Atomic Force Microscopy of Connexin40 Gap Junction Hemichannels Reveals Calcium-dependent Three-dimensional Molecular Topography and Open-Closed Conformations of Both the Extracellular and Cytoplasmic Faces

Abstract

Atomic force microscopy was used to study the three-dimensional molecular topography and calcium-sensitive conformational changes of Connexin40 hemichannels (connexons) reconstituted in 1,2-dioeloyl-sn-glycero-3-phosphatidylcholine lipid bilayers. Two classes of objects were observed that differed in their protrusion heights above the bilayer (2.6 versus 4.2 nm). Comparison to reconstituted connexons containing Connexin40 truncated to eliminate most of its C-terminal cytoplasmic domain showed that the two height classes corresponded to the shorter extracellular and taller cytoplasmic aspects of the hemichannels and that the C-terminal tail of Connexin40 contributes ϳ1.6 nm in thickness. Hemichannels imaged in solutions containing < 10 M Ca 2؉ showed 3.1-3.2 nm depressions (openings) in 30% of the cytoplasmic faces and 65% of the extracellular faces, and high-resolution three-dimensional topography of extracellular or cytoplasmic aspects of some connexons was observed. After addition of 3.6 mM Ca 2؉ , > 75% of the connexons in either orientation adopted closed conformations. In contrast, hemichannels imaged in the presence of 0.1 mM EDTA showed large (5.6-to 5.8-nm diameter) openings in nearly all hemichannels regardless of orientation, and detailed topography was visible in many connexons. Real-time imaging following the addition of 3.6 mM Ca 2؉ showed transitions of both extracellular and cytoplasmic orientations from "open" into "closed" conformations within several minutes. These studies provide the first high-resolution topographic information regarding a connexin with a large cytoplasmic domain and suggest that the extramembranous portions of Connexin40 contribute to a channel entrance that is relaxed by chelation of residual divalent cations.Gap junctions are specialized plasma membrane regions containing intercellular channels. They are made of apposing hemichannels (connexons) containing hexameric subunits from a family of proteins called connexins (Cx), 4 which has 21 members in the human and mouse genomes (1, 2). There is substantial similarity among the connexin sequences, especially in their transmembrane and extracellular regions. However, the major intracellular domains (cytoplasmic loop and carboxyl-terminal tail) are connexin-specific.Our understanding of the structures of these molecules (particularly the conserved regions) has recently been substantially advanced. An atomic resolution structure was determined from crystals of Cx26, a connexin with a very short C-terminal domain (3). These data confirmed and extended many of the observations that had previously been made in studies of a C-terminally truncated Cx43 (4). There has been substantially less information regarding the structures of connexins containing large cytoplasmic domains or connexons in dispersed (noncrystalline) arrangements. Cx40 and Cx43 are among such connexins. They are found in cardiac gap junctions that have been noted to have a "fuzzy layer" on their cytoplasmic surfaces in electron micrographs (5-7). Atomic force micros...

Structure and Permeability of Ion-channels by Integrated AFM and Waveguide TIRF Microscopy

“…The resolution of hemichannel structure is not high enough to decipher individual subunits of hexameric channels2025. However, the AFM images show distinct pores and surrounding subunits, the outer and inner diameters similar to published data2025.…”

Gating of connexin 43 gap junctions by a cytoplasmic loop calmodulin binding domain

“…The relative insensitivity of Cx40 gap junctions to the Ca 2ϩ /CaM gating mechanism suggests that atrial gap junctions may not close as completely in response to Ca 2ϩ overload conditions, which may be relevant to atrial fibrillation. The apparent insensitivity of Cx40 gap junctions to uncoupling by hundreds of nanomolar cytosolic [Ca 2ϩ ] in this study does not preclude blockade by submillimolar or millimolar [Ca 2ϩ ], as evidenced by the closing of Cx40 hemichannels in the presence of 3.6 mM Ca 2ϩ (3). The decline in Cx43 G j was inhibited by the omission of external CaCl 2 or pretreatment with the CaM inhibitor CDZ (Figs.…”

Voltage-dependent conformational changes in connexin channels

“…More recent studies, using atomic force microscopy (AFM) [23], reviewed by Sosinsky and Nicholson [24], showed that 0.5 mM Ca 2+ decreased the diameter of the extracellular entrance of Cx26 hemichannels from ~15 Å to ~6 Å. Conformational changes were also observed at the intracellular entrance of the pore, but these were more difficult to interpret as a consequence of the flexibility of the cytoplasmic surface, a feature subsequently confirmed by calculation of the temperature factors of the Cx26 crystal structure [10] and MD simulation [11]. More recently, Lal and co-workers reported Ca 2+ induced conformational changes at both the intracellular and extracellular entrance to Cx40 channels [25]. The original EM and subsequent AFM studies suggest that Ca 2+ gating involves substantial conformational changes of the entire channel.…”