Properties of heterologously expressed hTRP3 channels in bovine pulmonary artery endothelial cells

Abstract

1. We combined patch clamp and fura-2 fluorescence methods to characterize human TRP3 (hTRP3) channels heterologously expressed in cultured bovine pulmonary artery endothelial (CPAE) cells, which do not express the bovine trp3 isoform (btrp3) but express btrp1 and btrp4. 2. ATP, bradykinin and intracellular InsP3 activated a non-selective cation current (IhTRP3) in htrp3-transfected CPAE cells but not in non-transfected wild-type cells. During agonist stimulation, the sustained rise in [Ca2+]i was significantly higher in htrp3-transfected cells than in control CPAE cells. 3. The permeability for monovalent cations was PNa > PCs approximately PK >> PNMDG and the ratio PCa/PNa was 1.62 +/- 0.27 (n = 11). Removal of extracellular Ca2+ enhanced the amplitude of the agonist-activated IhTRP3 as well as that of the basal current The trivalent cations La3+ and Gd3+ were potent blockers of IhTRP3 (the IC50 for La3+ was 24.4 +/- 0.7 microM). 4. The single-channel conductance of the channels activated by ATP, assessed by noise analysis, was 23 pS. 5. Thapsigargin and 2,5-di-tert-butyl-1, 4-benzohydroquinone (BHQ), inhibitors of the organellar Ca2+-ATPase, failed to activate IhTRP3. U-73122, a phospholipase C blocker, inhibited IhTRP3 that had been activated by ATP and bradykinin. Thimerosal, an InsP3 receptor-sensitizing compound, enhanced IhTRP3, but calmidazolium, a calmodulin antagonist, did not affect IhTRP3. 6. It is concluded that hTRP3 forms non-selective plasmalemmal cation channels that function as a pathway for agonist-induced Ca2+ influx.

Figure 1. Nucleotide and deduced amino acid sequence of the bovine trp3 cDNA fragment aligned to the amino acid sequence of human trp3, and expression of bovine trp-related genes in CPAE cells

A, only those nucleotides outside the sequence of the degenerated primers used for amplification are shown. The numbering of htrp3 is according to Zhu et al. (1996). Identical amino acids are indicated by blanks. B, RT-PCR analysis using specific primer pairs, showing the expression of btrp1 and btrp4 but notbtrp3 in CPAE cells. The left and right lanes show the DNA site markers in base pairs as indicated.

Figure 2. Effects of hTRP3 expression on the ATP-induced electrical response and changes in [Ca²⁺]_i in CPAE cells

Comparison of the effects of ATP (1 μM) on membrane potential (top trace) and [Ca²⁺]_i (bottom trace) in non-transfected (A) and htrp3-transfected CPAE cells (B). Membrane potential was measured using the current clamp mode. The I-V curves in C (non-transfected cells) and D (htrp3-transfected cells) measured before (a) and during application of ATP (b) were obtained from a voltage ramp protocol applied in voltage clamp mode at the correspondingly labelled points in A and B. For Ca²⁺ measurements the dotted line refers to 0 μM [Ca²⁺]_i. Extracellular Ca²⁺ was 1·5 mM, except for the period marked by the horizontal bar in B (0 mM Ca²⁺).

Figure 3. Effects of hTRP3 channels on agonist-stimulated Ca²⁺ transients

A and B, changes in [Ca²⁺]_i as a result of ATP stimulation of a non-transfected (A) and an htrp3-transfected (B) CPAE cell clamped at 0 mV and perfused with Krebs solution. The internal solution was a high-K⁺ internal solution containing 0·1 mM EGTA. ATP (3 μM) was added to the bath as indicated by the horizontal bars. C, comparison of [Ca²⁺]_i measured at the resting, peak and plateau phases in non-transfected (□) and htrp3-transfected () cells. [Ca²⁺]_i was measured before and after application of 3 μM ATP. Plateau [Ca²⁺]_i was measured 3 min after the end of ATP application. *P < 0·01, significantly different from non-transfected cells.

Figure 4. Non-selective cation currents in htrp3-transfected cells

A and B, membrane currents at -50 mV in control (A) and htrp3-transfected (B) CPAE cells before and during stimulation with 3 μM ATP. The internal Cs⁺-containing pipette solution was buffered at 100 nM Ca²⁺. Bath solution was a 1·5 mM Ca²⁺-containing Krebs solution, which was replaced by a Ca²⁺-free Krebs solution (middle bar) before stimulating the cells with ATP, as indicated by the upper bar. The lower bar indicates the replacement of extracellular Na⁺ with NMDG⁺. Note the presence in both cell types of an inward current component under resting conditions, which is enhanced in Ca²⁺-free solution and eliminated by replacing extracellular Na⁺ with NMDG⁺. ATP did not induce any additional current in the control cell, but induced a huge current in the transfected cell, which again was abolished in Na⁺-free solution. C, I-V relationships of the htrp3-transfected cells obtained from voltage ramps applied at the time points marked in B.

Figure 5. Comparison of hTRP3 currents under various experimental conditions

Comparison of the densities of Na⁺ influx currents in htrp3-transfected CPAE cells after the addition of 2 or 10 μM InsP₃, 100 nM bradykinin or 10 μM ATP. The currents were measured at -50 mV in cells dialysed with the internal solutions, which were buffered with 5 mM EGTA at either 100 nM [Ca²⁺]_i or 1 μM [Ca²⁺]_i, not buffered at all (0·1 mM EGTA) or buffered with 12 mM BAPTA in the patch pipette. Numbers in parentheses, number of cells.

Figure 6. Block of I_hTRP3 by the trivalent cations La³⁺ and Gd³⁺

A and B, comparison of the I-V curves of the current activated by 3 μM ATP before and after application of 100 μM La³⁺ or 100 μM Gd³⁺. C, concentration-response curve for the La³⁺ block. The ordinate represents the percentage of the control current at -50 mV that is not affected by La³⁺ or Gd³. The continuous line represents the best fit of the La³⁺ data to eqn (5) with an IC₅₀ of 24·4 μM and n_H= 1·42.

Figure 7. Relationship between current fluctuations and current amplitude

A, time course of current variance (upper trace) and current amplitude (lower trace) at a holding potential of -50 mV during application of 3 μM ATP. [Ca²⁺]_i was buffered at 100 nM. B, current variance (σ²) as a function of current amplitude from the data in A. Parameters from the fit of the data to eqn (6) (continuous line) were i= 1·8 pA, N= 1420.

Figure 8. Effect of Ca²⁺-ATPase inhibitors on the membrane current of htrp3-transfected cells

A, effects of 20 μM BHQ (A) on a cell clamped at -50 mV using a pipette solution with Ca²⁺ buffered at 100 nM in a Na⁺-containing, Ca²⁺-free bath solution. B, same protocol but for 2 μM thapsigargin (TG). Note that 1 μM ATP could activate I_hTRP3 in both cells whereas both Ca²⁺-ATPase inhibitors had no effect. The lower trace in B shows the intracellular Ca²⁺ concentration during this experiment.

Figure 9. Effects of U-73122, a PLC inhibitor, and thimerosal, a sensitizer of the InsP₃ receptor, on agonist-activated current in transfected cells

A and B, effects of 2 μM U-73122 on I_hTRP3 activated by either 100 nM bradykinin or 3 μM ATP. The current was recorded at -50 mV; [Ca²⁺]_i was buffered at 100 nM [Ca²⁺]_i. The external bath solution contained Na⁺ as the major cation. C, effects of 5 μM thimerosal applied to the cell in a Ca²⁺-free solution. The membrane current was recorded at -50 mV with an internal high-Cs⁺ solution containing 0·1 mM EGTA and an external Na⁺ solution. Na⁺ was briefly replaced by NMDG⁺ to demonstrate the cationic nature of the current.