Charybdotoxin (ChTX), a K4 channel blocker, depolarizes human peripheral T lymphocytes and renders them insensitive to activation by mitogen. We observed four types of K+ channels in human T cells: one voltageactivated, and three Ca2+-activated. To discern the mehnism by which ChTX depolarizes T cells, we examined the sensitivity of both the voltage-activated and Ca2+-activated K+ channels to ChTX and other peptide channel blockers. All four types were blocked by ChTX, whereas noxiustoxin and margatoxin blocked only the voltage-activated channels. All three toxins, however, produced equivalent depolarization in human T cells. We conclude that the membrane potential of resting T cells is set by voltage-activated channels and that blockade of these channels is sufficient to depolarize resting human T cells and prevent activation.

Delayed-rectifier K؉ currents (I DR ) in pancreatic ␤-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. The voltagegated K ؉ channel, K V 2.1, is expressed in ␤-cells, and the biophysical characteristics of heterologously expressed channels are similar to those of I DR in rodent ␤-cells. A novel peptidyl inhibitor of K V 2.1/K V 2.2 channels, guangxitoxin (GxTX)-1 (half-maximal concentration ϳ1 nmol/l), has been purified, characterized, and used to probe the contribution of these channels to ␤-cell physiology. In mouse ␤-cells, GxTX-1 inhibits 90% of I DR and, as for K V 2.1, shifts the voltage dependence of channel activation to more depolarized potentials, a characteristic of gating-modifier peptides. GxTX-1 broadens the ␤-cell action potential, enhances glucose-stimulated intracellular calcium oscillations, and enhances insulin secretion from mouse pancreatic islets in a glucose-dependent manner. These data point to a mechanism for specific enhancement of glucose-dependent insulin secretion by applying blockers of the ␤-cell I DR , which may provide advantages over currently used therapies for the treatment of type 2 diabetes.

A novel nortriterpene, termed correolide, purified from the tree Spachea correae, inhibits Kv1.3, a Shaker-type delayed rectifier potassium channel present in human T lymphocytes. Correolide inhibits 86Rb+ efflux through Kv1.3 channels expressed in CHO cells (IC50 86 nM; Hill coefficient 1) and displays a defined structure-activity relationship. Potency in this assay increases with preincubation time and with time after channel opening. Correolide displays marked selectivity against numerous receptors and voltage- and ligand-gated ion channels. Although correolide is most potent as a Kv1.3 inhibitor, it blocks all other members of the Kv1 family with 4-14-fold lower potency. C20-29-[3H]dihydrocorreolide (diTC) was prepared and shown to bind in a specific, saturable, and reversible fashion (Kd = 11 nM) to a single class of sites in membranes prepared from CHO/Kv1.3 cells. The molecular pharmacology and stoichiometry of this binding reaction suggest that one diTC site is present per Kv1.3 channel tetramer. This site is allosterically coupled to peptide and potassium binding sites in the pore of the channel. DiTC binding to human brain synaptic membranes identifies channels composed of other Kv1 family members. Correolide depolarizes human T cells to the same extent as peptidyl inhibitors of Kv1.3, suggesting that it is a candidate for development as an immunosuppressant. Correolide is the first potent, small molecule inhibitor of Kv1 series channels to be identified from a natural product source and will be useful as a probe for studying potassium channel structure and the physiological role of such channels in target tissues of interest.

SummaryThe role that potassium channels play in human T lymphocyte activation has been investigated by using specific potassium channel probes. Charybdotoxin (ChTX), a blocker of small conductance Ca 2 +-activated potassium channels (Pc,c,) and voltage-gated potassium channels (PK,v) that are present in human T cells, inhibits the activation of these cells. ChTX blocks T cell activation induced by signals (e.g., anti-CD2, anti-CD3, ionomycin) that elicit a rise in intracellular calcium ([Ca2+]i) by preventing the elevation of [Ca2+]i in a dose-dependent manner. However, ChTX has no effect on the activation pathways (e.g., anti-CD28, interleukin 2 [IL-2]) that are independent of a rise in [Ca2+]i. In the former case, both proliferative response and lymphokine production (Ib2 and interferon 3') are inhibited by ChTX. The inhibitory effect of ChTX can be demonstrated when added simultaneously, or up to 4 h after the addition of the stimulants. Since ChTX inhibits both PK,C~ and Pg.v. we investigated which channel is responsible for these immunosuppressive effects with the use of two other peptides, noxiustoxin (NxTX) and margatoxin (MgTX), which are specific for Pg, v. Theses studies demonstrate that, similar to ChTX, both NxTX and MgTX inhibit lymphokine production and the rise in [Ca2+]i. Taken together, these data provide evidence that blockade of PK,V affects the Ca2+-dependent pathways involved in T lymphocyte proliferation and lymphokine production by diminishing the rise in [Ca2+]i that occurs upon T cell activation.

Voltage-gated potassium channels (Kv channels) are involved in repolarization of excitable cells. In pancreatic ␤-cells, prolongation of the action potential by block of delayed rectifier potassium channels would be expected to increase intracellular free calcium and to promote insulin release in a glucose-dependent manner. However, the specific Kv channel subtypes responsible for repolarization in ␤-cells, most importantly in humans, are not completely resolved. In this study, we have investigated the expression of 26 subtypes from Kv subfamilies in human islet mRNA. The results of the RT-PCR analysis were extended by in situ hybridization and/or immunohistochemical analysis on sections from human or Rhesus pancreas. Cell-specific markers were used to show that Kv2.1, Kv3.2, Kv6.2, and Kv9.3 are expressed in ␤-cells, that Kv3.1 and Kv6.1 are expressed in ␣-cells , and that Kv2.2 is expressed in ␦-cells. This study suggests that more than one Kv channel subtype might contribute to the ␤-cell delayed rectifier current and that this current could be formed by heterotetramers of active and silent subunits. Diabetes 53: 597-607, 2004