapid response to CCh stimulation. The conditions are the same as in A. (D) Comparable peak current densities at –60 mV and peak calcium fluorescence changes induced by TG or CCh for individual cells. (E) Comparable peak times of current and calcium; Blue boxes and bars show means±SEMs.

Fig. 4. Reduced current and calcium influx by TRPC1/4 heteromer formation.

(A) Simultaneous recording of calcium influx (grey) and inward current (black) induced by EA (100 nM) in cells transfected with mTRPC4β-GCaMP6s. (B) I-V curve indicated by the arrow in A. (C, D) The conditions are largely the same as in A~B, except that cells were cotransfected with hTRPC1α. (E) Images of GCaMP6s fluorescence in TRPC4 homomer and TRPC1/4 heteromer. (F) Peak current densities at –60 mV and calcium fluorescence changes evoked by EA in TRPC4 homotetramers and TRPC1/C4 heterotetramers.