Calcium Role in Gap Junction Channel Gating: Direct Electrostatic or Calmodulin-Mediated?

The chemical gating of gap junction channels is mediated by cytosolic calcium (Ca) at concentrations ([Ca]) ranging from high nanomolar (nM) to low micromolar (µM) range. Since the proteins of gap junctions, connexins/innexins, lack high-affinity Ca-binding sites, most likely gating is mediated by a Ca-binding protein, calmodulin (CaM) being the best candidate. Indeed, the role of Ca-CaM in gating is well supported by studies that have tested CaM blockers, CaM expression inhibition, testing of CaM mutants, co-localization of CaM and connexins, existence of CaM-binding sites in connexins/innexins, and expression of connexins (Cx) mutants, among others.

Gap Junction Channel Regulation: A Tale of Two Gates-Voltage Sensitivity of the Chemical Gate and Chemical Sensitivity of the Fast Voltage Gate.

Gap junction channels are regulated by gates sensitive to cytosolic acidification and trans-junctional voltage (Vj). We propose that the chemical gate is a calmodulin (CaM) lobe. The fast-Vj gate is made primarily by the connexin's NH-terminus domain (NT).

Nerve Structure-Function: Unusual Structural Details and Unmasking of Sulfhydryl Groups by Electrical Stimulation or Asphyxia in Axon Membranes and Gap Junctions.

This review describes and discusses unusual axonal structural details and evidence for unmasking sulfhydryl groups (-SH) in axoplasmic membranes resulting from electrical stimulation or asphyxia. Crayfish axons contain fenestrated septa (FS) that, in phase contrast, micrographs appear as repeated striations. In the electron microscope, each septum is made of two cross-sectioned membranes containing ~55 nm pores, each occupied by a microtubule.

Potential Role of Fenestrated Septa in Axonal Transport of Golgi Cisternae and Gap Junction Formation/Function.

Crayfish axons contain a system of parallel membranous cisternae spaced by ~2 μm and oriented perpendicularly to the axon's long axis. Each cisterna is composed of two roughly parallel membranes, separated by a 150-400 Å wide space. The cisternae are interrupted by 500-600 Å pores, each occupied by a microtubule.

Anesthetics and Cell-Cell Communication: Potential Ca-Calmodulin Role in Gap Junction Channel Gating by Heptanol, Halothane and Isoflurane.

Cell-cell communication via gap junction channels is known to be inhibited by the anesthetics heptanol, halothane and isoflurane; however, despite numerous studies, the mechanism of gap junction channel gating by anesthetics is still poorly understood. In the early nineties, we reported that gating by anesthetics is strongly potentiated by caffeine and theophylline and inhibited by 4-Aminopyridine. Neither Ca channel blockers nor 3-isobutyl-1-methylxanthine (IBMX), forskolin, CPT-cAMP, 8Br-cGMP, adenosine, phorbol ester or H7 had significant effects on gating by anesthetics.

Direct Cell-Cell Communication via Membrane Pores, Gap Junction Channels, and Tunneling Nanotubes: Medical Relevance of Mitochondrial Exchange.

The history of direct cell-cell communication has evolved in several small steps. First discovered in the 1930s in invertebrate nervous systems, it was thought at first to be an exception to the "cell theory", restricted to invertebrates. Surprisingly, however, in the 1950s, electrical cell-cell communication was also reported in vertebrates.

Calmodulin-Connexin Partnership in Gap Junction Channel Regulation-Calmodulin-Cork Gating Model.

In the past four decades numerous findings have indicated that gap junction channel gating is mediated by intracellular calcium concentrations ([Ca]) in the high nanomolar range via calmodulin (CaM). We have proposed a CaM-based gating model based on evidence for a direct CaM role in gating. This model is based on the following: CaM inhibitors and the inhibition of CaM expression to prevent chemical gating.

Gap Junction Channelopathies and Calmodulinopathies. Do Disease-Causing Calmodulin Mutants Affect Direct Cell-Cell Communication?

The cloning of connexins cDNA opened the way to the field of gap junction channelopathies. Thus far, at least 35 genetic diseases, resulting from mutations of 11 different connexin genes, are known to cause numerous structural and functional defects in the central and peripheral nervous system as well as in the heart, skin, eyes, teeth, ears, bone, hair, nails and lymphatic system. While all of these diseases are due to connexin mutations, minimal attention has been paid to the potential diseases of cell-cell communication caused by mutations of Cx-associated molecules.

Calmodulin-Cork Model of Gap Junction Channel Gating-One Molecule, Two Mechanisms.

The Calmodulin-Cork gating model is based on evidence for the direct role of calmodulin (CaM) in channel gating. Indeed, chemical gating of cell-to-cell channels is sensitive to nanomolar cytosolic calcium concentrations [Ca]. Calmodulin inhibitors and inhibition of CaM expression prevent chemical gating.

Chemical and Voltage Gating of Gap Junction Channels Expressed in Xenopus Oocytes.

In most tissues, cells in contact with each other directly intercommunicate via cell-to-cell channels aggregated at gap junctions. Direct cell-to-cell communication provides a fundamental mechanism for coordinating many cellular functions in mature and developing organs, as it enables free exchange of small cytosolic molecules. Gap junction channels are regulated by a chemical gating mechanism sensitive to cytosolic calcium concentration [Ca] in the nanomolar range mediated by Ca-activated calmodulin (CaM).

Connexin/Innexin Channels in Cytoplasmic Organelles. Are There Intracellular Gap Junctions? A Hypothesis!

This paper proposes the hypothesis that cytoplasmic organelles directly interact with each other and with gap junctions forming intracellular junctions. This hypothesis originated over four decades ago based on the observation that vesicles lining gap junctions of crayfish giant axons contain electron-opaque particles, similar in size to junctional innexons that often appear to directly interact with junctional innexons; similar particles were seen also in the outer membrane of crayfish mitochondria. Indeed, vertebrate connexins assembled into hexameric connexons are present not only in the membranes of the Golgi apparatus but also in those of the mitochondria and endoplasmic reticulum.

Calmodulin-Mediated Regulation of Gap Junction Channels.

Evidence that neighboring cells uncouple from each other as one dies surfaced in the late 19th century, but it took almost a century for scientists to start understanding the uncoupling mechanism (chemical gating). The role of cytosolic free calcium (Ca) in cell-cell channel gating was first reported in the mid-sixties. In these studies, only micromolar [Ca] were believed to affect gating-concentrations reachable only in cell death, which would discard Ca as a fine modulator of cell coupling.

Calmodulin association with connexin32-derived peptides suggests trans-domain interaction in chemical gating of gap junction channels.

Calmodulin plays a key role in the chemical gating of gap junction channels. Two calmodulin-binding regions have previously been identified in connexin32 gap junction protein, one in the N-terminal and another in the C-terminal cytoplasmic tail of the molecule. The aim of this study was to better understand how calmodulin interacts with the connexin32-binding domains.

Unusual slow gating of gap junction channels in oocytes expressing connexin32 or its COOH-terminus truncated mutant.

Gap junction channels are gated by a chemical gate and two transjunctional voltage (V (j))-sensitive gates: fast and slow. Slow V (j) gate and chemical gate are believed to be the same. The slow gate closes at the negative side of V (j) and is mostly inactive without uncouplers or connexin (Cx) mutations.

Interplay between cystic fibrosis transmembrane regulator and gap junction channels made of connexins 45, 40, 32 and 50 expressed in oocytes.

The cystic fibrosis transmembrane regulator (CFTR) is a Cl(-) channel known to influence other channels, including connexin (Cx) channels. To study the functional interaction between CFTR and gap junction channels, we coexpressed in Xenopus oocytes CFTR and either Cx45, Cx40, Cx32 or Cx50 and monitored junctional conductance (G (j)) and its sensitivity to transjunctional voltage (V (j)) by the dual voltage-clamp method. Application of forskolin induced a Cl(-) current; increased G (j) approximately 750%, 560%, 64% and 8% in Cx45, Cx40, Cx32 and Cx50, respectively; and decreased sensitivity to V (j ) gating, monitored by a change in the ratio between G (j) steady state and G (j) peak (G (j)SS/G (j)PK) at the pulse.

Interactions of connexins with other membrane channels and transporters.

Cell-to-cell communication through gap junctions exists in most animal cells and is essential for many important biological processes including rapid transmission of electric signals to coordinate contraction of cardiac and smooth muscle, the intercellular propagation of Ca(2+) waves and synchronization of physiological processes between adjacent cells within a tissue. Recent studies have shown that connexins (Cx) can have either direct or indirect interactions with other plasma membrane ion channels or membrane transport proteins with important functional consequences. For example, in tissues most severely affected by cystic fibrosis (CF), activation of the CF Transmembrane Conductance Regulator (CFTR) has been shown to influence connexin function.

Functional interaction between CFTR and Cx45 gap junction channels expressed in oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl(-)) channel known to influence the function of other channels, including connexin channels. To further study potential functional interactions between CFTR and gap junction channels, we have co-expressed CFTR and connexin45 (Cx45) in Xenopus oocytes and monitored junctional conductance and voltage sensitivity by dual voltage clamp electrophysiology. In single oocytes expressing CFTR, an increase in cAMP caused by forskolin application induced a Cl(-) current and increased membrane conductance; application of diphenylamine carboxylic acid (CFTR blocker) readily blocked the Cl(-) current.

Opposite Cx32 and Cx26 voltage-gating response to CO2 reflects opposite voltage-gating polarity.

Previous studies have shown that the V(j)-dependent gating behavior of gap junction channels is altered by CO(2) exposure. V(j)-dependent channel closure is increased by CO(2) in some connexin channels and decreased in others. Since the former type of channels gate on the relatively negative side by V(j) (negative gaters) and the latter at the positive side (positive gaters), it has been hypothesized that gating polarity determines the way CO(2) affects V(j) closure.

Inversion of both gating polarity and CO2 sensitivity of voltage gating with D3N mutation of Cx50.

The effect of CO(2)-induced acidification on transjunctional voltage (V(j)) gating was studied by dual voltage-clamp in oocytes expressing mouse connexin 50 (Cx50) or a Cx50 mutant (Cx50-D3N), in which the third residue, aspartate (D), was mutated to asparagine (N). This mutation inverted the gating polarity of Cx50 from positive to negative. CO(2) application greatly decreased the V(j) sensitivity of Cx50 channels, and increased that of Cx50-D3N channels.

CO(2) sensitivity of voltage gating and gating polarity of gapjunction channels--connexin40 and its COOH-terminus-truncated mutant.

The CO(2) sensitivity of transjunctional voltage ( V(j)) gating was studied by dual voltage clamp in oocytes expressing mouse Cx40 or its COOH terminus (CT)-truncated mutant (Cx40-TR). V(j) sensitivity, determined by a standard V(j) protocol (20 mV V(j) steps, 120 mV maximal), decreased significantly with exposure to 30% CO(2). The Boltzmann values of control versus CO(2)-treated oocytes were: V(0) = 36.

Chemical gating of gap junction channels; roles of calcium, pH and calmodulin.

Both Ca(2+) and H(+) play a role in chemical gating of gap junction channels, but, with the possible exception of Cx46 hemichannels, neither of them is likely to induce gating by a direct interaction with connexins. Some evidence suggests that low pH(i) affects gating via an increase in [Ca(2+)](i); in turn, Ca(2+) is likely to induce gating by activation of CaM, which may act directly as a gating particle. The effective concentrations of both Ca(2+) and H(+) vary depending on cell type, type of connexin expressed and procedure employed to increase their cytosolic concentrations; however, pH(i) as high as 7.

Calmodulin colocalizes with connexins and plays a direct role in gap junction channel gating.

The direct calmodulin (CaM) role in chemical gating was tested with CaM mutants, expressed in oocytes, and CaM-connexin labeling methods. CaMCC, a CaM mutant with greater Ca-sensitivity obtained by replacing the N-terminal EF hand pair with a duplication of the C-terminal pair, drastically increased the chemical gating sensitivity of Cx32 channels and decreased their Vj sensitivity. This only occurred when CaMCC was expressed before Cx32, suggesting that CaMCC, and by extension CaM, interacts with Cx32 before junction formation.