The maitotoxin (MTX)-induced cationic current (I-mtx) from human skin fibroblasts was characterized using the patch-clamp technique in whole-cell configuration. Under resting conditions (absence of MTX), the main current observed is produced by an outwardly rectifying K+ channel which is inhibited by 1 mM TEA. The current reversal potential was -86 mV (n = 12). MTX (500 pM) activated a current with a linear current-voltage relationship and a reversal potential of -10 mV (n = 10). Replacing the extracellular Na+ and K+ with N-methyl-D-glucamine (NMDG) caused a shift of the reversal potential to a value below -100 mV, indicating that Na+ and K+, but not NMDG, carry I-mtx. Further ion selectivity experiments showed that Ca2+ carries I-mtx also. The resulting permeability sequence obtained with the Goldman-Hodgkin-Katz equation yielded Na+ (1) approximate to K+ (1) > Ca2+ (0.87). The I-mtx activation time course reflected the changes in intracellular Ca2+ and Na+ measured with the fluorescent indicators fura-2 and SBFI, respectively, suggesting that the activation of I-mtx brings about an increment in intracellular Ca2+ and Na+. Reducing the extracellular Ca2+ concentration below 1.8 mM prevented the activation of I-tmx and the increment in intracellular Na+ induced by MTX. Mn2+ and Mg2+ could not replace Ca2+, but Ba2+ could replace Ca2+. MTX activation of current in 10 mM Ba2+ was approximately 50% of that induced in the presence of 1.8 mM Ca2+. When 5 mm of the Cast chelator BAPTA was included in the patch pipette, MTX either failed to activate the current or induced a small current (less than 15% of the control), indicating that intracellular Ca2+ is also required for the activation of I-tmx. Intracellular Ba2+ can replace Ca2+ as an activator of I-tmx. However, in the presence of 10 mM Ba2+ the activation by MTX of the current was 50% less than the activation with nM concentrations of free intracellular Ca2+.
Última actualización: 23/09/2016