The D-Diastereomer of ShK Toxin Selectively Blocks Voltage-gated K+ Channels and Inhibits T Lymphocyte Proliferation

Abstract

The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which are crucial in the activation of human effector memory T cells (T EM ); selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by T EM cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. The critical motif on the toxin for potassium channel blockade consists of neighboring lysine and tyrosine residues. Because this motif is sufficient for activity, an ShK analogue was designed based on D-amino acids. D-Allo-ShK has a structure essentially identical with that of ShK and is resistant to proteolysis. It blocked Kv1.3 with K d 36 nM (2,800-fold lower affinity than ShK), was 2-fold selective for Kv1.3 over Kv1.1, and was inactive against other K ؉ channels tested. D-Allo-ShK inhibited human T EM cell proliferation at 100-fold higher concentration than ShK. Its circulating half-life was only slightly longer than that of ShK, implying that renal clearance is the major determinant of its plasma levels. D-AlloShK did not bind to the closed state of the channel, unlike ShK. Models of D-allo-ShK bound to Kv1.3 show that it can block the pore as effectively as ShK but makes different interactions with the vestibule, some of which are less favorable than for native ShK. The finding that an all-D analogue of a polypeptide toxin retains biological activity and selectivity is highly unusual. Being resistant to proteolysis and nonantigenic, this analogue should be useful in K ؉ channel studies; all-D analogues with improved Kv1.3 potency and specificity may have therapeutic advantages.

Potassium Channel Modulation by a Toxin Domain in Matrix Metalloprotease 23

“…Electrophysiology-All experiments were conducted in the whole-cell configuration of the patch clamp technique, as described previously (1,43,44). Data acquisition and analysis was performed using pClamp software.…”

K+ Channel Modulators for the Treatment of Neurological Disorders and Autoimmune Diseases

“…324 Alternatively, it may be possible that once daily suppression of activated effector memory T cells is enough to inhibit an ongoing immune reaction. Interestingly, the recently published D-diastereomer of ShK, 329 which is resistant to proteolysis, has the same short half-life of 20-30 min, demonstrating that plasma clearance of peptide toxins is predominantly renal. With a MW of roughly 4000 Da these peptides are freely filtered (size exclusion of the filter ∼ 60 kDa).…”

“…Models of D-allo-ShK in the K V 1.3 pore suggest that it makes different contacts with the vestibule, some of which are less favorable than for native ShK. 329 The high affinity of peptide toxins to K V 1.3 also makes these toxins attractive as fluorophore-tagged tools for channel visualization in living cells. This is especially useful for flow cytometry because there currently is no good monoclonal antibody that recognizes an extracellular epitope of K V 1.3 and that could be used to identify K V 1.3 high effector memory T cells.…”

d-Maurocalcine, a Pharmacologically Inert Efficient Cell-penetrating Peptide Analogue

“…In contrast, the effects of PDI (which catalyzes breakage and reformation of disulfide bridges) and PPIase (which controls isomerization between the cis and trans configurations of prolyl imidic peptide bonds) on in vitro oxidation/folding of maurotoxin were stereo-selective. In another example, the 35-amino acid residue sea anemone ShK toxin that blocks potassium channels was also synthesized with amino acids that have a D-configuration at ␣-carbon (43). This peptide also folded as a mirror image of L-ShK.…”