Extended Data Fig. 6: Local structural shifts of Ca_v3.1-Δ8b upon Z944 binding.

a, Lys1462, which is conserved in T-type channels only, is important for Z944 inhibition. State-dependent blockade by Z944 at indicated concentrations in cells expressing Ca_v3.1-Δ8b (left), Ca_v3.1-Δ8b (K1462F) (middle) and Ca_v3.1-Δ8b (K1462G) (right) are tested. n values indicate the number of independent cells; mean ± s.e.m. The sample sizes (n) tested from low to high concentrations are: n = 4, 5, 5, 3, 8, 6, 3 for Ca_v3.1-∆8b; n = 3, 4, 8, 8, 8, 8, 3 for Ca_v3.1-∆8b (K1462F); and n = 8, 8, 10, 10, 8, 6, 3 for Ca_v3.1-∆8b (K1462G). b, Several lipid and CHS molecules are resolved in the structure of Ca_v3.1-Δ8b. Shown here is an extracellular view. The lipids, the precise identities of which remain unclear, are shown as sticks. Phosphatidylethanolamine (PE) molecules were tentatively modelled into these densities. Three densities are reminiscent of cholesteryl hemisuccinate (CHS1–CHS3), although they may also belong to the detergent glyco-diosgenin (GDN). c, Structures of Ca_v3.1-Δ8b alone and in complex with Z944 can be superimposed, with a r.m.s.d. of 0.45 Å over 851 Cα atoms. Two perpendicular views of the superimposed structures are shown. Ca_v3.1-Δ8b alone is coloured by domain and the complex is coloured light blue. d, Change of lipid distribution in the pore domain in the presence of Z944. Left: an extracellular view of the superimposed pore domain of Ca_v3.1-Δ8b with or without Z944. The bound lipids, shown as thin sticks, are coloured dark grey for the apo structure and light blue for the complex. Z944 is shown as silver sticks. An extra lipid molecule, highlighted with a red rectangle, was resolved in the pore domain of the complex. Right: the densities for Z944 and the nearby transverse lipid are contoured at 4.5σ. It is noted that the densities that were tentatively assigned with two Ca²⁺ ions are contiguous with that for the transverse lipid. Although we cannot entirely exclude the possibility that the densities in the selectivity filter (SF) may belong to a lipid, they are more likely to be bound ions because: (1) If the density belongs to the head group of a lipid, the SF is too narrow to accommodate any known positively charged linear head group with the length corresponding to the density; if the density belongs to a tail, then the hydrophobic property is incompatible with the polar environment within the SF. (2) Lipid-like densities have been observed traversing the pore domain in nearly all structures of Na_v and Ca_v channels with fenestrations. In these channels, a highly conserved inner site constituted by backbone C=O groups has been demonstrated to coordinate Na⁺ or Ca²⁺ by X-ray crystallographic and molecular dynamics simulation analyses. Taken together, two Ca²⁺ ions, instead of a lipid moiety, were tentatively assigned to the density in the SF. e, Half-map densities for the SF from two diagonal repeats, contoured at 4.5σ.

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