Electrical synapses can undergo activity-dependent plasticity. The calcium/calmodulin-dependent kinase II (CaMKII) appears to play a critical role in this phenomenon, but the underlying mechanisms of how CaMKII affects the neuronal gap junction protein connexin36 (Cx36) are unknown. Here we demonstrate effective binding of 35 S-labeled CaMKII to 2 juxtamembrane cytoplasmic domains of Cx36 and in vitro phosphorylation of this protein by the kinase. Both domains reveal striking similarities with segments of the regulatory subunit of CaMKII, which include the pseudosubstrate and pseudotarget sites of the kinase. Similar to the NR2B subunit of the NMDA receptor both Cx36 binding sites exhibit phosphorylation-dependent interaction and autonomous activation of CaMKII. CaMKII and Cx36 were shown to be significantly colocalized in the inferior olive, a brainstem nucleus highly enriched in electrical synapses, indicating physical proximity of these proteins. In analogy to the current notion of NR2B interaction with CaMKII, we propose a model that provides a mechanistic framework for CaMKII and Cx36 interaction at electrical synapses.brain ͉ electrical synapse ͉ gap junction ͉ protein-protein interaction ͉ synaptic plasticity
The macroscopic and single channel gating characteristics of connexin (Cx) 50 gap junction channels between pairs of N2A neuroblastoma cells transfected with mouse Cx50 DNA were investigated using the dual whole‐cell voltage clamp technique. The macroscopic junctional current (Ij) of Cx50‐transfected cells decayed exponentially with time in response to transjunctional voltage (Vj) steps (time constant (τ) of ≈4 s at a Vj of 30–40 mV and 100–200 ms at a Vj of 80–100 mV). The steady‐state junctional conductance (gj) was well described by a two‐state Boltzmann equation. The half‐inactivation voltage (V0), the ratio of minimal to maximal gj (gmin/gmax) and the equivalent gating charge were ± 37 mV, 0.21 and 4, respectively. The conductance of single Cx50 channels measured using patch pipettes containing 130 mM CsCl was 220 ± 13.1 pS (12 cell pairs). A prominent residual or subconductance state corresponding to 43 ± 4.2 pS (10 cell pairs) was also observed at large Vj s. The relationship between channel open probability (Po) and Vj was well described by a Boltzmann relationship with parameters similar to those obtained for macroscopic gj (V0= 34 mV, gating charge = 4.25, maximum P= 0.98). The ensemble average of single channel currents at Vj= 50 mV declined in a monoexponential manner (τ= 905 ms), a value similar to the decline of the macroscopic Ij of Cx50 channels at the same voltage. Ion substitution experiments indicated that Cx50 channels have a lower permeability to anions than to cations (transjunctional conductance of KCl vs. potassium glutamate (γj,KCl/γj,KGlut), 1.2; 6 cell pairs). The results have important implications for understanding the role of connexins in tissues where Cx50 is a major gap junction component, including the lens.
Gap junctions are formed of at least 20 connexin proteins in mammals and possibly pannexins as well. Of the connexins, at least 5 (Cx30.2, Cx37, Cx40, Cx43 and Cx45) are prominently expressed in the heart and each shows regional and cell type specific expression. Contributions of each of these connexins to heart function has been in many cases illuminated by connexin null mice. The cardiac connexin genes whose genomic organization and transcriptional controls have been studied most thoroughly indicate more complex possibilities for alternate promoter usage than originally thought as well a multiple transcription factor binding sites; presumably, such complexity governs developmental timing and regional connexin expression patterns. The structure of cardiac connexin proteins indicate four primarily alpha-helical transmembrane domains, cytoplasmic amino and carboxyl termini and a cytoplasmic loop, all of which contain some regions of alpha-helix, and extracellular loops that are primarily Beta-structure. A number of proteins that bind to cardiac connexins are known, and more are certain to be discovered, linking the connexin into an intercellular signaling complex, the nexus. Binding sites may either correspond to structured regions within the connexin molecules or be unstructured, leading to presumably low-affinity and dynamic interactions.
Vertebrate gap junction channels are formed by a family of more than 20 connexin proteins. These gap junction proteins are expressed with overlapping cellular and tissue specificity, and coding region mutations can cause human hereditary diseases. Here we present a summary of what has been learned from voltage clamp studies performed on cell pairs either endogenously expressing gap junctions or in which connexins are exogenously expressed. General protocols presented here are currently used to transfect mammalian cells with connexins and to study the biophysical properties of the heterologously expressed connexin channels. Transient transfection is accomplished overnight with maximal expression occurring at about 36 h; stable transfectants normally can be generated within three or four weeks through colony selection. Electrophysiological protocols are presented for analysis of voltage dependence and single-channel conductance of gap junction channels as well as for studies of chemical gating of these channels.
Trafficking of the proteins that form gap junctions (connexins) from the site of synthesis to the junctional domain appears to require cytoskeletal delivery mechanisms. Although many cell types exhibit specific delivery of connexins to polarized cell sites, such as connexin32 (Cx32) gap junctions specifically localized to basolateral membrane domains of hepatocytes, the precise roles of actin-and tubulin-based systems remain unclear. We have observed fluorescently tagged Cx32 trafficking linearly at speeds averaging 0.25 m/s in a polarized hepatocyte cell line (WIF-B9), which is abolished by 50 M of the microtubule-disrupting agent nocodazole. To explore the involvement of cytoskeletal components in the delivery of connexins, we have used a preparation of isolated Cx32-containing vesicles from rat hepatocytes and assayed their ATP-driven motility along stabilized rhodamine-labeled microtubules in vitro. These assays revealed the presence of Cx32 and kinesin motor proteins in the same vesicles. The addition of 50 M ATP stimulated vesicle motility along linear microtubule tracks with velocities of 0.4 -0.5 m/s, which was inhibited with 1 mM of the kinesin inhibitor AMP-PNP (adenylyl-imidodiphosphate) and by anti-kinesin antibody but only minimally affected by 5 M vanadate, a dynein inhibitor, or by anti-dynein antibody. These studies provide evidence that Cx32 can be transported intracellularly along microtubules and presumably to junctional domains in cells and highlight an important role of kinesin motor proteins in microtubule-dependent motility of Cx32.Gap junction channels form direct intercellular passageways that allow the exchange of ions and second messenger molecules between cells, thereby spreading signals and unifying cellular responses within a tissue. Gap junctions are formed by the connexin family of proteins that are co-translationally inserted into endoplasmic reticulum membranes (1) and assembled into hexameric connexons or hemichannels within the endoplasmic reticulum or Golgi compartments depending on connexin, cell type, or both (2-6). Connexins are tetra-spanning transmembrane proteins with both termini (N termini and C termini) facing the cytoplasm. This conformation results in two extracellular loops required for connexon docking and one cytoplasmic loop.Knowing the mechanisms of delivery is crucial to understanding connexin regulation because insertion into the plasma membrane is a prerequisite to connexin function. The route of delivery of connexons from the trans-Golgi to the surface membrane has been the subject of many studies, most recently focusing on exogenously expressed connexins with carboxylterminally tagged fluorescent proteins. These studies have indicated that the vesicular structures in which connexins are delivered to the surface occur in a variety of sizes and are motile. Moreover, although delivery appears to be facilitated by microtubules, intact cytoskeleton is not absolutely required for trafficking, as evidenced by the variable effects reported for microtubule-disrupt...
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