As the 2 2 subunit is located on the extracellular side and the subunit is located on the cytoplasmic side17,18, the membrane topology of DHPR can be determined from the subunit-specific antibody labelling as shown here, which is consistent with the direction and orientation of ion-conduction channel shown in Figure 2b

As the 2 2 subunit is located on the extracellular side and the subunit is located on the cytoplasmic side17,18, the membrane topology of DHPR can be determined from the subunit-specific antibody labelling as shown here, which is consistent with the direction and orientation of ion-conduction channel shown in Figure 2b. K+, Na+, and Cl? channels have been revealed. However, the molecular architecture of Ca2+ channels, which play pivotal roles in a variety of biological processes, such as muscle contraction, secretion, integration of synaptic input in neurons and synaptic transmission, remains elusive. Recently, a bacterial voltage-gated Ca2+ channel, CavAb, which is a homotetramer composed of a single peptide, was constructed from its Na+ channel homologue, NavAb, and the crystal structure was solved by X-ray crystallography. From which, the Ca2+ selectivity and conduction mechanism was proposed1. However, the molecular architecture of a eukaryotic, multiple-subunit Ca2+ channel complex is LDN-57444 still not available. The skeletal dihydropyridine receptor (DHPR) is an L-type Ca2+ channel (Cav1.1). It is a 450?kDa protein complex composed of five subunits LDN-57444 (1, 176?kDa; 2, 147?kDa; , 24?kDa; , 56?kDa; and , 34?kDa) in a molar ratio of 1 1:1:1:1:1 (Ref. 2). The 2/ are encoded by the same gene and linked by a disulphide bond. The 2/ functions by enhancing membrane trafficking and increasing current amplitude3,4. The subunit is in the cytoplasmic side and affects the channel gating properties and the trafficking of the 1 subunit4,5. The crystal structure of subunit reveals that it interacts with 1 through a conserved 1-interaction domain (AID)6,7. These subunits function as auxiliary for the main part of the channel 1 subunit, which is the voltage sensor and also forms the Ca2+ channel2. Structural determination of DHPR/L-type Ca2+ channel complex has been hampered since its first purification in 1987 due to the extreme difficulty to obtain chemically pure and physically homogenous protein sample for X-ray crystallography or electron microscopy studies. The structure has been stuck at beyond 20-? resolution since 1990s. At such resolution, only the morphology of DHPR is obtained, the ion-conduction channel, the membrane topology, even the location of subunits, remain unresolved. By improving the purification procedure, we made a breakthrough in obtaining chemically pure and physically homogenous DHPR sample, enabling us to break the 20-? resolution barrier and obtain a higher resolution structure of DHPR/L-type Ca2+ channel complex. Here, we present a 15-? cryo-electron microscopy (cryo-EM) structure of the skeletal DHPR/L-type Ca2+ channel complex. Combining with antibody labelling and cryo-EM identification of the location of key subunits, we unambiguously determined the membrane topology and resolved the ion-conduction channel. This structure revealed the molecular architecture of a eukaryotic, multiple-subunit Ca2+ channel complex. Furthermore, this structure provides structural insights into the key elements of DHPR involved in physical coupling with the ryanodine receptor (RyR)/Ca2+ release channel and shed light onto the mechanism of excitation-contraction coupling (ECC coupling). Results and Discussion Improvement of DHPR sample for cryo-EM Due to the relatively smaller size (450?kDa) and lack of any asymmetry, it would be difficult to obtain high-resolution structure by cryo-EM and single particle analysis if the sample is IL23R antibody inhomogeneous. One of the possible reasons that previous structural studies did not obtain a high-resolution structure of DHPR is due to sample heterogeneity. To overcome this difficulty, we first improved DHPR purification procedure (see Methods) and purified DHPR using this new method. The protein purified by our new procedure consists of 5 bands with molecular weights 176-, 147-, 56-, 34- and 24-kDa, respectively, as identified by SDS-PAGE in reducing conditions, which correspond to the 1, LDN-57444 2, , and subunits, respectively (Fig. 1a, left panel). In non-reducing conditions, however, the 2 2 and bands vanished; instead, a band slightly above 1 with an apparent molecular weight ~180-kDa appeared which corresponds to the 2/ complex connected by a disulphide bond. The identities of subunits were confirmed by Western blotting analysis (Fig. 1a, right panel). The chemical purity of the protein sample can be confirmed by the cleanness of the SDS-PAGE gel apart from the five bands belonging to the DHPR complex, there are no other bands apparently recognizable. Western blot identified the band below 2 being part of 1 1 (Fig. 1a, right panel, lane 1), presumably resulted from endogenous protease cleavage. Native PAGE gel of the purified protein sample showed only a single band with.