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Research Article

MAITOTOXIN CONVERTS THE PLASMALEMMAL Ca²⁺ PUMP (PMCA) INTO A Ca²⁺-PERMEABLE NON-SELECTIVE CATION CHANNEL

¹Case Western Reserve University ²University of Cincinnati College of Medicine ³MetroHealth Medical Center

Submitted 11 June 2009 ; revised 17 September 2009 ; accepted in final form 28 September 2009

Maitotoxin (MTX) activates Ca²⁺-permeable non-selective cation channels and causes a dramatic increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in every cell examined to date, but the molecular identity of the channels involved remains unknown. A clue came from studies of a structurally related marine toxin called palytoxin (PTX). PTX binds to the plasmalemmal Na⁺,K⁺-ATPase (NKA) and converts the Na⁺ pump into a non-selective cation channel. Given the high permeability of the MTX channel for Ca²⁺, we considered the possibility that MTX may bind to the plasmalemmal Ca²⁺-ATPase (PMCA) pump, and like PTX, convert the pump into a channel. To test this hypothesis, the PMCA was over-expressed in Sf9 insect cells and in HEK 293 cells. In both cell types, enhanced expression of the PMCA was associated with a significant increase in MTX-induced whole-cell membrane currents. The effect of MTX on whole-cell currents in both wild-type and PMCA over-expressing HEK cells was sensitive to pump ligands including Ca²⁺ and ATP. MTX-induced currents were significantly reduced by knockdown of PMCA1 in HEK cells using siRNA, or in mouse embryonic fibroblasts from genetically-modified mice with the PMCA1(+/-) PMCA4(-/-) genotype. Lastly, PMCA catalytic activity (i.e., Ca²⁺-ATPase) in isolated membranes, or in purified PMCA preparations, was inhibited by MTX. Together, these results suggest that MTX binds to and converts the PMCA pump into a Ca²⁺-permeable non-selective cation channel.

Pump-Channels; Patch Clamp; Ca2+-ATPase; siRNA