Multiple binding sites for melatonin on Kv1.3

Melatonin is a small amino acid derivative hormone of the pineal gland. Melatonin quickly and reversibly blocked Kv1.3 channels, the predominant voltage-gated potassium channel in human T-lymphocytes, acting from the extracellular side. The block did not show state or voltage dependence and was associated with an increased inactivation rate of the current. A half-blocking concentration of 1.5 mM was obtained from the reduction of the peak current. We explored several models to describe the stoichiometry of melatonin-Kv1.3 interaction considering one or four independent binding sites per channel. The model in which the occupancy of one of four binding sites by melatonin is sufficient to block the channels gives the best fit to the dose-response relationship, although all four binding sites can be occupied by the drug. The dissociation constant for the individual binding sites is 8.11 mM. Parallel application of charybdotoxin and melatonin showed that both compounds can simultaneously bind to the channels, thereby localizing the melatonin binding site out of the pore region. However, binding of tetraethylammonium to its receptor decreases the melatonin affinity, and vice versa. Thus, the occupancy of the two separate receptor sites allosterically modulates each other.