L-type Ca2+ channel and Ca2+-permeable nonselective cation channel as a Ca2+ conducting pathway in myocytes isolated...

The Ca2+-conducting pathway of myocytes isolated from the cricket lateral oviduct was investigated by means of the whole-cell patch clamp technique. In voltage-clamp configuration, two types of whole cell inward currents were identified. One was voltage-dependent, initially activated at -40 mV and reaching a maximum at 10 mV with the use of 140 mM Cs2+-aspartate in the patch pipette and normal saline in the bath solution. Replacement of the external Ca2+ with Ba2+ slowed the current decay. Increasing the external Ca2+ or Ba2+ concentration increased the amplitude of the inward current and the current-voltage (I-V) relationship was shifted as expected from a screening effect on negative surface charges. The inward current could be carried by Na+ in the absence of extracellular Ca2+. Current carried by Na+ (INa) was almost completely blocked by the dihydropyridine Ca2+ channel antagonist, nifedipine, suggesting that the INa is through voltage-dependent L-type Ca2+ channels. The other inward current is voltage-independent and its I-V relationship was linear between -100 mV to 0 mV with a slight inward rectification at more hyperpolarizing membrane potentials when 140 mM Cs+-aspartate and 140 mM Na+-gluconate were used in the patch pipette and in the bath solution, respectively. A similar current was observed even when the external Na+ was replaced with an equimolar amount of K+ or Cs+, or 50 mM Ca2+ or Ba2+. When the osmolarity of the bath solution was reduced by removing mannitol from the bath solution, the inward current became larger at negative potentials. The I-V relationship for the current evoked by the hypotonic solution also showed a linear relationship between -100 mV to 0 mV. Bath application of Gd3+ (10 microM) decreased the inward current activated by membrane hyperpolarization. These results clearly indicate that the majority of current activated by a membrane hyperpolarization is through a stretch-activated Ca2+-permeable nonselective cation channel (NSCC). Here, for the first time, we have identified voltage-dependent L-type Ca2+ channel and stretch-activated Ca2+-permeable NSCCs from enzymatically isolated muscle cells of the cricket using the whole-cell patch clamp recording technique.