Interactions between lipids and voltage sensor paddles detected with tarantula toxins

Voltage-activated ion channels open and close in response to changes in voltage, a property that is essential for generating nerve impulses. Studies on voltage-activated potassium (Kv) channels show that voltage-sensor activation is sensitive to the composition of lipids in the surrounding membrane. Here we explore the interaction of lipids with S1-S4 voltage-sensing domains and find that the conversion of the membrane lipid sphingomyelin to ceramide-1-phosphate alters voltage-sensor activation in an S1-S4 voltage-sensing protein lacking an associated pore domain, and that the S3b-S4 paddle motif determines the effects of lipid modification on Kv channels. Using tarantula toxins that bind to paddle motifs within the membrane, we identify mutations in the paddle motif that weaken toxin binding by disrupting lipid-paddle interactions. Our results suggest that lipids bind to voltage-sensing domains and demonstrate that the pharmacological sensitivities of voltage-activated ion channels are influenced by the surrounding lipid membrane

Free intracellular Mg2+ concentration and inhibition of NMDA responses in cultured rat neurons

Intracellular Mg2+ (Mgi2+) blocks single-channel currents and modulates the gating kinetics of NMDA receptors. However, previous data suggested that Mgi2+ inhibits whole-cell current less effectively than predicted from excised-patch measurements. We examined the basis of this discrepancy by testing three hypothetical explanations.To test the first hypothesis, that control of free Mgi2+ concentration ([Mg2+]i) during whole-cell recording was inadequate, we measured [Mg2+]i using mag-indo-1 microfluorometry. The [Mg2+]i measured in cultured neurons during whole-cell recording was similar to the pipette [Mg2+] measured in vitro, suggesting that [Mg2+]i was adequately controlled.To test the second hypothesis, that open-channel block by Mgi2+ was modified by patch excision, we characterised the effects of Mgi2+ using cell-attached recordings. We found the affinity and voltage dependence of open-channel block by Mgi2+ similar in cell-attached and outside-out patches. Thus, the difference between Mgi2+ inhibition of whole-cell and of patch currents cannot be attributed to a difference in Mgi2+ block of single-channel current.The third hypothesis tested was that the effect of Mgi2+ on channel gating was modified by patch excision. Results of cell-attached recording and modelling of whole-cell data suggest that the Mgi2+-induced stabilisation of the channel open state is four times weaker after patch excision than in intact cells. This differential effect of Mgi2+ on channel gating explains why Mgi2+ inhibits whole-cell NMDA responses less effectively than patch responses