William H. Fletcher

Mutations of the Connexin43 gap-junction gene in patients with heart malformations and defects of laterality.

BACKGROUND Gap junctions are thought to have a crucial role in the synchronized contraction of the heart and in embryonic development. Connexin43, the major protein of gap junctions in the heart, is targeted by several protein kinases that regulate myocardial cell-cell coupling. We hypothesized that mutations altering sites critical to this regulation would lead to functional or developmental abnormalities of the heart. METHODS Connexin43 DNA from 25 normal subjects and 30 children with a variety of congenital heart diseases was amplified by the polymerase chain reaction and sequenced. Mutant DNA was expressed in cell culture and examined for its effect on the regulation of cell-cell communication. RESULTS The 25 normal subjects and 23 of the 30 children with heart disease had no amino acid substitutions in connexin43. All six children with syndromes that included complex heart malformations had substitutions of one or more phosphorylatable serine or threonine residues. Four of these children had two independent mutations, suggesting an autosomal recessive disorder. Five of these children had substitutions of proline for serine at position 364. A seventh child, with a different heart condition, also had a point mutation in connexin43. Transfected cells expressing the Ser364Pro mutant connexin43 sequence showed abnormalities in the regulation of cell-cell communication, as compared with cells expressing normal connexin43. CONCLUSIONS Mutations in the connexin43 gap-junction gene, which lead to abnormally regulated cell-cell communication, are associated with visceroatrial heterotaxia.

The connexin43 gap junction protein is phosphorylated by protein kinase A and protein kinase C: In vivo and in vitro studies

There is general agreement that the connexin43 gap junction protein is a substrate for phosphorylation by protein kinase C but there is no similar consensus regarding the action of protein kinase A. Our previous studies demonstrated that channels formed by connexin43 were reversibly gated in response to microinjected protein kinase A and protein kinase C, but we did not determine whether these effects involved direct action on the connexin43 protein. Using a combination of in vivo metabolic labeling and in vitro phosphorylation of recombinant protein and synthetic peptides, we now find that connexin43 is a relatively poor substrate for purified protein kinase A compared to protein kinase C, but that phosphorylation can be accelerated by 8-Br-cAMP (8-bromoadenosine 3′,5′-cyclic monophosphate) which also enhances connexin43 synthesis but at a much slower rate than phosphorylation. Phosphorylation of a critical amino acid, Ser364, by protein kinase A, appears to be necessary for subsequent multiple phosphorylations by protein kinase C. However, protein kinase C can phosphorylate connexin43 at a reduced level in the absence of prior phosphorylation. The results suggest that the correct regulation of channels formed by connexin43 may require sequential phosphorylations of this protein by protein kinase A and protein kinase C.

α1 connexin (connexin43) gap junctions and activities of cAMP-dependent protein kinase and protein kinase C in developing mouse heart

α1 connexin (connexin43) is the dominant gap junction protein of the developing and mature heart where it forms channels that mediate intercellular electrical and metabolic coupling events that are critical for heart function. α1 connexin channels are rapidly and reversibly gated by actions of cAMP‐dependent protein kinase (PKA) and protein kinase C (PKC), and disruption of consensus sites for these phosphorylations are associated with severe heart malformations. However, there have been no reports on the relative activities of PKA or PKC in early heart formation. Nor has the presence and phosphorylation state of α1 connexin been documented in these same developmental stages. To begin these studies, we used hearts from 8.5–18.5 dpc (days postcoitus) mouse embryos, postpartum pups, and adults. Membrane or supernatant fractions were used for immunoblots to assess the amounts and distribution of α1 connexin protein and each protein kinase. Phosphotransferase assays were done to document the endogenous activities of PKA and PKC. Three species of α1 connexin at 44, 46, and 49 kDa were evident in 8.5‐ and 9.5‐dpc embryos and adult hearts, but the 49‐kDa band was not consistently found in 10.5 dpc or embryos through 18.5 dpc, although it was robust in adult heart. The amount of PKA was minimal in 8.5‐dpc hearts but rose thereafter and was maximal by 10.5 dpc and remained stable throughout development. Catalytic activity of this enzyme was minimal in 8.5‐dpc hearts then rose thereafter and was maximal by 10.5 dpc of development. PKCδ was confined mainly to membrane fractions, whereas PKCϵ had supernatant‐ and membrane‐associated forms. Both enzyme isoforms showed large fluctuations throughout development. In 8.5‐ and 9.5‐dpc hearts, PKC catalytic activity was maximal but, by 10.5 dpc, activity dramatically declined and remained low thereafter. The results demonstrate that α1 connexin is present at the heart tube stage (8.5 dpc) of development onward and provide evidence suggesting that channels formed by this protein are dynamically regulated by PKA and PKC, especially in 8.5‐ and 9.5‐day embryonic hearts, which are crucial times for heart formation and left/right patterning in general.

Effect of Leukotriene Inhibitor on Salicylate Induced Morphologic Changes of Isolated Cochlear Outer Hair Cells

Our previous studies have shown that salicylate ototoxicity is associated with decreased levels of prostaglandins (PGs) and increased levels of leukotrienes (LTs) in the perilymph. Other studies have demonstrated that salicylate ototoxicity is associated with decreased cochlear blood flow, reversible changes in isolated cochlear outer hair cells (OHCs), and decreased otoacoustic emission. We have shown that pretreatment with an LT inhibitor prevents salicylate induced hearing loss, a decrease in cochlear blood flow and changes in otoacoustic emissions. The objectives of the current study were to determine the effect of exposure of salicylate and LTs on the morphology of isolated OHSc and to determine the effect of LT inhibitors on salicylate induced morphologic changes of isolated OHCs. Isolated OHCs from chinchilla cochlea were exposed to different test solutions. The groups included sodium salicylate (10 mM) with or without pretreatment with an LT inhibitor (L-663, 536, 30 microM), 0.1 or 1.0 microM solution of LTC4, LTD4, LTE4, and two control solutions, standard bathing solution (SBS) or leukotriene inhibitor alone. Osomolality of all solutions were kept at 305 +/- 5 mmolkg-1. The OHCs were observed under an inverted microscope. Images were stored onto a computer and analyzed later. OHCs exposed to the salicyalate developed a decrease in mean cell length. The exposure of OHCs to LTC4, LTD4, and LTE4 also demonstrated a similar decrease in mean cell length. Cells in the control SBS or LT inhibitor alone groups did not show any change. OHCs exposed to salicylate in the presence of the LT inhibitor did not exhibit morphologic changes. This study suggest that arachidonic acid metabolites, especially an increase in the concentration of LTs, seem to play an important role in the pathogenesis of salicylate ototoxicity.

Effect of Corticosteroid on Salicylate-Induced Morphological Changes of Isolated Cochlear Outer Hair Cells

Our previous studies showed that pretreatment with corticosteroids, which inhibits release of arachidonic acid (precursor of prostaglandins and leukotrienes), partially prevented salicylate-induced hearing loss in vivo. The purpose of this study was to determine the effect of pretreatment with corticosteroid (dexamethasone sodium phosphate) on isolated cochlear outer hair cells (OHCs) exposed to salicylate in vitro. Isolated OHCs from the chinchilla cochlea were exposed to salicylate with or without pretreatment with dexamethasone. Images were stored and analyzed on the Image program. The OHCs exposed to salicylate demonstrated a significant shortening in cell length. The OHCs exposed to salicylate after pretreatment with dexamethasone exhibited no significant change in cell length. We conclude that corticosteroid treatment of isolated OHCs is effective in blocking the morphological changes induced by salicylate. This study gives additional evidence that salicylate ototoxicity is mediated by alteration in the levels of arachidonic acid metabolites.

Physiological Roles of Gap Junctional Communication in Reproduction

The uterus exhibits a unique structural and physiological plasticity when responding to ovarian cues with orderly cycles of tissue differentiation, glandular secretion, and regional turnover of terminally differentiated cells. These cyclic changes are programmed to be developmentally synchronous with blastocyst development, such that arrival of a blastocyst in the uterus, followed by interaction of blastocyst trophectoderm with uterine epithelial cells, results in the progressive phases of implantation.

[24] Direct cytochemical localization of regulatory subunit of cAMP-dependent protein kinase using fluoresceinated catalytic subunit

Publisher Summary The concept that the catalytic and regulatory subunits may separately carry out specific actions would imply that the subunits may differentially move and sequester in distinct cellular sites, once dissociated. Upon removal of the cAMP signal, the high-affinity interaction of the regulatory and catalytic subunits would then terminate the separate actions of each, but the timing of this may require subunit relocalization. To examine the fate and function of regulatory subunits upon dissociation of the protein kinase, a direct cytochemical procedure using fluoresceinated catalytic subunit (F:C), which binds to free regulatory subunit, has been developed. The basic rationale of this method is identical to that given in the preceding chapter, underlying the use of fluoresceinated inhibitor protein to locate intracellular sites of the free catalytic subunit. The binding of the catalytic to the regulatory subunit is strictly dependent on cAMP. Thus when cytochemical staining is performed in the absence versus the presence of exogenous cAMP, the differential between these two reflects specific recognition of the regulatory subunit. This probe, in the presence of cAMP, may also be able to cytochemically detect the inhibitor protein, although this needs to be substantiated.

The highly conserved Gln49 and Ser50 of mammalian connexin43 are present in chick connexin43 and essential for functional gap junction channels.

In an attempt to compare the regulation of chick connexin43 channels to those of mammalian connexin43, we found that the nucleotide sequence reported for chick connexin43 differs from that of the chick connexin gene by two codons that had been entered as histidine49 (H49) and valine50 (V50) (accession no. M29003), but are in fact glutamine49 (Q49) and serine50 (S50). Neuro2A cells were transfected with corrected wild-type (Q49/S50) chick connexin43 (accession no. AF233738), the double-replacement Q49H/S50V connexin43, or the single replacement of Q49H or S50V. All clones had gap junctions in membrane based on immunocytochemistry and immunoblots of the triton-resistant membrane fraction. Wild-type transfectants had three conductance states with a predominant channel conductance of 85 - 5 pS. Cells producing the Q49H-Cx43 or the double-replacement Q49H/S50V-Cx43 protein had no detectable connexin43 channels. In contrast, cells expressing S50V-Cx43 gap junctions had channels with reduced conductances (75 - 8 pS) compared to wild-type controls. Low or high pH of the bathing solution had no effect on the Q49H-Cx43 channels. We conclude that glutamine49 is important for channel function, and replacement of this residue with histidine most likely distorts secondary structure of the first extracellular loop, possibly by changing the orientation of conserved cysteines, and this inhibits channel function. The S50V substitution may also cause similar but less severe structural changes.