Ou, Yijun

A Na+ current is present in human jejunal circular smooth muscle cells. The aim of the present study was to determine the role of the cytoskeleton in the regulation of the Na+ current. Whole cell currents were recorded by using standard patch-clamp techniques with Cs+ in the pipette to block K+currents. Cytochalasin D and gelsolin were used to disrupt the actin cytoskeleton and phalloidin to stabilize it. Colchicine was used to disassemble the microtubule cytoskeleton (and intermediate filaments) and paclitaxel to stabilize it. Acrylamide was used to disrupt the intermediate filament cytoskeleton. Perfusion of the recording chamber at 10 ml/min increased peak Na+ current recorded from jejunal smooth muscle cells by 27 ± 3%. Cytochalasin D and gelsolin abolished the perfusion-induced increase in Na+current, whereas incubation with phalloidin, colchicine, paclitaxel, or acrylamide had no effect. In conclusion, the Na+ current expressed in human jejunal circular smooth muscle cells appears to be regulated by the cytoskeleton. An intact actin cytoskeleton is required for perfusion-induced activation of the Na+ current.

Smooth muscle exhibits mechanosensitivity independent of neural input, suggesting that mechanosensitive pathways reside within smooth muscle cells. The native L-type calcium current recorded from human intestinal smooth muscle is modulated by stretch. To define mechanosensitive mechanisms involved in the regulation of smooth muscle calcium entry, we cloned the α1C L-type calcium channel subunit (CaV1.2) from human intestinal smooth muscle and expressed the channel in a heterologous system. This channel subunit retained mechanosensitivity when expressed alone or coexpressed with a β2 calcium channel subunit in HEK-293 or Chinese hamster ovary cells. The heterologously expressed human cardiac α1C splice form also demonstrated mechanosensitivity. Inhibition of kinase signaling did not affect mechanosensitivity of the native channel. Truncation of the α1C COOH terminus, which contains an inhibitory domain and a proline-rich domain thought to mediate mechanosensitive signaling from integrins, did not disrupt mechanosensitivity of the expressed channel. These data demonstrate mechanical regulation of calcium entry through molecularly identified L-type calcium channels in mammalian cells and suggest that the mechanosensitivity resides within the pore forming α1C-subunit.