Caribbean maitotoxin elevates [Ca(2+)]i and activates non-selective cation channels in HIT-T15 cells

Abstract

Aim: To investigate the cytotoxic mechanism of caribbean maitotoxin (MTX-C) in mammalian cells.

Methods: We used whole-cell patch-clamp techniques and fluorescence calcium imaging to determine the cellular toxic mechanisms of MTX-C in insulin secreting HIT-T15 cells, which is a system where the effects of MTX have been observed. HIT-T15 cells stably express L-type calcium current, making it a suitable model for this study. Using the fluorescence calcium indicator Indo-1 AM, we found that there is a profound increase in HIT-T15 intracellular free calcium 3 min after application of 200 nmol/L MTX-C.

Results: About 3 min after perfusion of MTX-C, a gradual increase in free calcium concentration was observed. This elevation was sustained throughout the entire recording period. Application of MTX-C did not elicit the L-type calcium current, but large cationic currents appeared after applying MTX-C to the extracellular solution. The current-voltage relationship of the cation current is approximately linear within the voltage range from -60 to 50 mV, but flattened at voltages at -80 and -100 mV. These results indicate that MTX-C induces a non-voltage activated, inward current under normal physiological conditions, which by itself or through a secondary mechanism results in a large amount of cationic influx. The biophysical mechanism of MTX-C is different to its isoform, pacific maitotoxin (MTX-P), when the extracellular calcium is removed.

Conclusion: We conclude that MTX-C causes the opening of non-selective, non-voltage-activated ion channels, which elevates level of intracellular calcium concentration and leads to cellular toxicities.

Keywords: Calcium fluorescence; High voltage gated Ca2+ channels; Insulin secreting cells; Maitotoxin; Whole cell patch clamp.

Figure 1

Indo-1 calcium fluorescence measurements of caribbean maitotoxin, pacific maitotoxin and 40 mmol/L of KCl in HIT-T15 cells. The vertical axis represents relative free intracellular calcium concentration as estimated by the ratio of fluorescent emissions at 405 and 485, respectively. The vertical bars in the figure indicate the time when the toxins or KCl were perfused.

Figure 2

Effect of caribbean maitotoxin (A) and pacific maitotoxin (C) on voltage gated calcium channels in HIT-T15 cells. Representative barium current traces recorded at 10 mV when held at -70 mV in patch clamp. Extracellular solution contains 40 mmol/L BaCl₂. B and D show I-V relationship of voltage gated calcium currents elicited by caribbean maitotoxin (B) and pacific maitotoxin (D), respectively. Open and solid circles represent values of current before and after application of maitotoxins, respectively.

Figure 3

Effect of caribbean maitotoxin and pacific maitotoxin on non-voltage activated cation currents in HIT-T15 cells. Representative traces of cation current elicited recorded at -80, -40, 0 and 40 mV before and after adding caribbean maitotoxin (MTX-C) (A) or pacific maitotoxin (MTX-P) (C). The holding potential was at 0 mV. I-V relationships of MTX-C- and MTX-P-elicited currents are shown in B and D, respectively. The solid circles represent current amplitudes measured after MTX-C (B) or MTX-P (D) administration. The open circles represent the current recorded under the control condition.

Figure 4

Non-voltage activated cation current induced by caribbean maitotoxin or pacific maitotoxin in the calcium free extracellular solution. A: Current traces measured before and after caribbean maitotoxin (MTX-C) or pacific maitotoxin (MTX-P) administration at -80, -40, 0 and 40 mV when held at 0 mV; B and C: I-V relationships of MTX-C (B) and MTX-P (C) elicited currents. The solid circles represent current amplitudes measured after MTX-C (B) or MTX-P (C) administration. The open circles represent the current recorded under the control condition; D: Current conductance measured at different test potentials in cells treated with MTX-C or MTX-P.

Figure 5

Current and voltage (I-V) relationships of caribbean maitotoxin (A) and pacific maitotoxin (B) opened cation currents. The pipette solution contains 130 mmol/L CsCl; the bath solutions contain 120 mmol/L of CsCl (open circle), NaCl (solid circle) or KCl (triangle).