Plant defensins are a family of small Cys-rich antifungal proteins that play important roles in plant defense against invading fungi. Structures of several plant defensins share a Cys-stabilized a/b-motif. Structural determinants in plant defensins that govern their antifungal activity and the mechanisms by which they inhibit fungal growth remain unclear. Alfalfa (Medicago sativa) seed defensin, MsDef1, strongly inhibits the growth of Fusarium graminearum in vitro, and its antifungal activity is markedly reduced in the presence of Ca 21. By contrast, MtDef2 from Medicago truncatula, which shares 65% amino acid sequence identity with MsDef1, lacks antifungal activity against F. graminearum. Characterization of the in vitro antifungal activity of the chimeras containing portions of the MsDef1 and MtDef2 proteins shows that the major determinants of antifungal activity reside in the carboxy-terminal region (amino acids 31-45) of MsDef1. We further define the active site by demonstrating that the Arg at position 38 of MsDef1 is critical for its antifungal activity. Furthermore, we have found for the first time, to our knowledge, that MsDef1 blocks the mammalian L-type Ca 21 channel in a manner akin to a virally encoded and structurally unrelated antifungal toxin KP4 from Ustilago maydis, whereas structurally similar MtDef2 and the radish (Raphanus sativus) seed defensin Rs-AFP2 fail to block the L-type Ca 21 channel. From these results, we speculate that the two unrelated antifungal proteins, KP4 and MsDef1, have evolutionarily converged upon the same molecular target, whereas the two structurally related antifungal plant defensins, MtDef2 and Rs-AFP2, have diverged to attack different targets in fungi.

The benzothiazepine diltiazem blocks ionic current through L-type Ca(2+) channels, as do the dihydropyridines (DHPs) and phenylalkylamines (PAs), but it has unique properties that distinguish it from these other drug classes. Wild-type L-type channels containing alpha(1CII) subunits, wild-type P/Q-type channels containing alpha(1A) subunits, and mutants of both channel types were transiently expressed in tsA-201 cells with beta(1B) and alpha(2)delta subunits. Whole-cell, voltage-clamp recordings showed that diltiazem blocks L-type Ca(2+) channels approximately 5-fold more potently than it does P/Q-type channels. Diltiazem blocked a mutant P/Q-type channel containing nine amino acid changes that made it highly sensitive to DHPs, with the same potency as L-type channels. Thus, amino acids specific to the L-type channel that confer DHP sensitivity in an alpha(1A) background also increase sensitivity to diltiazem. Analysis of single amino acid mutations in domains IIIS6 and IVS6 of alpha(1CII) subunits confirmed the role of these L-type-specific amino acid residues in diltiazem block, and also indicated that Y1152 of alpha(1CII), an amino acid critical to both DHP and PA block, does not play a role in diltiazem block. Furthermore, T1039 and Y1043 in domain IIIS5, which are both critical for DHP block, are not involved in block by diltiazem. Conversely, three amino acid residues (I1150, M1160, and I1460) contribute to diltiazem block but have not been shown to affect DHP or PA block. Thus, binding of diltiazem to L-type Ca(2+) channels requires residues that overlap those that are critical for DHP and PA block as well as residues unique to diltiazem.

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