Miduturu Srinivas

Quinine blocks specific gap junction channel subtypes

We demonstrate that the antimalarial drug quinine specifically reduces currents through gap junctions formed by some connexins (Cx) in transfected mammalian cells, but does not affect other gap junction types. Quinine blocked Cx36 and Cx50 junctional currents in a reversible and concentration-dependent manner with half maximal blocking concentrations of 32 and 73 μM, respectively; Hill coefficients for block by quinine were about 2 for both connexins. In contrast, quinine did not substantially block gap junction channels formed by Cx26, Cx32, Cx40, and Cx43, and only moderately affected Cx45 junctions. To determine the location of the binding site of quinine (pKa = 8.7), we investigated the effect of quinine at various external and internal pH values and the effect of a permanently charged quaternary derivative of quinine. Our results indicate that the binding site for quinine is intracellular, possibly within the pore. Single-channel studies indicated that exposure to quinine induced slow transitions between open and fully closed states that decreased open probability of the channel. Quinine thus offers a potentially useful method to block certain types of gap junction channels, including those between neurons that are formed by Cx36. Moreover, quinine derivatives that are excluded from other types of membrane channels may provide molecules with connexin-specific as well as connexin-selective blocking activity.

New possible roles for aquaporin-4 in astrocytes: cell cytoskeleton and functional relationship with connexin43

Aquaporin‐4 (AQP4), the main water channel in the brain, is expressed in the perivascular membranes of mouse, rat, and human astrocytes. In a previous study, we used small interfering RNA (siRNA) to specifically knock down AQP4 in rat astrocyte primary cultures and found that together with reduced osmotic permeability, AQP4 knockdown (KD) led to altered cell morphology. However, a recent report on primary cultured astrocytes from AQP4 null mice (KO) showed no morphological differences compared with wild types. In this study, we compared the effect of AQP4 KD in mouse, rat, and human astrocyte primary cultures and found that AQP4 KD in human astrocytes resulted in a morphological phenotype similar to that found in rat. In contrast, AQP4 KD in mouse astrocytes caused only very mild morphological changes. The actin cytoskeleton of untreated astrocytes exhibited strong species‐specific differences, with F‐actin being organized in cortical bands in mouse and in stress fibers in rat and human astrocytes. Surprisingly, as a consequence of AQP4 KD, F‐actin cytoskeleton was depolymerized in rat and human whereas it was completely rearranged in mouse astrocytes. Although AQP4 KD induced alterations of the cell cytoskeleton, we found that the expression of dystrophin (DP71), β‐dystroglycan, and α‐syntrophin was not altered. AQP4 KD in cultured mouse astrocytes produced strong down‐regulation of connexin43 (Cx43) with a concomitant reduction in cell coupling while no major alterations in Cx43 expression were found in rat and human cells. Taken together, these results demonstrate that with regard to these properties, human astrocytes in culture are more similar to rat than to mouse astrocytes. Moreover, even though AQP4 KD in mouse astrocytes did not result in a dramatic morphological phenotype, it induced a remarkable rearrangement of F‐actin, not related to disruption of the dystrophin complex, indicating a primary role of this water channel in the cytoskeleton changes observed. Finally, the strong down‐regulation of Cx43 and cell coupling in AQP4 KD mouse astrocytes indicate that a functional relationship likely exists between water channels and gap junctions in brain astrocytes.

Differentially altered Ca2+ regulation and Ca2+ permeability in Cx26 hemichannels formed by the A40V and G45E mutations that cause keratitis ichthyosis deafness syndrome

Mutations in GJB2, which encodes Cx26, are one of the most common causes of inherited deafness in humans. More than 100 mutations have been identified scattered throughout the Cx26 protein, most of which cause nonsyndromic sensorineural deafness. In a subset of mutations, deafness is accompanied by hyperkeratotic skin disorders, which are typically severe and sometimes fatal. Many of these syndromic deafness mutations localize to the amino-terminal and first extracellular loop (E1) domains. Here, we examined two such mutations, A40V and G45E, which are positioned near the TM1/E1 boundary and are associated with keratitis ichthyosis deafness (KID) syndrome. Both of these mutants have been reported to form hemichannels that open aberrantly, leading to “leaky” cell membranes. Here, we quantified the Ca2+ sensitivities and examined the biophysical properties of these mutants at macroscopic and single-channel levels. We find that A40V hemichannels show significantly impaired regulation by extracellular Ca2+, increasing the likelihood of aberrant hemichannel opening as previously suggested. However, G45E hemichannels show only modest impairment in regulation by Ca2+ and instead exhibit a substantial increase in permeability to Ca2+. Using cysteine substitution and examination of accessibility to thiol-modifying reagents, we demonstrate that G45, but not A40, is a pore-lining residue. Both mutants function as cell–cell channels. The data suggest that G45E and A40V are hemichannel gain-of-function mutants that produce similar phenotypes, but by different underlying mechanisms. A40V produces leaky hemichannels, whereas G45E provides a route for excessive entry of Ca2+. These aberrant properties, alone or in combination, can severely compromise cell integrity and lead to increased cell death.

Temporal expression of neuronal connexins during hippocampal ontogeny

Communication through gap junction channels provides a major signaling mechanism during early brain histogenesis, a developmental time during which neural progenitor cells are inexcitable and do not express ligand-gated channel responses to the major CNS neurotransmitters. Expression of different gap junction types during neurogenesis may therefore define intercellular pathways for transmission of developmentally relevant molecules. To better understand the molecular mechanism(s) by which growth and differentiation of neurons are modulated by gap junction channels, we have been examining the developmental effects of a specific set of cytokines on differentiation and gap junction expression in a conditionally immortalized mouse embryonic hippocampal neuronal progenitor cell line (MK31). When multipotent MK31 cells are in an uncommitted state, they uniformly express the neuroepithelial intermediate filament class VI marker, nestin, are strongly coupled by gap junctions composed of connexin43 (Cx43) and express connexin45 (Cx45) at the mRNA level. As these cells undergo neuronal lineage commitment and exit from cell cycle, they begin to express the early neurofilament marker, NF66, and coupling strength and expression of Cx43 begin to decline with concurrent expression of other connexin proteins, including Cx26, Cx33, Cx36, Cx40 and Cx45. Terminal neuronal differentiation is heralded by the expression of more advanced neurofilament proteins, increased morphologic maturation, the elaboration of inward currents and action potentials that possess mature physiological properties, and changing profiles of expression of connexin subtypes, including upregulation of Cx36 expression. These important developmental transitions are regulated by a complex network of cell cycle checkpoints. To begin to examine the precise roles of gap junction proteins in traversing these developmental checkpoints and in thus regulating neurogenesis, we have focused on individual members of two classes of genes involved in these seminal events: ID (inhibitor of differentiation)-1 and GAS (growth arrest-specific gene)5. When MK31 cells were maintained in an uncommitted state, levels of ID-1 mRNA were high and GAS5 transcripts were essentially undetectable. Application of cytokines that promote neuronal lineage commitment and cell cycle exit resulted in down-regulation of ID-1 and upregulation of GAS5 transcripts, whereas additional cytokine paradigms that promoted terminal neuronal differentiation resulted in the delayed down-regulation of GAS5 mRNA. Stable MK31 transfectants were generated for ID-1 and GAS5. In basal conditions, cellular proliferation was enhanced in the ID-1 transfectants and inhibited in the GAS5 transfectants when compared with control MK31 cells. When cytokine-mediated neurogenesis was examined in these transfected cell lines, constitutive expression of ID-1 inhibited and constitutive expression of GAS5 enhanced initial and terminal stages of neuronal differentiation, with evidence that terminal neuronal maturation in both transfectant lines was associated with decreased cellular viability, possibly due to the presence of conflicting cell cycle-associated developmental signals. These experimental reagents will prove to be valuable experimental tools to help define the functional interrelationships between changing profiles of connexin protein expression and cell cycle regulation during neuronal ontogeny in the mammalian brain. The present review summarizes the current state of research involving the temporal expression of such connexin types in differentiating hippocampal neurons and speculates on the possible role of these intercellular channels in the development and plasticity of the nervous system. In addition, we describe the functional properties and expression pattern of the newly discovered neuronal-specific gap junctional protein, Cx36, in the developing mouse fetal hippocampus and in the rat retina and brain.

Voltage dependence of macroscopic and unitary currents of gap junction channels formed by mouse connexin50 expressed in rat neuroblastoma cells

1 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. 2 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. 3 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. 4 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. 5 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). 6 The results have important implications for understanding the role of connexins in tissues where Cx50 is a major gap junction component, including the lens.

Block of specific gap junction channel subtypes by 2-aminoethoxydiphenyl borate (2-APB)

2-Aminoethoxydiphenyl borate (2-APB), an inositol 1,4,5-triphosphate receptor modulator, inhibits capacitive current transients measured in normal rat kidney and human embryonic kidney 293 cells, an indication of blocking gap junction channels between these cells. Here, we used the dual whole-cell patch-clamp method to study the actions of 2-APB on gap junction channels formed by selected connexins expressed in a communication-deficient neuroblastoma cell line (N2A). 2-APB dose-dependently and reversibly blocked junctional currents of connexin (Cx) 50 gap junction channels. The concentration-inhibition curve of 2-APB on the junctional current indicated an IC50 of 3.7 μM, lower than that of most gap junction inhibitors. At a concentration of 20 μM, 2-APB also significantly blocked junctional conductance in cell pairs coupled by Cx26, Cx30, Cx36, Cx40, and Cx45 but did not appreciably affect coupling in cell pairs expressing Cx32, Cx43, and Cx46. Although concentration inhibition curves of 2-APB on Cx36 channels were similar to Cx50 (Cx36; IC50, 3.0 μM), IC50 values were higher for Cx43 (51.6 μM), Cx45 (18.1 μM), and Cx46 (29.4 μM). The blocking action of 2-APB did not substantially alter transjunctional voltage-dependent gating of Cx50 gap junction channels, and recordings from poorly coupled pairs of Cx50-transfected N2A cells indicated that 2-APB reduced gap junction channel open probability without changing the main state single-channel conductance. The differential efficacy of block by 2-APB of gap junction channels formed by different connexins may provide a useful tool that could be exploited in gap junction research to selectively block certain gap junction channel subtypes.

Loop Gating of Connexin Hemichannels Involves Movement of Pore-lining Residues in the First Extracellular Loop Domain

Unapposed connexin hemichannels exhibit robust closure in response to membrane hyperpolarization and extracellular calcium. This form of gating, termed “loop gating,” is largely responsible for regulating hemichannel opening, thereby preventing cell damage through excessive flux of ions and metabolites. The molecular components and structural rearrangements underlying loop gating remain unknown. Here, using cysteine mutagenesis in Cx50, we demonstrate that residues at the TM1/E1 border undergo movement during loop gating. Replacement of Phe43 in Cx50 with a cysteine resulted in small or no appreciable membrane currents. Bath application of dithiothreitol or TPEN (N,N,N′,N′-tetrakis(2-pyridylmethyl) ethylenediamine), reagents that exhibit strong transition metal chelating activity, led to robust currents indicating that the F43C substitution impaired hemichannel function, producing “lock-up” in a closed or poorly functional state due to formation of metal bridges. In support, Cd2+ at submicromolar concentrations (50–100 nm) enhanced lock-up of F43C hemichannels. Moreover, lock-up occurred under conditions that favored closure, indicating that the sulfhydryl groups come close enough to each other or to other residues to coordinate metal ions with high affinity. In addition to F43C, metal binding was also found for G46C, and to a lesser extent, D51C substitutions, positions found to be pore-lining in the open state using the substituted-cysteine accessibility method, but not for A40C and A41C substitutions, which were not found to reside in the open pore. These results indicate that metal ions access the cysteine side chains through the open pore and that closure of the loop gate involves movement of the TM1/E1 region that results in local narrowing of the large aqueous connexin pore.

Prospects for Rational Development of Pharmacological Gap Junction Channel Blockers

Connexin-null mice and human genetic gap junction diseases illustrate the important roles that gap junction channels play under normal conditions, and the neuro- and cardioprotective effects of gap junction blocking agents demonstrate that closure of these channels may be beneficial in certain pathological situations. This overview summarizes studies in which gap junction modifying reagents have been characterized, highlighting examples of agents for which selectivity for gap junction subtypes has been demonstrated. In addition, strategies for targeting connexin domains through peptide inhibitors are outlined, which may ultimately provide agents that are not only connexin-selective in their actions, but also affect only a subset of a gap junction channels gating responses.

Properties of Gap Junction Channels Formed by Cx46 Alone and in Combination with Cx50

Gap junctions formed of connexin46 (Cx46) and connexin50 (Cx50) in lens fiber cells are crucial for maintaining lens transparency. We determined the functional properties of homotypic Cx46, heterotypic Cx46/Cx50, and heteromeric Cx46/Cx50 channels in a communication-deficient neuroblastoma (N2A) cell line, using dual whole-cell recordings. N2A cultures were stably and/or transiently transfected with Cx46, Cx50, and green fluorescent protein (EGFP). The macroscopic voltage sensitivity of homotypic Cx46 conformed to the two-state model (Boltzmann parameters: G(min) = 0.11, V(0) = +/- 48.1 mV, gating charge = 2). Cx46 single channels showed a main-state conductance of 140 +/- 8 pS and multiple subconductance states ranging from < or =10 pS to 60 pS. Conservation of homotypic properties in heterotypic Cx46/Cx50 cell pairs allowed the determination of a positive relative gating polarity for the dominant gating mechanisms in Cx46 and Cx50. Observed gating properties were consistent with a second gating mechanism in Cx46 connexons. Moreover, rectification was observed in heterotypic cell pairs. Some cell pairs in cultures simultaneously transfected with Cx46 and Cx50 exhibited junctional properties not observed in other preparations, suggesting the formation of heteromeric channels. We conclude that different combinations of Cx46 and Cx50 within gap junction channels lead to unique biophysical properties.

Divalent Cations Regulate Connexin Hemichannels by Modulating Intrinsic Voltage-dependent Gating

Connexin hemichannels are robustly regulated by voltage and divalent cations. The basis of voltage-dependent gating, however, has been questioned with reports that it is not intrinsic to hemichannels, but rather is derived from divalent cations acting as gating particles that block the pore in a voltage-dependent manner. Previously, we showed that connexin hemichannels possess two types of voltage-dependent gating, termed Vj and loop gating, that in Cx46 operate at opposite voltage polarities, positive and negative, respectively. Using recordings of single Cx46 hemichannels, we found both forms of gating persist in solutions containing no added Mg2+ and EGTA to chelate Ca2+. Although loop gating persists, it is significantly modulated by changing levels of extracellular divalent cations. When extracellular divalent cation concentrations are low, large hyperpolarizing voltages, exceeding −100 mV, could still drive Cx46 hemichannels toward closure. However, gating is characterized by continuous flickering of the unitary current interrupted by occasional, brief sojourns to a quiet closed state. Addition of extracellular divalent cations, in this case Mg2+, results in long-lived residence in a quiet closed state, suggesting that hyperpolarization drives the hemichannel to close, perhaps by initiating movements in the extracellular loops, and that divalent cations stabilize the fully closed conformation. Using excised patches, we found that divalent cations are only effective from the extracellular side, indicative that the binding site is not cytoplasmic or in the pore, but rather extracellular. Vj gating remains essentially unaffected by changing levels of extracellular divalent cations. Thus, we demonstrate that both forms of voltage dependence are intrinsic gating mechanisms in Cx46 hemichannels and that the action of external divalent cations is to selectively modulate loop gating.