Heteromerization and functional properties of placental CALHM paralogs. (A) Western blot (detecting the myc-tag attached to the C-termini of respective constructs) of CALHM subunits expressed upon co-transfection of HEK293 GnTI− cells with pairs of CALHM subunits. CALHM channels were isolated by affinity purification of one subunit containing a fusion to Venus and an SBP-tag (bait, shown at higher molecular weight). The resulting samples contain a mix of homomers of the purified subunit and heteromers (with the second subunit not containing Venus and SBP tags, prey at lower molecular weight). The ratio of pray/bait is displayed based on the integration of the intensity of the displayed Western blots. The experiment has been carried out once. (B-E) Represented patch-clamp electrophysiology recordings (whole cell configuration) of indicated CALHM subunits expressed in HEK-293 cells measured in buffers containing 2 mM Ca2+ on the extracellular side. (B) Comparison of currents from mock transfected cells (left) and cells expressing CALHM1 (right). Inset shows voltage protocol. (C) Current response from cells co-transfected with DNA coding for CALHM1 and CALHM3 subunits. (D) Current response from cells transfected with CALHM2 (left) or CALHM4 (right) subunits. (E) Current response from cells co-transfected with CALHM2 and CALHM4 subunits. (F) Mean current density of recordings of different CALHM constructs (measured at 100 mV, 400 ms after the voltage step), errors are s.e.m. for n > 3 and differences from the mean in case of n=2, values from individual recordings are shown as circles, values from individual recordings are shown as circles.

Figure supplement 1. Electrophysiology data of CALHM paralogs at low extracellular Ca2+.

Structural characterization of the CALHM4/SbC4 complex. (A) Cryo-EM density at 3.7 Å (after application of D10 symmetry) of a pair of decameric CALHM4/SbC4 complexes interacting via their cytoplasmic regions. (B) Ribbon representation of the same complex viewed parallel (left) and perpendicular (right) to the membrane plane. Membrane boundaries are indicated (left). A, B, CALHM4 subunits are colored in red and green, SbC4 in yellow. (C) Cryo-EM density (left) and ribbon representation (right) of a single subunit of the CALHM4/SbC4 complex. Transmembrane helices are labeled and shown in unique colors. (D) Organization of TM helices in the decameric channel. The view is from the extracellular side, the coloring is as in C. (E) Slab through a low-pass filtered map of the CALHM4/SbC4 complex (viewed from within the membrane) shows bilayer-like density in the lumen of the wide pore.

Figure supplement 1. Sybody selection and characterization.

Figure supplement 2. Cryo-EM reconstruction of CALHM4 in complex with sybody SbC4.

Structural characterization of a CALHM2/SbC2 complex. (A) Cryo-EM density of the undecameric CALHM2/SbC2 complex at 3.1 Å (after application of C11 symmetry). (B) Ribbon representation of the same complex viewed parallel to the membrane plane (left) and from the extracellular side (right). Membrane boundaries are indicated (left). Space filling model shows a bound lipid. A, B, CALHM2 subunits are colored in yellow and blue, SbC2 in green, the bound lipid in magenta. (C) Cryo-EM density (left) and ribbon representation (right) of a single subunit of the CALHM2/SbC2 complex. Transmembrane helices are labeled and shown in unique colors. (D) Subunit (with orientation relative to C indicated) and blow-up of the sybody-interaction region. The C-terminus of the adjacent subunits contributing to the interaction epitope is colored in yellow and labeled (CT+1). (E) Slab through a low-pass filtered map of the CALHM2/SbC2 complex (viewed from within the membrane) does not show bilayer-like density in the lumen of the pore.

Figure supplement 1. Cryo-EM reconstruction of CALHM2 in complex with sybody SbC2.

Classification of CALHM2/CALHM4 heteromers. Classes correspond to distinct populations of oligomers in the CALHM2/4/SbC2/SbC4 complex. (A) Assemblies containing an excess of CALHM4 subunits. Shown is the well-resolved halve of interacting channel pairs (with weak density corresponding to the second channel). (B, C) Assemblies with an excess of CALHM2 subunits. (C) Dodecameric channels containing three and four CALHM4 subunits. (D) Undecameric channels containing two to four CALHM4 subunits. A-C, Shown are low resolution maps (processed in C1) with bound SbC2 and SbC4 colored in green and yellow , respectively. Numbering refers to CALHM4 subunits. The relationship between views is indicated. Top panels show inclined view towards the membrane plane, bottom panels a view from the extracellular side.

Figure supplement 1. Cryo-EM reconstruction of CALHM2/4 in complex with sybody SbC4.

Figure supplement 2. Cryo-EM reconstruction of CALHM2/4 in complex with sybodies SbC2 and SbC4.

CALHM2/CALHM4 heteromer structure. (A) Schematic depiction of the subunit distribution in CALHM2/4 heteromers. 4 refers to CALHM4, 2 to CALHM2 in ‘up’ conformation, 2d to CALHM2 in ‘down’ conformation, 2u/d to a mixed population of both conformations of CALHM2 subunits, 4/2 and 2/4 to mixed populations of CALHM2 and 4 with the first number indicating the predominant subunit. (B) Cryo-EM density of the CALHM2/4/SbC4 complex at 3.8 Å viewed with an inclination towards the membrane plane. (C) Ribbon representation of the CALHM2/4/SbC4 complex viewed from the same perspective as in B (left) and from the extracellular side (right). (D) Cryo-EM density of the CALHM2/4/SbC2/SbC4 complex at 3.3 Å viewed with an inclination towards the membrane plane (top) and from the intracellular side (bottom). (C) Ribbon representation of the CALHM2/4/SbC2/SbC4 complex viewed from the same perspective as in B (left, top) and from the extracellular side (right). B-E, Colors and numbers of channel subunits are as in A. SbC2 and SbC4 are colored in yellow and green, respectively.

Conformational properties of CALHM subunits in CALHM2/4 heteromers. (A) Cryo-EM density of CALHM2 subunits in ‘down’ (position 3, left) and ‘up’ (position 6, top) and of CALHM4 (in ‘down’ conformation, position 1, bottom). (B) Ribbon representation of a superposition of CALHM2 subunits in ‘up’ and ‘down’ conformations. Inset (right) shows blowup of the intracellular region around TM1-3. (C) Molecular surface of the intracellular region relating TM3 and TM4 viewed from within the membrane plane. A gap between both helices (left, asterisk), which is occupied by a lipid (space-filling representation) in the ‘up’ conformation (center), is closed in the ‘down’ conformation (right). A-C Transmembrane helices and attached sybodies are shown in unique colors. (D) Low-pass filtered map of the CALHM2/4/SbC2/SbC4 complex depicting residual bilayer-like density in the pore lumen. Slice through the channel in a view parallel to the membrane (left), and from the extracellular side (right). Subunit conformations are indicated (d, ‘down’, u, ‘up’). CALHM4 subunits are shown in red and magenta, CALHM2 subunits in blue and cyan.

Structural features of subunit interfaces. (A-C) Interaction interfaces between pairs of CALHM2 subunits (A), CALHM4 subunits (B) and a CALHM2 and a CALHM4 subunit (C). Shown are ribbon representations of neighboring subunits viewed from the inside of the channel parallel to the membrane and their molecular surfaces that are buried in the interaction interface. (D-F) Subunit interactions in the cytoplasmic domain of CALHM channels. Shown is a central subunit (x) and its interaction partners in clockwise and counterclockwise position (top). The region of the intracellular domain that is contacted by neighboring subunits is mapped on the molecular surface of the central subunit. Center, view from the extracellular side (out), bottom from the cytoplasm (in). A-F The relative position of subunits within the assembly is indicated. Paralogs are represented in unique colors.

Discussion. (A, B) Superposition of single subunits of different CALHM paralogs on CALHM2 viewed from within the membrane (top) and of their cytosolic domain viewed from the outside (bottom). (A) Superposition with CALHM2-like subunits CALHM4-6 (CALHM5 PDB:7D60, CALHM6 PDB:6YTV). (B) Superposition with CALHM1 (PDB:8GMQ). The difference in the conformation of the intracellular domain is apparent. (C) Segregation of subunits in heteromeric channels of the LRRC8 family (left) and of CALHM2/4 channels (right). Proteins are shown as space-filling models. The view is from the extracellular side. (D) Schematic depiction of how the conformation of CALHM subunits influences the properties of the bilayer-like density inside the channel. The conformation of helices is indicated.

Cryo-EM Data collection, refinement and validation statistics.

Electrophysiology data of CALHM paralogs recorded at low extracellular Ca2+. (A-D) Patch clamp electrophysiology recordings (whole cell configuration) of representative traces of indicated CALHM subunits expressed in HEK-293 cells measured in buffers not containing Ca2+ on the extracellular side. (A) Comparison of currents from mock transfected cells (left) and cells expressing CALHM1 (right). Inset shows voltage protocol. (B) Current response from cells co-transfected with DNA coding for CALHM1 and CALHM3 subunits. (C) Current response from cells transfected with CALHM2 (left) or CALHM4 (right) subunits. (D) Current response from cells co-transfected with CALHM2 and CALHM4 subunits. (E) Mean current density of recordings of different CALHM constructs (measured at 100 mV, 400 ms after voltage step), errors are s.e.m. for n > 3 and differences from the mean in case of n=2, values from individual recordings are shown as circles, values from individual recordings are shown as circles.

Sybody selection and characterization. (A) Sequence alignment of the two sybodies SbC4 and SbC2 specifically targeting CALHM4 and CALHM2, respectively. (B) Size exclusion chromatography profile of the CALHM4/SbC4 complex. The elution peak of the equivalent amount of CALHM4 is displayed as dashed line in comparison. Inset (right) shows SDS-PAGE gel of the peak fraction. SPR experiments of SbC4 binding to immobilized CALHM4 illustrating specific binding (C) and to CALHM2 not showing specific interactions (D). Dashed line in D corresponds to a fit to a 1:1 binding model yielding a Kd of 100 nM. (E) Size exclusion chromatography profile of the CALHM2/SbC2 complex. The elution peak of the equivalent amount of CALHM2 is displayed as dashed line in comparison. Inset (right) shows SDS-PAGE gel of the peak fraction. SPR experiments of SbC2 binding to immobilized CALHM2 illustrating specific binding (F) and CALHM4 not showing specific interactions (G). B-G the experiments werw carried out once.

Cryo-EM reconstruction of CALHM4 in complex with sybody SbC4. (A) Representative cryo-EM micrograph acquired with a Titan Krios microscope equipped with a K3 camera. (B) Representative 2D class averages of the CALHM4/Sbc4 complex. (C) Data processing workflow. Particles were picked and subjected to 2D classification. High resolution 2D class averages were used as input for initial 3D model generation and subsequent 3D classification. The 3D classification allowed to isolate CALHM4-SbC4 particles, which were further refined with iterative polishing, and 3D and CTF refinements. The distribution of particles (%) of each class is indicated. (D) FSC plot of the final refined undecameric unmasked (blue), masked (green), randomized (orange) and corrected for mask convolution effects (purple) cryo-EM density map of the CALHM4/SbC4 complex. The resolution at which the FSC curve drops below the 0.143 threshold is indicated. (E) Final 3D reconstruction of CALHM4/SbC4 complex colored according to local resolution. The angular distribution of the particles included in the final reconstruction is indicated. Each cylinder represents one view and the height corresponds to the number of particles in that view.

Cryo-EM reconstruction of CALHM2 in complex with sybody SbC2. (A) Representative cryo-EM micrograph acquired with a Titan Krios microscope equipped with an energy filter and a K3 camera. (B) 2D class averages of CALHM2-SbC2 complex. (C) Data processing workflow. Particles were picked and subjected to 2D classification. High resolution 2D class averages were used as an input for ab-initio reconstructions, which allowed to isolate two populations of particles (containing decameric and undecameric assemblies) from a decoy class. The distribution of particles (%) of each class is indicated. Particles were further sorted by heterogenous refinement and undecameric assemblies were improved in several rounds of iterative non-uniform and CTF refinement. To increase the resolution in the sybody binding site, a local refinement of the masked interaction region was performed. (D) FSC plot of the final refined cryo-EM density map of undecameric channels (unmasked (blue), loose (green) and tight (red) mask, and corrected for mask convolution effects (purple)) of the CALHM2/SbC2 complex. The resolution at which the FSC curve drops below the 0.143 threshold is indicated. (E) Final 3D reconstruction of the CALHM2/sybody complex colored according to local resolution.

Cryo-EM reconstruction of CALHM2/4 in complex with sybody SbC4. (A) Representative cryo-EM micrograph acquired with a Titan Krios microscope equipped with a K3 camera. (B) Data processing workflow. Particles were picked and subjected to 2D classification. 2D class averages allowed for the initial separation of populations of single channels and their dimers. The distribution of particles (%) of each class is indicated. Further 3D classification has led to the separation of different assemblies. Ab-initio reconstructions of particles featuring single channels resulted in low resolution maps of undecameric assemblies. Further steps of heterogenous refinement have separated undecamers and dodecamers. The smaller channels were refined to high resolution via several cycles of hetero- and non-uniform refinement. (C) FSC plot of the final refined unmasked (blue), masked (green), randomized (orange) and corrected for mask convolution effects (purple) cryo-EM density maps of an undecameric CALHM4/SbC4 complex. (D) Final 3D reconstruction of a CALHM2/4/SbC4 complex colored according to local resolution.

Cryo-EM reconstruction of CALHM2/4 in complex with sybodies SbC2 and SbC4. (A) Representative cryo-EM micrograph acquired with a Titan Krios microscope equipped with a K3 camera. (B) Data processing workflow. Particles were picked and subjected to 2D classification. High resolution 2D class averages revealed populations of single channels and dimers of channels. The distribution of particles (%) of each class is indicated. Subsequent 3D classification of all the particles allowed for the separation of different assemblies. Ab-initio reconstructions of particles featuring single channels yielded undecameric assemblies. The subsequent heterogenous refinement separated undecameric and dodecameric channels. The undecameric assemblies were further improved to high resolution by hetero, CTF and non-uniform refinement. (C) Fourier shell correlation (FSC) curves of the final refined unmasked (blue), masked (green), randomized (orange) and corrected for mask convolution effects (purple) cryo-EM density maps of an undecameric CALHM2/4/SbC2/SbC4 complex. (D) Final 3D reconstruction of the CALHM2/4/SbC2/SbC4 complex colored according to local resolution. Shown is the map of the channel viewed from the side (left) and the slice through the channel (right).