Members of the transient receptor potential (TRP) ion channels conduct cations into cells. They mediate functions ranging from neuronally mediated hot and cold sensation to intracellular organellar and primary ciliary signaling. Here we report a cryo-electron microscopy (cryo-EM) structure of TRPC4 in its unliganded (apo) state to an overall resolution of 3.3 Å. The structure reveals a unique architecture with a long pore loop stabilized by a disulfide bond. Beyond the shared tetrameric six-transmembrane fold, the TRPC4 structure deviates from other TRP channels with a unique cytosolic domain. This unique cytosolic N-terminal domain forms extensive aromatic contacts with the TRP and the C-terminal domains. The comparison of our structure with other known TRP structures provides molecular insights into TRPC4 ion selectivity and extends our knowledge of the diversity and evolution of the TRP channels.

Micromolar Ca2؉ facilitates ϳ10-fold enhancement of pyruvate dehydrogenase phosphatase (PDP) activity by aiding the association of PDP with the dihydrolipoyl acetyltransferase (E2) component. Connected by linker regions, E2 consists of two lipoyl domains, the NH 2 -lipoyl domain (L1) and the interior lipoyl domain (L2), and a pyruvate dehydrogenase component binding domain surrounding a 60-mer inner core. Using recombinant constructs of L1 or L2, E2-enhanced PDP activity was markedly decreased by L2 but not by L1, effectively competing with intact E2 in Ca 2؉-dependent binding of PDP (half-maximal reduction at 2.0 M L2 versus 6.7 M E2 subunit). Using L2 fused to glutathione S-transferase resulted in direct Ca 2؉-dependent binding of PDP to L2 (K d , ϳ1.7 M L2). Affinity-bound glutathione S-transferase-L2 was used to purify PDP to homogeneity by selective binding and elution by Ca 2؉ chelation. The large activity enhancement of PDP by E2 was eliminated by enzymatic removal of lipoates from E2 and restored by their enzymatic reintroduction. The critical role of the L2 lipoate is not in binding of PDP to E2, since PDP was still bound by delipoylated L2, and delipoylated L2 inhibited E2-enhanced PDP activity, although lipoylated L2 was more effective in each of these tests. Thus, pyruvate dehydrogenase complex activity is increased by enhanced availability of PDP to its E2-bound, phosphorylated pyruvate dehydrogenase substrate as a consequence of the Ca 2؉ -facilitated interchange of PDP among the mobile L2 domains and an essential (undetermined) step engaging the L2 lipoate. Mammalian pyruvate dehydrogenase complex (PDC)1 is organized around a 60-subunit dihydrolipoyl acetyltransferase (E2) structure, which consists of four independently folded domains connected to each other by mobile linker regions 20 -30 amino acids in length (1-3). Association of 20 trimer units of the COOH-terminal domain of E2 produces a central cavity in the shape of a dodecahedron. Exterior to this inner core assemblage, after the first linker region, each E2 has a 5-kDa domain that binds the pyruvate dehydrogenase (E1) component (4, 5); 20 -30 E1 tetramers (␣ 2 ␤ 2 ) bind per E2 60 . Then, set off by two more linker regions are two ϳ10-kDa lipoyl domains, an interior one (L2), and an NH 2 -terminal one (L1). An E3-binding protein (E3BP) is similar to E2 in consisting of three linker connected domains (6) in which the distinct inner domain of E3BP binds the inner domain of E2 (7, 8) apparently inside the dodecahedron cavity 2 and connects by a linker region to an exterior E3 binding domain (10), followed by a linker-connected lipoyl domain.PDC is regulated by interconversion of E1 between a nonphosphorylated, active form and a phosphorylated, inactive form (E1b). Pyruvate dehydrogenase phosphatase (PDP) catalyzes the Mg 2ϩ -requiring, Ca 2ϩ -stimulated dephosphorylation and activation of E1 (11)(12)(13)(14). Removal of phosphates from the ␣ subunit of E1 can occur with resolved E1b but is enhanced manyfold when E1b and PDP associate with th...

20Members of the transient receptor potential (TRP) ion channels conduct cations into cells. They 21 mediate functions ranging from neuronally-mediated hot and cold sensation to intracellular 22 organellar and primary ciliary signaling. Structures belonging to the TRPV, TRPM, TRPP, 23 TRPA and TRPML subfamilies have been solved, but to date, none of the founding canonical 24 (TRPC) structures. Here we report an electron cryo-microscopy (cryo-EM) structure of TRPC4 25 in its apo state to an overall resolution of 3.3 Å. The structure reveals an unusually complex 26 architecture with a long pore loop stabilized by a disulfide bond. Beyond the shared tetrameric 27 six-transmembrane fold, the TRPC4 structure deviates from other TRP channels with a unique 28 cytosolic domain, this unique cytosolic N-terminal domain forms extensive aromatic contacts 29 with the TRP and the C-terminal domains. The comparison of our structure with other known 30 TRP structures provides molecular insights into TRPC4 ion selectivity and extends our 31 knowledge of the diversity and evolution of the TRP channels. 32 peer-reviewed)

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