Inhibitory interneurons are critical to sensory transformations, plasticity and synchronous activity in the neocortex There are many types of inhibitory neurons, but their synaptic organization is poorly understood Here we describe two functionally distinct inhibitory networks comprising either fast-spiking (FS) or low-threshold spiking (LTS) neurons Paired-cell recordings showed that inhibitory neurons of the same type were strongly interconnected by electrical synapses, but electrical synapses between different inhibitory cell types were rare The electrical synapses were strong enough to synchronize spikes in coupled interneurons Inhibitory chemical synapses were also common between FS cells, and between FS and LTS cells, but LTS cells rarely inhibited one another Thalamocortical synapses, which convey sensory information to the cortex, specifically and strongly excited only the FS cell network The electrical and chemical synaptic connections of different types of inhibitory neurons are specific, and may allow each inhibitory network to function independently

Two networks of electrically coupled inhibitory neurons in neocortex

Adjacent cells share ions, second messengers and small metabolites through intercellular channels which are present in gap junctions. This type of intercellular communication permits coordinated cellular activity, a critical feature for organ homeostasis during development and adult life of multicellular organisms. Intercellular channels are structurally more complex than other ion channels, because a complete cell-to-cell channel spans two plasma membranes and results from the association of two half channels, or connexons, contributed separately by each of the two participating cells. Each connexon, in turn, is a multimeric assembly of protein subunits. The structural proteins comprising these channels, collectively called connexins, are members of a highly related multigene family consisting of at least 13 members. Since the cloning of the first connexin in 1986, considerable progress has been made in our understanding of the complex molecular switches that control the formation and permeability of intercellular channels. Analysis of the mechanisms of channel assembly has revealed the selectivity of inter-connexin interactions and uncovered novel characteristics of the channel permeability and gating behavior. Structure/function studies have begun to provide a molecular understanding of the significance of connexin diversity and demonstrated the unique regulation of connexins by tyrosine kinases and oncogenes. Finally, mutations in two connexin genes have been linked to human diseases. The development of more specific approaches (dominant negative mutants, knockouts, transgenes) to study the functional role of connexins in organ homeostasis is providing a new perception about the significance of connexin diversity and the regulation of intercellular communication.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1996.0001q.x

Connections with connexins: the molecular basis of direct intercellular signaling

Saez, Juan C., Viviana M. Berthoud, Maria C. Branes, Agustin D. Martinez, and Eric C. Beyer. Plasma Membrane Channels Formed by Connexins: Their Regulation and Functions. Physiol Rev 83: 1359-1400,...

Plasma Membrane Channels Formed by Connexins: Their Regulation and Functions

DNAs coding for seven murine connexins (Cx) (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43, and Cx45) are functionally expressed in human HeLa cells that were deficient in gap junctional communication. We compare the permeabilities of gap junctions comprised of different connexins to iontophoretically injected tracer molecules. Our results show that Lucifer yellow can pass through all connexin channels analyzed. On the other hand, propidium iodide and ethidium bromide penetrate very poorly or not at all through Cx31 and Cx32 channels, respectively, but pass through channels of other connexins. 4,6 Diamidino-2-phenylindole (DAPI) dihydrochloride shows less transfer among Cx31 or Cx43 transfectants. Neurobiotin is weakly transferred among Cx31 transfectants. Total junctional conductance in Cx31 or Cx45 transfected cells is only about half as high as in other connexin transfectants analyzed and does not correlate exactly with any of the tracer permeabilities. Permeability through different connexin channels appears to be dependent on the molecular structure of each tracer, i.e. size, charge and possibly rigidity. This supports the hypothesis that different connexin channels show different permeabilities to second messenger molecules as well as metabolites and may fulfill in this way their specific role in growth control and differentiation of cell types. In addition, we have investigated the function of heterotypic gap junctions after co-cultivation of two different connexin transfectants, one of which had been prelabeled with fluorescent dextran beads. Analysis of Lucifer yellow transfer reveals that HeLa cells expressing Cx31 (beta-type connexin) do not communicate with any other connexin transfectant tested but only with themselves. Two other beta-type connexin transfectants, HeLa-Cx26 and -Cx32, do not transmit Lucifer yellow to any of the alpha-type connexins analyzed. Among alpha-type connexins, Cx40 does not communicate with Cx43. Thus, connexins differ in their ability to form functional heterotypic gap junctions among mammalian cells.

/pdf/specific-permeability-and-selective-formation-of-gap-1dyzab06ql.pdf

Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells.

This summary is a proposed synthesis of available information for the non-specialist. It does not incorporate all the published data, is inconsistent with some, and reflects the biases of the author. Connexin proteins have a common transmembrane topology, with four alpha-helical transmembrane domains, two extracellular loops, a cytoplasmic loop, and cytoplasmic N- and C-terminal domains. The sequences are most conserved in the transmembrane and extracellular domains, yet many of the key functional differences between connexins are determined by amino-acid differences in these largely conserved domains. Each extracellular loop contains three cysteines with invariant spacing (save one isoform) that are required for channel function. The junctional channel is composed of two end-to-end hemichannels, each of which is a hexamer of connexin subunits. Hemichannels formed by some connexin isoforms can function as well-behaved, single-membrane-spanning channels in plasma membrane. In junctional channels, the cysteines in the extracellular loops form intra-monomer disulfide bonds between the two loops, not intermonomer or inter-hemichannel bonds. The end-to-end homophilic binding between hemichannels is via non-covalent interactions. Mutagenesis studies suggest that the docking region contains beta structures, and may resemble to some degree the beta-barrel structure of porin channels. The two hemichannels that compose a junctional channel are rotationally staggered by approximately 30 degrees relative to each other so that the alpha-helices of each connexin monomer are axially aligned with the alpha-helices of two adjacent monomers in the apposed hemichannel. At present there is a published 3D map with 7.5 A resolution in the plane of the membrane, based on electron cryomicroscopy of 2D crystals of junctional channels formed by C-terminal truncated Cx43. The correspondence between the imaged transmembrane alpha-helices and the known transmembrane amino-acid sequences is a matter of debate. Each of the approximately 20 connexin isoforms produces channels with distinct unitary conductances, molecular permeabilities, and electrical and chemical gating sensitivities. The channels can be heteromeric, and subfamilies among connexins largely determine heteromeric specificity, similar to the specificities within the voltage-dependent potassium channel superfamily. The second extracellular loop contains the primary determinants of the specificity of hemichannel-hemichannel docking (analogous to the tetramerization domain of potassium channels). The 7.5 A map shows that each monomer exposes only two transmembrane alpha-helices to the pore lumen. However the conductance state of the imaged structure and the effects of the C-terminal truncation are unknown, so it is possible that other transmembrane domains contribute to the lumen in other functional states of the channel. In the transmembrane region, SCAM and mutagenesis data suggest that parts of the first three transmembrane alpha-helices are exposed to the lumen. Some of these data are contradictory, but may reflect conformational or isoform differences. There is reason to think that the first part of the N-terminal cytoplasmic domain can line the pore in some conformations. In the extracellular part of junctional channels, the N-terminal portion of the first extracellular loop is exposed to the lumen. The unitary conductances through connexin channels vary over an order of magnitude, from 15 pS to over 300 pS. There is a range of charge selectivities among atomic ions, from slightly anion selective to highly cation selective, which does not correlate with unitary conductance. There appear to be substantial ion-ion interactions within the pore, making the GHK model of assessing selectivities of limited value. Pores formed by different connexins have a range of limiting diameters as assessed by uncharged and charged probes, which also does not correlate with unitary conductance (i.e. some have high conductance but have a narrow limiting diameter, and vice versa). Channels formed by different connexins have different permeabilities to various cytoplasmic molecules. Where it has been assessed, the selectivity among cytoplasmic molecules is substantial and does not correlate in an obvious manner with the size selectivity data derived from fluorescent tracer studies, suggesting there are chemical specificities within the pore that enhance or reduce permeability to specific cytoplasmic molecules, functionally analogous to the ability of some porins to facilitate transport of specific substrates. For example, heteromeric channels with different stoichiometries or arrangements of isoforms can distinguish among second messengers. The differences in permeability to cytoplasmic molecules have biological consequences; in most cases one connexin cannot fully substitute for another. Voltage and chemical gating mechanisms largely operate within each hemichannel, though there is evidence for inter-hemichannel allosteric effects as well. There are at least two distinct gating mechanisms. One (Vj-gating) is a voltage-driven mechanism that governs rapid transitions between conducting states. Its voltage sensor involves charges in the first several positions of the cytoplasmic N-terminal domain and possibly in the N-terminal part of the first extracellular loop, which may both be exposed to the lumen of the pore in some states. The polarity of Vj-gating sensitivity is connexin-specific, closing with depolarization for some connexins and with hyperpolarization for others. The polarity can be reversed by point mutations at the second position. The lower conductance states induced by Vj-gating correspond to physical restrictions of the pore, and thus restricted or eliminated molecular permeation. Since the channels are not fully closed by Vj-gating, it can be seen as a way to eliminate molecular signaling while leaving electrical signaling operational. A second, independent gating mechanism mediates slow transitions (approximately 10-30 ms) into and out of non-conducting state(s). These transitions can occur in response to voltage ('loop gating'), chemical factors such as pH and lipophiles ('chemical gating'), and the docking of two hemichannels (sometimes called the 'docking gate'). These slow transitions may reflect a common structural change induced by these several effectors (electrical, chemical and homodimerization). Alternatively, they could reflect distinct gating processes responding to one or more of these effectors, that are indistinguishable at the single-channel level and have yet to be resolved mechanistically. The slow or loop gate closes with hyperpolarization. As a result, where Vj-gating closes with depolarization, individual hemichannels can close in response to both polarities of voltage (but only to a subconductance state for the Vj-gating polarity). Because of this, it is difficult to assign a macroscopic voltage sensitivity, or its modification due to mutagenesis, chemical modification or heteromeric interactions, to one or the other of these very distinct voltage-sensitive processes. This distinction can be made reliably only at the single-channel level. The Vj-gating voltage sensor and the loop-gating voltage sensor appear to be independent structures, since the Vj-gating voltage sensitivity can modified without effect on loop gating. For some connexins, certain modifications of the C-terminal domain seem to interfere with the operation of the Vj-gate while leaving loop gating unaffected. In some connexins, but not all, the chemical sensitivity to pH can involve interactions between regions of the C-terminal domain and cytoplasmic loop. Whether these regions exert their effects directly by physically blocking the pore, or by allosteric mechanisms (which may be more consistent with the relatively long time-course of closure) is not clear. For several connexins, truncation of the C-terminal domain eliminates the pH sensitivity, and co-expressing the domain with the truncated connexin restores the pH sensitivity. This has a functional resemblance to the particle-receptor mechanism for N-type inactivation of Shaker channels. What is being protonated is not clear, and may involve cytoplasmic factors, such as endogenous aminosulfonates. For other connexins, the action of pH does not involve the C-terminal domain and seems due to direct protonation of connexin. PKC phosphorylation of serine(s) in the C-terminal domain can affect the substate occupancy of at least one connexin. Phosphorylation of series in the C-terminal domain by MAP kinase appears to facilitate an interaction between it and an unknown receptor domain to eliminate coupling. This process has yet to be studied at the single-channel level. It also has a functional analogy to the particle-receptor model of channel inactivation. Both MAP kinase phosphorylation-induced and pH-induced inhibition can be mediated in truncated connexins by the corresponding free peptide. However, the relation between these two mechanisms are unexplored, as are specific mechanisms of direct endogenous regulation of connexin channel activity. (ABSTRACT TRUNCATED)

/pdf/emerging-issues-of-connexin-channels-biophysics-fills-the-3o9vrnrqk5.pdf

Emerging issues of connexin channels: biophysics fills the gap.

Gap junction channels have long been viewed as static structures containing a large-diameter, aqueous pore. This pore has a high permeability to hydrophilic molecules of approximately 900 daltons in molecular weight and a weak ionic selectivity. The evidence leading to these conclusions is reviewed in the context of more recent observations primarily coming from unitary channel recordings from transfected connexin channels expressed in communication-deficient cell lines. What is emerging is a more diverse view of connexin-specific gap junction channel structure and function where electrical conductance, ionic selectivity, and dye permeability vary by one full order of magnitude or more. furthermore, the often held contention that channel conductance and ionic or molecular selectivity are inversely proportional is refuted by recent evidence from five distinct connexin channels. The molecular basis for this diversity of channel function remains to be identified for the connexin family of gap junction proteins.

Size and selectivity of gap junction channels formed from different connexins

Connexin37 forms high conductance gap junction channels with subconductance state activity and selective dye and ionic permeabilities

The relative permeability sequences of the rat connexin 43 (rCx43) gap junction channel to seven cations and chloride were examined by double whole cell patch clamp recording of single gap junction channel currents in rCx43 transfected neuroblastoma 2A (N2A) cell pairs. The measured maximal single channel slope conductances (gammaj, in pS) of the junctional current-voltage relationships in 115 mM XCI were RbC1 (103) > or = CsC1 (102) > KC1 (97) > NaC1 (79) > or = LiC1 (78) > TMAC1 (65) > TEAC1 (53) and for 115 mM KY were KBr (105) > KC1 (97) > Kacetate (77) > Kglutamate (61). The single channel conductance- aqueous mobility relationships for the test cations and anions were linear. However, the predicted minimum anionic and cationic conductances of these plots did not accurately predict the rCx43 channel conductance in 115 mM KC1. Instead, the conductance of the rCx43 channel in 115 mM KC1 was accurately predicted from cationic and anionic conductance-mobility plots by applying a mobility scaling factor Dx/Do, which depends upon the relative radii of the permeant ions to an estimated pore radius. Relative permeabilities were determined for all of the monovalent catious and anions tested from asymmetric salt reversal potential measurements and the Goldman-Hodgkin-Katz voltage equation. These experiments estimate the relative chloride to potassium permeability to be 0.13. The relationship between the relative cation permeability and hydrated radius was modeled using the hydrodynamic equation assuming a pore radius of 6.3 +/- 0.4 A. Our data quantitatively demonstrate that the rCx43 gap junction channel is permeable to monovalent atomic and organic cations and anions and the relative permeability sequences are consistent with an Eisenman sequence II or I, respectively. These predictions about the rCx43 channel pore provide a useful basis for future investigations into the structural determinants of the conductance and permeability properties of the connexin channel pore.

/pdf/monovalent-ion-selectivity-sequences-of-the-rat-connexin43-3125lzte5m.pdf

Monovalent Ion Selectivity Sequences of the Rat Connexin43 Gap Junction Channel

The unitary conductances and permeability sequences of the rat connexin40 (rCx40) gap junction channels to seven monovalent cations and anions were studied in rCx40-transfected neuroblastoma 2A (N2A) cell pairs using the dual whole cell recording technique. Chloride salt cation substitutions (115 mM principal salt) resulted in the following junctional maximal single channel current-voltage relationship slope conductances (gamma 1 in pS): CsC1 (153), RbC1 (148), KC1 (142), NaC1 (115), LiC1 (86), TMAC1 (71), TEAC1 (63). Reversible block of the rCx40 channel was observed with TBA. Potassium anion salt gamma j are: Kglutamate (160), Kacetate (160), Kaspartate (158), KNO3 (157), KF (148), KC1 (142), and KBr (132). Ion selectivity was verified by measuring reversal potentials for current in rCx40 gap junction channels with asymmetric salt solutions in the two electrodes and using the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The permeabilities relative to Li+ are: Cs+ (1.38), Rb+ (1.32), K+ (1.31), Na+ (1.16), TMA+ (0.53), TEA+ (0.45), TBA+ (0.03), Cl- (0.19), glutamate+ (0.04), and NO(3)- (0.14), assuming that the monovalent anions permeate the channel by forming ion pairs with permeant monovalent cations within the pore thereby causing proportionate decreases in the channel conductance. This hypothesis can account for why the predicted increasing conductances with increasing ion mobilities in an essentially aqueous channel were not observed for anions in the rCx40 channel. The rCx40 effective channel radius is estimated to be 6.6 A from a theoretical fit of the relationship of relative permeability and cation radius.

https://rupress.org/jgp/article-pdf/109/4/509/1188755/gp-7262.pdf

Monovalent cation permeation through the connexin40 gap junction channel: cs, rb, k, na, li, tea, tma, tba, and effects of anions br, cl, f, acetate, aspartate, glutamate, and no3

Cardiac Gap Junction Channel Regulation. Synchronization of pacemaker activity and cardiac action potential propagation is dependent upon the maintenance of electrical communication via gap junctions. Gap junction channels in mammalian heart are formed by a 43-kilodalton (kD) protein, connexin43, and have a unitary conductance of 50 picosiemens (pS). In embryonic chick heart, three gap junction proteins of 42, 43, and 45 kD have been cloned, the functional properties of which have not been individually identified. Unitary channel conductances ranging from 40-240 pS have been reported, but conductances of 40-80 and 160 pS are most frequently observed in embryonic chick heart. Developmental changes in transjunctional voltage dependence are known to occur and may be correlated with differential expression of the three chick connexins during development. Gap junction conductance (gj) is highly regulated, being maintained or increased by ATP and cAMP-dependent pathways. Conversely, gj is reduced by treatment with various lipophilic agents (e.g., n-alkanols, arachidonic acid, halothane), large transjunctional potentials, increasing intracellular cation (e.g., H+, Ca2+) concentrations, and tyrosine phophorylation of rat connexin43. It is unclear at this time if any of the regulatory mechanisms can mediate beat-to-beat fluctuations in gj of normal myocardium. Most available evidence indicates that this modulation occurs by the open-closed gating of individual channels rather than effects on the unitary channel conductance. The precise molecular mechanisms are not yet known, but the functional expression of gap junction proteins should allow for determination of the functional domains responsible for direct and indirect modulation of the cardiac gap junction proteins.

Richard D. Veenstra

Papers

Size and selectivity of gap junction channels formed from different connexins

Connexin37 forms high conductance gap junction channels with subconductance state activity and selective dye and ionic permeabilities

Monovalent Ion Selectivity Sequences of the Rat Connexin43 Gap Junction Channel

Monovalent cation permeation through the connexin40 gap junction channel: cs, rb, k, na, li, tea, tma, tba, and effects of anions br, cl, f, acetate, aspartate, glutamate, and no3

Physiological Modulation of Cardiac Gap Junction Channels