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 NH2-terminus domain (NT). The chemical gate closes the channel slowly and completely, while the fast-Vj gate closes the channel rapidly but incompletely. The chemical gate closes with increased cytosolic calcium concentration [Ca2+]i and with Vj gradients at Vj's negative side. In contrast, the fast-Vj gate closes at the positive or negative side of Vj depending on the connexin (Cx) type. Cxs with positively charged NT close at Vj's negative side, while those with negatively charged NT close at Vj's positive side. Cytosolic acidification alters in opposite ways the sensitivity of the fast-Vj gate: it increases the Vj sensitivity of negative gaters and decreases that of positive gaters. While the fast-Vj gate closes and opens instantaneously, the chemical gate often shows fluctuations, likely to reflect the shifting of the gate (CaM's N-lobe) in and out of the channel's pore.

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. Thin filaments, which we believe are made of kinesin, bridge the microtubule to the edge of the pore. FS are believed to play a role in axoplasmic flow. The axons also display areas in which axon and sheath glial cell plasma membranes are sharply curved and project into the axoplasm. In freeze-fractures, the protoplasmic leaflet (P-face) of the projections appears as elongated indentations containing parallel chains of particles. The sheath glial cell plasma membrane also contains particles, but they are irregularly aggregated. The axons also display areas where axonal and glial plasma membranes fuse, creating intercellular pores. In axons fixed during electrical stimulation, the plasma membrane, the outer membrane of mitochondria, membranes of other cytoplasmic organelles, and gap junctions increase in electron opacity and thickness, resulting from unmasking of sulfhydryl groups (-SH). Similar changes occur in asphyxiated nerve cords.

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. Significantly, filaments, likely made of kinesin, often bridge the gap between the microtubule and the edge of the pore. Neighboring cisternae are linked by longitudinal membranous tubules. In small axons, the cisternae seem to be continuous across the axon, while in large axons they are intact only at the axon's periphery. Due to the presence of pores, we have named these structures "Fenestrated Septa" (FS). Similar structures are also present in vertebrates, including mammals, proving that they are widely expressed in the animal kingdom. We propose that FS are components of the "anterograde transport" mechanism that moves cisternae of the Golgi apparatus (GA) toward the nerve ending by means of motor proteins, likely to be kinesins. In crayfish lateral giant axons, we believe that vesicles that bud off FS at the nerve ending contain gap junction hemichannels (innexons) for gap junction channel and hemichannel formation and function.

Ca2+-Dependent and -Independent Calmodulin Binding to the Cytoplasmic Loop of Gap Junction Connexins.

Ca2+/calmodulin (Ca2+/CaM) interaction with connexins (Cx) is well-established; however, the mechanistic basis of regulation of gap junction function by Ca2+/CaM is not fully understood. Ca2+/CaM is predicted to bind to a domain in the C-terminal portion of the intracellular loop (CL2) in the vast majority of Cx isoforms and for a number of Cx-s this prediction has proved correct. In this study, we investigate and characterise both Ca2+/CaM and apo-CaM binding to selected representatives of each of the α, ß and γ connexin family to develop a better mechanistic understanding of CaM effects on gap junction function. The affinity and kinetics Ca2+/CaM and apo-CaM interactions of CL2 peptides of ß-Cx32, γ-Cx35, α-Cx43, α-Cx45 and α-Cx57 were investigated. All five Cx CL2 peptides were found to have high affinity for Ca2+/CaM with dissociation constants (Kd(+Ca)) from 20 to 150 nM. The limiting rate of binding and the rates of dissociation covered a broad range. In addition, we obtained evidence for high affinity Ca2+-independent interaction of all five peptides with CaM, consistent with CaM remaining anchored to gap junctions in resting cells. However, for the α-Cx45 and α-Cx57 CL2 peptides, Ca2+-dependent association at resting [Ca2+] of 50-100 nM is indicated in these complexes as one of the CaM Ca2+ binding sites displays high affinity with Kd of 70 and 30 nM for Ca2+, respectively. Furthermore, complex conformational changes were observed in peptide-apo-CaM complexes with the structure of CaM compacted or stretched by the peptide in a concentration dependent manner suggesting that the CL2 domain may undergo helix-to-coil transition and/or forms bundles, which may be relevant in the hexameric gap junction. We demonstrate inhibition of gap junction permeability by Ca2+/CaM in a dose dependent manner, further cementing Ca2+/CaM as a regulator of gap junction function. The motion of a stretched CaM-CL2 complex compacting upon Ca2+ binding may bring about the Ca2+/CaM block of the gap junction pore by a push and pull action on the CL2 C-terminal hydrophobic residues of transmembrane domain 3 (TM3) in and out of the membrane.

Anesthetics and Cell-Cell Communication: Potential Ca2+-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 Ca2+ 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. In our publication, we concluded that neither cytosolic Ca2+i nor pHi were involved, and suggested a direct effect of anesthetics on gap junction channel proteins. However, while a direct effect cannot be excluded, based on the potentiating effect of caffeine and theophylline added to anesthetics and data published over the past three decades, we are now reconsidering our earlier interpretation and propose an alternative hypothesis that uncoupling by heptanol, halothane and isoflurane may actually result from a rise in cytosolic Ca2+ concentration ([Ca2+]i) and consequential activation of calmodulin linked to gap junction proteins.

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. Once more, it was thought to be an exception restricted to excitable cells. In contrast, in the mid-1960s, two startling publications proved that virtually all cells freely exchange small neutral and charged molecules. Soon after, cell-cell communication by gap junction channels was reported. While gap junctions are the major means of cell-cell communication, in the early 1980s, evidence surfaced that some cells might also communicate via membrane pores. Questions were raised about the possible artifactual nature of the pores. However, early in this century, we learned that communication via membrane pores exists and plays a major role in medicine, as the structures involved, "tunneling nanotubes", can rescue diseased cells by directly transferring healthy mitochondria into compromised cells and tissues. On the other hand, pathogens/cancer could also use these communication systems to amplify pathogenesis. Here, we describe the evolution of the discovery of these new communication systems and the potential therapeutic impact on several uncurable diseases.

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 ([Ca2+i]) 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. A CaM mutant with higher Ca2+ sensitivity greatly increases gating sensitivity. CaM co-localizes with connexins. Connexins have high-affinity CaM-binding sites. Connexin mutants paired to wild type connexins have a higher gating sensitivity, which is eliminated by the inhibition of CaM expression. Repeated trans-junctional voltage (Vj) pulses progressively close channels by the chemical/slow gate (CaM's N-lobe). At the single channel level, the gate closes and opens slowly with on-off fluctuations. Internally perfused crayfish axons lose gating competency but recover it by the addition of Ca-CaM to the internal perfusion solution. X-ray diffraction data demonstrate that isolated gap junctions are gated at the cytoplasmic end by a particle of the size of a CaM lobe. We have proposed two types of CaM-driven gating: "Ca-CaM-Cork" and "CaM-Cork". In the first, the gating involves Ca2+-induced CaM activation. In the second, the gating occurs without a [Ca2+]i rise.

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. An important Cx accessory protein is calmodulin (CaM), which is the major regulator of gap junction channel gating and a molecule relevant to gap junction formation. Recently, diseases caused by CaM mutations (calmodulinopathies) have been identified, but thus far calmodulinopathy studies have not considered the potential effect of CaM mutations on gap junction function. The major goal of this review is to raise awareness on the likely role of CaM mutations in defects of gap junction mediated cell communication. Our studies have demonstrated that certain CaM mutants affect gap junction channel gating or expression, so it would not be surprising to learn that CaM mutations known to cause diseases also affect cell communication mediated by gap junction channels.

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 [Ca2+]i in the nanomolar range mediated by Ca2+-activated calmodulin (CaM). Evidence for the relevance of chemical regulation of gap junctional communication to cell function in health and disease prompted the development of methodologies aimed at quantitatively monitoring channel gating. A widely used method is the double voltage clamp of Xenopus laevis oocytes. Basically, this method involves pairing at the vegetal pole devitellinized oocytes in a conical well of a culture dish, inserting in each of them a current and a voltage microelectrode, establishing double voltage clamp and measuring junctional conductance (Gj) from voltage and current records.

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 [Ca2+]i. Calmodulin inhibitors and inhibition of CaM expression prevent chemical gating. CaMCC, a CaM mutant with higher Ca2+-sensitivity greatly increases chemical gating sensitivity (in CaMCC the NH2-terminal EF-hand pair (res. 9-76) is replaced by the COOH-terminal pair (res. 82-148). Calmodulin colocalizes with connexins. Connexins have high-affinity CaM binding sites. Several connexin mutants paired to wild-type connexins have a high gating sensitivity that is eliminated by inhibition of CaM expression. Repeated transjunctional voltage (Vj) pulses slowly and progressively close a large number of channels by the chemical/slow gate (CaM lobe). At the single-channel level, the chemical/slow gate closes and opens slowly with on-off fluctuations. The model proposes two types of CaM-driven gating: "Ca-CaM-Cork" and "CaM-Cork". In the first, gating involves Ca2+-induced CaM-activation. In the second, gating takes place without [Ca2+]i rise. The Ca-CaM-Cork gating is only reversed by a return of [Ca2+]i to resting values, while the CaM-Cork gating is reversed by Vj positive at the gated side.

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. It seems possible, therefore, that cytoplasmic organelles may be able to exchange small molecules with each other as well as with organelles of coupled cells via gap junctions.

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 (Ca2+i) in cell-cell channel gating was first reported in the mid-sixties. In these studies, only micromolar [Ca2+]i were believed to affect gating-concentrations reachable only in cell death, which would discard Ca2+i as a fine modulator of cell coupling. More recently, however, numerous researchers, including us, have reported the effectiveness of nanomolar [Ca2+]i. Since connexins do not have high-affinity calcium sites, the effectiveness of nanomolar [Ca2+]i suggests the role of Ca-modulated proteins, with calmodulin (CaM) being most obvious. Indeed, in 1981 we first reported that a CaM-inhibitor prevents chemical gating. Since then, the CaM role in gating has been confirmed by studies that tested it with a variety of approaches such as treatments with CaM-inhibitors, inhibition of CaM expression, expression of CaM mutants, immunofluorescent co-localization of CaM and gap junctions, and binding of CaM to peptides mimicking connexin domains identified as CaM targets. Our gating model envisions Ca2+-CaM to directly gate the channels by acting as a plug ("Cork" gating model), and probably also by affecting connexin conformation.

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. Lobe-specific interactions of calmodulin with connexin32 peptides were studied by stopped flow kinetics, using Ca(2+) binding-deficient mutants. Peptides corresponding to the N-terminal tail (residues 1-22) of connexin32 engaged both the N- and C-terminal lobes (N- and C-lobes) of calmodulin, binding with higher affinity to the C-lobe of calmodulin (Ca(2+) dissociation rate constants k(3,4), 1.7+/-0.5 s(-1)) than to the N-lobe (k(1,2), 10.8+/-1.3 s(-1)). In contrast, peptides representing the C-terminal tail domain (residues 208-227) of connexin32 bound either the C- or the N-lobe but only one calmodulin lobe at a time (k(3,4), 2.6+/-0.1 s(-1) or k(1), 13.8+/-0.5 s(-1) and k(2), 1000 s(-1)). The calmodulin-binding domains of the N- and C-terminal tails of connexin32 were best defined as residues 1-21 and 216-227, respectively. Our data, showing separate functions of the N- and C-lobes of calmodulin in the interactions with connexin32, suggest trans-domain or trans-subunit bridging by calmodulin as a possible mechanism of gap junction gating.

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. In contrast, our present data indicate otherwise. Oocytes expressing Cx32 were subjected to series of -100 mV V (j) pulses (12-s duration, 30-s intervals). Both peak (PK) and steady-state (SS) junctional conductances (G (j)), measured at each pulse, decreased exponentially by 50-60% (tau = approximately 1.2 min). G (j)PK dropped more dramatically, such that G (j)SS/G (j)PK increased from 0.4 to 0.6, indicating a drop in V (j) sensitivity. Less striking effects were obtained with -60 mV pulses. During recovery, G (j), measured by applying 20 mV pulses (2-s duration, 30-s intervals), slowly returned to initial values (tau = approximately 7 min). With reversal of V (j) polarity, G (j)PK briefly increased and G (j)SS/G (j)PK decreased, suggesting that V (j)-dependent hemichannel reopening is faster than hemichannel closing. Similar yet more dramatic results were obtained with COOH-terminus truncated Cx32 (Cx32-D225), a mutant believed to lack fast V (j) gating. The data indicate that the slow gate of Cx32 is active in the absence of uncouplers or mutations and displays unusual V (j) behavior. Based on previous evidence for direct calmodulin (CaM) involvement in chemical/slow gating, this may also be CaM-mediated.

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. Moreover, a direct interaction between Cx45.6 and the Major Intrinsic Protein/AQP0 in lens appears to influence the process of cell differentiation whereas interactions between aquaporin 4 (AQP4) and Cx43 in mouse astrocytes may coordinate the intercellular movement of ions and water between astrocytes. In this review, we discuss evidence supporting interactions between Cx and membrane channels/transporters including CFTR, aquaporins, ionotropic glutamate receptors, and between pannexin1, another class of putative gap-junction-forming proteins, and Kvbeta3, a regulatory beta-subunit of voltage gated potassium channels. Although the precise molecular nature of these interactions has yet to be defined, their consequences may be critical for normal tissue homeostasis.

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. In oocyte pairs expressing just Cx45 in one oocyte (#1) and both Cx45 and CFTR in the other (#2), with negative pulses applied to oocyte #1 forskolin application still increased G (j) and decreased the sensitivity to V (j) gating, indicating that CFTR activation is effective even when it affects only one of the two hemichannels and that the G (j) and V (j) changes are not artifacts of decreased membrane resistance in the pulsed oocyte. COOH-terminus truncation reduced the forskolin effect on Cx40 (Cx40TR) but not on Cx32 (Cx32TR) channels. The data suggest a cross-talk between CFTR and a variety of gap junction channels. Cytoskeletal scaffolding proteins and/or other intermediate cytoplasmic proteins are likely to play a role in CFTR-Cx interaction.

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) also affected the kinetics of V(j) dependent inactivation of junctional current (I(j)), decreasing the gating speed of Cx50 channels and increasing that of Cx50-D3N channels. In addition, the D3N mutation increased the CO(2) sensitivity of chemical gating such that even CO(2) concentrations as low as 2.5% significantly lowered junctional conductance (G(j)). With Cx50 channels G(j) dropped by 78% with a drop in intracellular pH (pH(i)) to 6.83, whereas with Cx50-D3N channels G(j) dropped by 95% with a drop in pH(i) to just 7.19. We have previously hypothesized that the way in which V(j) gating reacts to CO(2) might be related to connexin's gating polarity. This hypothesis is confirmed here by evidence that the D3N mutation inverts the gating polarity as well as the effect of CO(2) on V(j) gating sensitivity and speed.

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 7.2 and [Ca(2+)](i) as low as 150 nM or lower have been reported to be effective in some cells. Some data suggest that Ca(2+) and H(+) affect gating by acting synergistically, but other data do not support synergism. Chemical gating follows the activation of a slow gate distinct from the fast V(j)-sensitive gate, and there is evidence that the chemical/slow gate is V(j)-sensitive. At the single channel level, the chemical/slow gate closes the channels slowly and completely, whereas the fast V(j) gate closes the channels rapidly and incompletely. At least three molecular models of channel gating have been proposed, but all of them are mostly based on circumstantial evidence.

The voltage gates of connexin channels are sensitive to CO(2).

Cx45 channel sensitivity to CO(2), transjunctional voltage (V(j)) and inhibition of calmodulin (CaM) expression was tested in oocytes by dual voltage-clamp. Cx45 channels are very sensitive to V(j) and close preferentially by the slow gate, likely the same as the chemical gate. With CO(2)-induced drop in junctional conductance (G(j)), the speed of V(j)-dependent inactivation of junctional current (I(j)) and V(j) sensitivity increased. With 40 mV V(j), the tau of single exponential I(j) decay reversibly decreased by approximately 40% with CO(2), and G(j steady state)/G(j peak) decreased multiphasically, indicating that kinetics and V(j) sensitivity of chemical/slow-V(j) gating are altered by changes in [H(+)](i) and/or [Ca(2+)](i). With 15 min exposure to CO(2), G(j) dropped to 0% in controls and by approximately 17% following CaM expression inhibition; similarly, V(j) sensitivity decreased significantly. This indicates that the speed and sensitivity of V(j)-dependent inactivation of Cx45 channels are increased by CO(2), and that CaM plays a role in gating. Cx32 channels behaved similarly, but the drop in both G(j steady state)/G(j peak) and tau with CO(2) matched more closely that of G(j peak). In contrast, sensitivity and speed of V(j) gating of Cx40 and Cx26 channels decreased, rather than increased, with CO(2) application.