Lysophosphatidyl choline modulates mechanosensitive L-type Ca2+ current in circular smooth muscle cells from human jejunum

Abstract

The L-type Ca2+ channel expressed in gastrointestinal smooth muscle is mechanosensitive. Direct membrane stretch and shear stress result in increased Ca2+ entry into the cell. The mechanism for mechanosensitivity is not known, and mechanosensitivity is not dependent on an intact cytoskeleton. The aim of this study was to determine whether L-type Ca2+ channel mechanosensitivity is dependent on tension in the lipid bilayer in human jejunal circular layer myocytes. Whole cell currents were recorded in the amphotericin-perforated-patch configuration, and lysophosphatidyl choline (LPC), lysophosphatidic acid (LPA), and choline were used to alter differentially the tension in the lipid bilayer. Shear stress (perfusion at 10 ml/min) was used to mechanostimulate L-type Ca2+ channels. The increase in L-type Ca2+ current induced by shear stress was greater in the presence of LPC (large head-to-tail proportions), but not LPA or choline, than in the control perfusion. The increased peak Ca2+ current also did not return to baseline levels as in control conditions. Furthermore, steady-state inactivation kinetics were altered in the presence of LPC, leading to a change in window current. These findings suggest that changes in tension in the plasmalemmal membrane can be transmitted to the mechanosensitive L-type Ca2+ channel, leading to altered activity and Ca2+ entry in the human jejunal circular layer myocyte.

Fig. 1.

Time line showing 7-step experimental protocol of mechanical stimulus and application of amphipathic compounds. LPC, lysophosphatidyl choline.

Fig. 2.

Molecular space-fill models of LPC, lysophosphatidic acid (LPA), and choline. Charge and shape of amphipathic compounds direct location of incorporation into the lipid bilayer and induce a subsequent conformational change of proteins embedded in the bilayer.

Fig. 3.

Shear stress increased L-type Ca²⁺ current in circular smooth muscle cells of human jejunum. A: representative whole cell Ca²⁺ (arrow) currents before, during, and after 2 consecutive rounds of shear stress induced by perfusion. Arrowhead shows faster Na⁺ current. Inset: visual identification of Na⁺ (−30 mV, I_FAST) and Ca²⁺ (0 mV, I_SLOW) currents by step voltage and kinetics. B: current-voltage plots of peak inward currents from representative cell in A. I_FAST peaked at −30 to −25 mV; I_SLOW peaked at −5 to 5 mV. C: normalized peak Ca²⁺ currents averaged from 6 cells. *P < 0.05 vs. baseline (B). P1, 1st perfusion; PP1, 1st postperfusion; P2, 2nd perfusion; PP2, 2nd postperfusion.

Fig. 4.

LPC increased L-type Ca²⁺ current during and after mechanoactivation. A: representative whole cell Ca²⁺ currents responding to 2 consecutive mechanical stimuli before and after application of 10 μM LPC. Faster Na⁺ current is also shown. LPC had no effect on Na⁺ current. B: current-voltage plots of peak inward currents from representative cell in A. I_FAST peaked at −30 to −25 mV; I_SLOW peaked at −5 to 5 mV. C: normalized peak Ca²⁺ currents averaged from 6 cells. *P < 0.05 vs. B. **P < 0.05 vs. B and P1.

Fig. 5.

LPA (10 μM) and choline (C, 10 μM) did not increase L-type Ca²⁺ current mechanoactivation. A and B: inward Ca²⁺ currents normalized to baseline current from each cell. Values are means ± SE (n = 6). *P < 0.05 vs. B.

Fig. 6.

LPC altered steady-state L-type Ca²⁺ channel kinetics. Steady-state activation and inactivation kinetics of human jejunal L-type Ca²⁺ channel in the presence of LPC (A, n = 12), LPA (B, n = 11), or choline (C, n = 4). Controls were recorded in each cell before addition of the amphipathic compound.