Rat Connexin Research Articles

Effect of WenXin KeLi on Improvement of Arrhythmia after Myocardial Infarction by Intervening PI3K-AKT-mTOR Autophagy Pathway.

Background Myocardial infarction (MI) is an acute and serious cardiovascular disease. Arrhythmia after MI can lead to sudden cardiac death, which seriously affects the survival outcome of patients. WenXin KeLi is a Chinese patent medicine for the treatment of arrhythmia in a clinic, which can significantly improve symptoms of palpitation and play an important role in reducing the risk of arrhythmia after MI. In this study, we aimed to explore the pharmacological mechanism of WenXin KeLi in protecting the heart. Methods The MI model was established by ligating the left coronary artery and the ventricular fibrillation threshold (VFT) was measured by electrical stimulation. The expression of connexin43 (CX43) and autophagy-related protein were measured by Western Blot, and correlation analysis was conducted to study the relationship between cardiac autophagy, CX43, and arrhythmia in rats after MI. The effects of WenXin KeLi on arrhythmia, cardiac structure, and function in MI rats were respectively observed by electrical stimulation, cardiac gross section, Masson staining, and cardiac ultrasound. The effects of WenXin KeLi on the expression of phosphoinositide 3 kinase-protein kinase B-mammalian targets of rapamycin (PI3K-AKT-mTOR) autophagy pathway and CX43 were observed by Western Blot. Results After 4 weeks of MI, the VFT in the model group was significantly reduced, the expression levels of yeast ATG6 homolog (Beclin1), microtubule-associated protein 1A/1B-light chain 3 (LC3II/LC3I), and p-CX43 (S368) significantly increased, the expression of sequestosome-1(P62) and CX43 significantly decreased. LC3II/LC3I and Beclin1 expression were significantly negatively correlated with the VFT, and the expression of P62 and CX43 were significantly positively correlated with the VFT. LC3II/LC3I and Beclin1 expression were negatively correlated with CX43 expression, while P62 expression was positively correlated with CX43 expression. WenXin KeLi could significantly increase the VFT, reduce the deposition of collagen fibers, and increase the index levels of the left ventricular end-diastolic anterior wall (LVEDAW), interventricular septum end-diastolic (IVSED), left ventricular end-systolic anterior wall (LVESAW), interventricular septum end-systolic (IVSES), left ventricular end-diastolic posterior wall (LVEDPW), left ventricular end-systolic posterior wall (LVESPW), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), and reduce the index levels of the left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD), left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV). WenXin KeLi could increase the expression of CX43, P62, AKT, p-PI3K, p-AKT (308), p-AKT (473), and p-mTOR and decrease the expression of LC3II/LC3I and Beclin1. Conclusion WenXin KeLi can activate the PI3K-AKT-mTOR signaling pathway, improve cardiac autophagy and Cx43 expression in rats after MI, reduce the risk of arrhythmia after MI, and play a cardioprotective role.

TGF‐β1 and connexin‐43 expression in neurogenic bladder from rats with sacral spinal cord injury

Sacral spinal cord injury (SCI) could induce underactive bladder (UAB). Malfunction of connexin 43 (CX43) regulated by TGF-β1 might involve in urinary bladder dysfunction. We studied the changes of CX43 and TGF-β1/Smad3 signaling in detrusor of neurogenic bladder (NB) in sacral SCI rats. Sacral SCI was produced by hemisection (SSCH) or transection (SSCT) of spinal cord between L4 and L5 in female Wistar rats. BBB scores, residual urine volume and bladder weight as well as characteristic cystometric parameters at 6th week were used to confirm the successful establishment of NB. Western blotting and qRT-PCR were used to exam the protein and mRNA expression levels of CX43, CX45, TGF-β1, and Smad3 in detrusor. BBB scores were significantly decreased, with the lowest in SSCT rats (P < 0.01). The residual urine volume, mean bladder weight, and cystometric parameters were increased, with the highest in SSCT rats. CX43 and phospho-CX43 protein levels were significantly decreased, but those of TGF-β1, Smad3, and phospho-Smad3 were significantly increased. It was the protein and mRNA levels of CX43 but not those of CX45 which were decreased in negative accordance with those of TGF-β1 and Smad3. Those changes were more significant in SSCT than in SSCH rats. This study indicates that voiding dysfunction is related to the decreased CX43 function in detrusor from NB. TGF-β1/Smad3 signaling might be involved in the down-regulation of CX43 in SCI rats. Early regulation of CX43 might be beneficial to patients with voiding dysfunction.

Cardiac connexin-43 and PKC signaling in rats with altered thyroid status without and with omega-3 fatty acids intake.

Thyroid hormones are powerful modulators of heart function and susceptibility to arrhythmias via both genomic and non-genomic actions. We aimed to explore expression of electrical coupling protein connexin-43 (Cx43) in the heart of rats with altered thyroid status and impact of omega-3 polyunsaturated fatty acids (omega-3) supplementation. Adult male Lewis rats were divided into following six groups: euthyroid controls, hyperthyroid (treated with T(3)) and hypothyroid (treated with methimazol) with or without six-weeks lasting supplementation with omega-3 (20 mg/100 g/day). Left and right ventricles, septum and atria were used for immunoblotting of Cx43 and protein kinase C (PKC). Total expression of Cx43 and its phosphorylated forms were significantly increased in all heart regions of hypothyroid rats compared to euthyroid controls. In contrast, the total levels of Cx43 and its functional phosphorylated forms were decreased in atria and left ventricle of hyperthyroid rats. In parallel, the expression of PKC epsilon that phosphorylates Cx43, at serine 368, was increased in hypothyroid but decreased in hyperthyroid rat hearts. Omega-3 intake did not significantly affect either Cx43 or PKC epsilon alterations. In conclusion, there is an inverse relationship between expression of cardiac Cx43 and the levels of circulating thyroid hormones. It appears that increased propensity of hyperthyroid while decreased of hypothyroid individuals to malignant arrhythmias may be in part attributed to the changes in myocardial Cx43.

Role of Akt and Ca2 + on cell permeabilization via connexin43 hemichannels induced by metabolic inhibition

Connexin hemichannels are regulated under physiological and pathological conditions. Metabolic inhibition, a model of ischemia, promotes surface hemichannel activation associated, in part, with increased surface hemichannel levels, but little is known about its underlying mechanism. Here, we investigated the role of Akt on the connexin43 hemichannel's response induced by metabolic inhibition. In HeLa cells stably transfected with rat connexin43 fused to EGFP (HeLa43 cells), metabolic inhibition induced a transient Akt activation necessary to increase the amount of surface connexin43. The increase in levels of surface connexin43 was also found to depend on an intracellular Ca2+ signal increase that was partially mediated by Akt activation. However, the metabolic inhibition-induced Akt activation was not significantly affected by intracellular Ca2+ chelation. The Akt-dependent increase in connexin43 hemichannel activity in HeLa43 cells also occurred after oxygen-glucose deprivation, another ischemia-like condition, and in cultured cortical astrocytes (endogenous connexin43 expression system) under metabolic inhibition. Since opening of hemichannels has been shown to accelerate cell death, inhibition of Akt-dependent phosphorylation of connexin43 hemichannels could reduce cell death induced by ischemia/reperfusion.

Immunoelectron microscopic observation of connexin43 in rat odontoblasts

Gap junctions play an important role in differentiation of odontoblasts. Gap junction protein, connexin 43 is expressed in odontoblast. However, the detailed localization in odontoblasts has yet to be fully investigated. We investigated the localization of connexin43 in rat odontoblasts immuno-electron microscopically. The rats were transcardially fixed with 1% paraformaldehyde in 0.1M phosphate buffer, and mandibles were decalcified with 10% ethylenediamine tetraacetic acid. Pre-embedding method was carried out for immuno-electron microscopic analysis. Microscopically, gap junctions were localized between bodies of odontoblasts, and between bodies and processes of odontoblasts. The gap junctions were labeled with gold particles that indicated connexin43. These results suggest that gap junctions between odontoblasts are definitely composed of connexin43 in rats, and our methods used in this study is useful to investigate localization of connexin43 immuno-electron microscopically.

Di-4-ANEPPS Slows Cardiac Conduction Velocity

One of the most widely used voltage-sensitive dyes for optically mapping cardiac conduction is di-4-ANEPPS. Previous studies suggest that di-4-ANEPPS broadens the QRS; however, little is known about its effects on myocardial conduction. We hypothesized that di-4-ANEPPS suppresses cardiac conduction velocity (CV). CV was quantified in Langendorff-perfused guinea pig hearts using unipolar electrode and optical recordings. Electrode recordings from the anterior epicardium revealed that di-4-ANEPPS (7.5 µM) slowed cardiac transverse CV significantly from 23±4 cm/s to 18±3 cm/s (p<0.05). To investigate a possible concentration dependent effect of di-4-ANEPPS, CV and anisotropy was quantified using optical signals recorded from the anterior epicardium of both the right and left ventricle (RV, LV) at different concentrations of di-4-ANEPPS. Increasing the concentration of di-4-ANEPPS from 1.9 to 15 µM reduced transverse CV by 7±2 cm/s in the RV (p<0.05) (n=4) and 4±2 cm/s in the LV (p<0.05) (n=4). The decrease in longitudinal CV trended towards significance in both the RV (14±7 cm/s, p=0.08) and the LV (15±9 cm/s, p=0.07). The anisotropic ratio of CV was not affected by di-4-ANEPPS concentration. Connexin43 conductance was not significantly changed by di-4-ANEPPS at 15 µM evident from dual patch clamp experiments on HeLa cell-pairs overexpressing rat connexin43 (n=5), suggesting that decreased gap junction conductance is not the underlying mechanism. These data suggest that the perfusion of di-4-ANEPPS into whole heart tissue slows CV in both the right and left ventricles and this effect does not appear to be connexin related. Investigators should take the effect of di-4-ANEPPS on conduction into account when interpreting data obtained with this dye.

Effects of Sympathetic Nerve Stimulation on Ischemia-induced Ventricular Arrhythmias by Modulating Connexin43 in Rats

Increased cardiac sympathetic nerve activity is thought to contribute to ventricular tachyarrhythmias during acute myocardial ischemia (MI). However, the mechanism is not completely understood. This study investigated the effects of sympathetic nerve stimulation (SNS) on ventricular tachyarrhythmias and connexin43 (Cx43) during acute MI in rats. Ninety five male Wistar rats were randomly assigned into four groups receiving the following treatments: myocardial ischemia with sympathetic nerve stimulation (MI-SNS, n=25), sham-operation treated with sham stimulation (SO, n=20), myocardial ischemia with sham stimulation (MI, n=25), myocardial ischemia pretreated with sympathetic nerve stimulation (pSNS-MI, n=25). During the 30-min ischemia, the incidence of ventricular tachyarrhythmias, i.e., ventricular tachycardia or ventricular fibrillation (VT/VF) was increased in the MI-SNS group and decreased in the pSNS-MI group compared to that in the MI group (p<0.05 for both). The total amount of Cx43 protein was significantly decreased in the MI-SNS group but not in the MI group and the pSNS-MI group. The amount of phosphorylated Cx43 in the MI-SNS group was significantly lower compared to that in the SO group and the MI group (p<0.05). However, the amount of phosphorylated Cx43 was significantly increased in the pSNS-MI group compared to that in the MI group and the MI-SNS group (p<0.05). SNS promoted the degradation of Cx43 protein, especially the phosphorylated Cx43 protein, whereas pSNS inhibited the ischemia-induced loss of phosphorylated Cx43 during acute MI. These changes may be related to the pro- or anti-arrhythmic effect of SNS or pSNS during acute MI.

Effects of chronic renal failure on the expression of connexin 43 in the rat's corpus cavernosum

To explore the mechanism of chronic renal failure (CRF)-related erectile dysfunction (ED). CRF experimental models were established by 5/6 nephrectomy from male Sprague-Dawley rats. Both the rats from the control group (NCRF group, n=6) and the experimental group (CRF group, n=30) were injected with a low dose (80 microg/kg) of apomorphine in the 12th week after resection surgery to measure corresponding penile erections. Western blot method was thereafter conducted to measure the expression of connexin 43 (CX43) in the rat corpus cavernosum in the 12th week after the resection surgery. There was one death in the NCRF group and five in the CRF group. The penile erection ratio of the CRF group was 28% (7/25), whereas that of the NCRF group was 100% (5/5), which presents a significant difference between the two groups (P < 0.05). In terms of penile erection frequency, the average of the CRF group was 1.0 +/- 0.0, which was significantly different from that of the NCRF group (2.2 +/- 0.8) (P < 0.05). As for the expression of CX43 in the rat corpus cavernosum, a notable difference existed between the CRF group (0.21 +/- 0.07) and the NCRF group (0.53 +/- 0.27) (P < 0.01). CRF significantly reduces the erectile function of rats. A close correlation exists between the expression of CX43 in rats' corpus cavernosum and CRF-related ED.

Effect of Transjunctional KCl Gradients on the Spermine Inhibition of Connexin40 Gap Junctions

Spermine inhibits rat connexin40 (Cx40) gap junctions. Glutamate residues at positions 9 and 13 and a basic amino acid (HKH) motif at positions 15–17 on the amino terminal domain are essential for this inhibitory activity. Questions remain as to whether spermine occludes the channel within the ion permeation pathway. To examine this question, cis or trans [KCl] was systematically lowered and the equilibrium dissociation constants (Kd) and kinetics of unilateral spermine block on wild-type Cx40 gap junctions were determined. Asymmetric reductions in the trans [KCl] produced noticeable asymmetric shifts in the V1/2 and Gmin values that progressively resembled Gj-Vj relationships observed in heterotypic connexin gap junction combinations. As cis or trans [KCl] was reduced by 25%, 50%, or 75% relative to the spermine-containing side, the transjunctional voltage (Vj)-dependent Kd values increased or decreased, respectively. The spermine on-rates and off-rates, calculated from the junctional current decay and recovery time constants, were similarly affected. Hill coefficients for the spermine dose-response curves were ∼0.58, indicative of negative cooperativity and possible multiple spermine inhibitory sites. The equivalent “electrical distance” (δ) ranged from 0.61 at 25% cis [KCl] to 1.4 at 25% trans [KCl], with a Hill coefficient of 1.0. Symmetrical reductions in [KCl] resulted in intermediate decreases in the spermine Kds, indicative of a minor electrostatic effect and a more significant effect of the transjunctional KCl electrodiffusion potential on the spermine association and dissociation rates. These data are consistent with a single spermine molecule being sufficient to occlude the Cx40 gap junction channel within the KCl permeation pathway.

Changes in phosphorylation of connexin43 in rats during acute myocardial hypoxia and effects of antiarrhythmic peptide on the phosphorylation

In order to confirm the hypothesis that during acute hypoxia, the antiarrhythmic peptide (AAP10) could improve conductance by changing the phosphorylation state of connexin43 (Cx43), isolated perfused rat hearts were randomly divided into three groups: control, hypoxia and AAP10 (n=9 in each group). The change in Cx43 phosphorylation was tested by Western-blot; the distribution of Cx43 was observed by confocal immunofluorescence microscopy. Western-blot analysis revealed that the expression of total Cx43 protein was significantly decreased during acute hypoxia, while nonphosphorylated Cx43 (NP-Cx43) was unchanged. AAP10 could increase the expression of total Cx43 protein, but had no effects on the NP-Cx43 protein. Immunofluorescence study showed that during acute hypoxia, both total Cx43 and NP-Cx43 proteins were greatly decreased, while AAP10 only increased the expression of total Cx43 protein, but had no effect of the NP-Cx43 protein expression. These findings suggested that the decrease of intercellular communication may be associated with the reduction of phosphorylated Cx43 (p-Cx43) and translocation of NP-Cx43 from the surface of gap junction into intracellular pools during acute hypoxia. AAP10 can improve intercellular communication by enhancing phosphorylation of Cx43.

Inhibition of connexin 43 expression and function in cultured rat dental pulp cells by antisense oligonucleotide

Connexins are gap-junction proteins forming hexameric structures in the plasma membranes of adjacent cells, thereby creating intercellular channels. Connexin 43 (CX43) is expressed in pulp tissue. However, its function in dental pulp tissue has yet to be fully investigated. We have employed antisense oligonucleotides (AS) against rat CX43 to study the role of CX43 in dental pulp cells. Cultured dental pulp cells were treated with AS or sense (S) oligonucleotides. The number of cells in the AS-treated groups was approximately 1.3-fold that in the S-treated controls. Growth rates were significantly different between the AS- and S-treated groups at 48 h (P < 0.01). An alkaline phosphatase assay revealed that AS-treated pulp cells dramatically decreased at 48 h after AS incorporation, whereas S-treated pulp cells showed no marked changes. Western blot analysis revealed that heat-shock protein 25 was highly expressed in S-treated cells but was only weakly expressed in AS-treated cells at 48 h. Furthermore, AS-treated cells highly expressed CX45, whereas S-treated cells exhibited high expression of CX32. These results suggest that CX43 is involved in cell growth, mineralization, and differentiation to odontoblasts in rat pulp cells, and that CX43 plays the opposite role to that of CX45.

An amino-terminal lysine residue of rat connexin40 that is required for spermine block.

Spermine blocks connexin40 (Cx40) gap junctions, and two cytoplasmic amino-terminal domain glutamate residues are essential for this inhibitory activity. To further examine the molecular basis for block, we mutated a portion of a basic amino acid (HKH) motif on the Cx40 amino-terminal domain. Replacement of the Cx40 H15 + K16 residues with the Q15 + A16 sequence native to spermine-insensitive connexin43 (Cx43) gap junctions increased the equilibrium dissociation constant (K(d)) and reduced the maximum inhibition by spermine. The corresponding electrical distance (delta) approximation was decreased by about 50%. The transjunctional voltage (V(j))-dependent gating of homotypic Cx40 H15Q + K16A mutant gap junctions was also significantly reduced. The minimum normalized steady-state junctional conductance (G(min)) increased from 0.17 to 0.72, with an increase in the half-inactivation voltage from 48 to 60 mV. However, the unitary junctional conductance (gamma(j); 160 pS) was only slightly altered, and the relative cation/anion conductance and permeability ratios were unchanged from wild-type Cx40 gap junction channels. The relative K(+)/Cl(-) permeability (P(K)/P(Cl)) ratio increased from six to ten when [KCl] was reduced to 25% of normal. These data suggest that the HKH motif at positions 15-17 is important to the conformational structure of the putative voltage sensor and spermine receptor of Cx40, without causing significant alteration of the electrostatic surface charge potentials that contribute to the ion selectivity of this gap junction channel.

Expression of the rat connexin 39 (rCx39) gene in myoblasts and myotubes in developing and regenerating skeletal muscles: an in situ hybridization study

We report a detailed analysis of the expression pattern of the recently identified rat connexin gene, named rat connexin 39 (rCx39), both during embryonic development and in adult life. Qualitative and quantitative reverse transcription/polymerase chain reaction analysis showed intense expression of rCx39 restricted to differentiating skeletal muscles, with a peak of expression detected at 18 days of embryonic life, followed by a rapid decline to undetectable levels within the first week of postnatal life. A combination of the in situ hybridization technique for the detection of rCx39 mRNA and immunohistochemistry for myogenin, a myoblast-specific marker, allowed us to establish that the mRNA for this connexin was expressed in myogenin-positive myoblasts and early myotubes but disappeared in mature myotubes. Moreover, in adult animals, rCx39 mRNA was expressed in myogenic cells involved in skeletal myofiber regeneration following a crush injury. This is the first case of a connexin being mainly expressed in the myogenic cell lineage. The information presented should pave the way to novel molecular approaches in studies on the role of connexin-based gap-junctional communication in skeletal muscle differentiation and regeneration.

Amino terminal glutamate residues confer spermine sensitivity and affect voltage gating and channel conductance of rat connexin40 gap junctions.

Connexin40 (Cx40) contains a specific binding site for spermine (affinity approximately 100 microm) whereas connexin43 (Cx43) is unaffected by identical concentrations of intracellular spermine. Replacement of two unique glutamate residues, E9 and E13, from the cytoplasmic amino terminal domain of Cx40 with the corresponding lysine residues from Cx43 eliminated the block by 2 mm spermine, reduced the transjunctional voltage (V(j)) gating sensitivity, and reduced the unitary conductance of this Cx40E9,13K gap junction channel protein. The single point mutations, Cx40E9K and Cx40E13K, predominantly affected the residual conductance state (G(min)) and V(j) gating properties, respectively. Heterotypic pairing of Cx40E9,13K with wild-type Cx40 in murine neuro2A (N2A) cells produced a strongly rectifying gap junction reminiscent of the inward rectification properties of the Kir (e.g. Kir2.x) family of potassium channels. The reciprocal Cx43K9,13E mutant protein exhibited reduced V(j) sensitivity, but displayed much less rectification in heterotypic pairings with wtCx43, negligible changes in the unitary channel conductance, and remained insensitive to spermine block. These data indicate that the connexin40 amino terminus may form a critical cytoplasmic pore-forming domain that serves as the receptor for V(j)-dependent closure and block by intracellular polyamines. Functional reciprocity between Cx40 and Cx43 gap junctions involves other amino acid residues in addition to the E or K 9 and 13 loci located on the amino terminal domain of these two connexins.

Cardiac connexins Cx43 and Cx45: formation of diverse gap junction channels with diverse electrical properties.

HeLa cells expressing rat connexin43 (Cx43) and/or mouse Cx45 were studied with the dual voltage-clamp technique. Different types of cell pairs were established and their gap junction properties determined, i.e. the dependence of the instantaneous and steady-state conductances (gj,inst, gj,ss) on the transjunctional voltage (Vj) and the kinetics of inactivation of the gap junction current (Ij). Pairs of singly transfected cells showed homogeneous behaviour at both Vj polarities. Homotypic Cx43-Cx43 and Cx45-Cx45 cell pairs yielded distinct symmetrical functions gj,inst=f(Vj) and gj,ss=f(Vj). Heterotypic Cx43-Cx45 preparations exhibited asymmetric functions gj,inst=f(Vj) and gj,ss=f(Vj) suggesting that connexons Cx43 and Cx45 gate with positive and negative Vj, respectively. Preparations containing a singly (Cx43 or Cx45) or doubly (Cx43/45) transfected cell showed quasi-homogeneous behaviour at one Vj polarity and heterogeneous behaviour at the other polarity. The former yielded Boltzmann parameters intermediate between those of Cx43-Cx43, Cx45-Cx45 and Cx43-Cx45 preparations; the latter could not be explained by homotypic and heterotypic combinations of homomeric connexons. Each pair of doubly transfected cells (Cx43/Cx45) yielded unique functions gj,inst=f(Vj) and gj,ss=f(Vj). This can not be explained by combinations of homomeric connexons. We conclude that Cx43 and Cx45 form homomeric-homotypic, homomeric-heterotypic channels as well as heteromeric-homotypic and heteromeric-heterotypic channels. This has implications for the impulse propagation in specific areas of the heart.

Analysis of the rat connexin 43 proximal promoter in neonatal cardiomyocytes

Altered transcriptional control is likely to contribute to the down-regulation of connexin 43 (Cx43) expression observed in many forms of heart disease. However, little is known about the factors regulating Cx43 transcription in the heart under (patho)physiological conditions. Therefore, a systematic study of rat Cx43 (rCx43) proximal promoter regulation in rat primary neonatal ventricular cardiomyocytes (NCM) and, for comparison, different cell types was initiated. Luciferase assays revealed that, in NCM, the proximal promoter is preserved in a conserved region extending from 148 nucleotides upstream towards 281 nucleotides downstream relative to the transcription initiation site (TIS). Further deletional analysis suggested the involvement of four putative Sp- and two AP1-binding sites. The binding of both Sp1 and Sp3 to the Sp-binding elements and AP1 to the AP1-binding elements was demonstrated by electrophoretic mobility shift assays (EMSA). Promoter–luciferase assays using the natural rCx43 proximal promoter and mutated derivatives in NCM, HL-1 and A7r5 cells revealed that all sites contribute to basal promoter activity. Trans-activation of the Cx43 proximal promoter with Sp1 and Sp3 in Drosophila Schneider line 2 (SL2) cells demonstrated that Sp1 and, to a lesser extent, Sp3 determine rCx43 promoter activation. Thus Sp1, Sp3 and AP1 determine basal Cx43 expression. In addition, we studied the effect of the cardiac transcription factor Nkx2.5 on Cx43 regulation. NCM were infected with adenovirus encoding either β-galactosidase (control) or Nkx2.5. Cx43 protein and mRNA were significantly decreased after Nkx2.5 infection as shown by Western and Northern blot analyses. Promoter–reporter assays demonstrated that the rCx43 promoter was down-regulated approximately twofold upon Nkx2.5 overexpression. Therefore, in NCM, Nkx2.5 appears to play a role in the regulation of Cx43 expression.

No ischemic preconditioning in heterozygous connexin43-deficient mice.

Protein kinase Cepsilon (PKCepsilon) plays a central role in ischemic preconditioning (IP) in mice and rabbits, and activated PKCepsilon colocalizes with and phosphorylates connexin43 (Cx43) in rats and humans. Whether or not Cx43 contributes to the mechanism(s) of IP in vivo is yet unknown. Therefore, wild-type (n = 8) and heterozygous Cx43-deficient mice (n = 8) were subjected to 30 min occlusion and 120 min reperfusion of the left anterior descending coronary artery. IP was induced by one cycle of 5 min occlusion and 10 min reperfusion (n = 8/8 mice) before the sustained occlusion. Infarct size was reduced by IP in wild-type mice [11.3 +/- 3.4% vs. 23.7 +/- 7.2% of the left ventricle (LV), P < 0.05] but not in Cx43-deficient mice (26.0 +/- 6.0% vs. 25.1 +/- 3.8% of LV). Also, three cycles of 5 min occlusion and 10 min reperfusion (n = 5) did not induce protection in Cx43-deficient mice (27.6 +/- 5.5 % of LV). Thus Cx43 contributes to the protection of IP in mice in vivo.

PH gating of lens fibre connexins

Chemical gating of gap junction channels by intracellular pH may be an important mechanism for the physiological regulation of cell-cell coupling. In the ocular lens, pH gating of gap junction channels has been implicated as a possible cause of cataract in diabetics. To address this question further, I determined the pH dependence of the rat connexin (Cx)-46 and ovine Cx49 in transfected HeLa cells using the pH-clamp technique during dual whole-cell recording. pH gating for both connexins was fast and reversible. The apparent p K(a) (p K(a,app)) was 6.66 +/- 0.01 and the Hill coefficient ( n) 6.8 +/- 1.8 for Cx49, and for Cx46 6.8 +/- 0.01 and 2.2 +/- 0.15, respectively. C-terminal truncation of Cx46 by 163 aa did not abolish the pH sensitivity but shifted the p K(a,app) to 6.6 +/- 0.01. This finding is inconsistent with the ball-and-chain model proposed for Cx43. Voltage gating of Cx46 channels was also not altered by truncation or acidic pH, indicating that the two gating mechanisms are functionally and possibly structurally separate. The data also imply a significant role of pH gating for lens pathophysiology. For the normal pH range in the lens cortex (pH 6.8-7.2) most gap junction channels will be open. However, mild acidification will reduce gap junctional coupling significantly, especially for Cx50 channels. Localized closure of gap junction channels will disrupt lens transport and thus may contribute to the tissue damage observed in diabetic lenses.

Ionic Blockade of the Rat Connexin40 Gap Junction Channel by Large Tetraalkylammonium Ions

The rat connexin40 gap junction channel is permeable to monovalent cations including tetramethylammonium and tetraethylammonium ions. Larger tetraalkyammonium (TAA +) ions beginning with tetrabutylammonium (TBA +) reduced KCl junctional currents disproportionately. Ionic blockade by tetrapentylammonium (TPeA +) and tetrahexylammonium (THxA +) ions were concentration- and voltage-dependent and occurred only when TAA + ions were on the same side as net K + efflux across the junction, indicative of block of the ionic permeation pathway. The voltage-dependent dissociation constants ( K m( V j)) were lower for THxA + than TPeA +, consistent with steric effects within the pore. The K m –V j relationships for TPeA + and THxA + were fit with different reaction rate models for a symmetrical (homotypic) connexin gap junction channel and were described by either a one- or two-site model that assumed each ion traversed the entire V j field. Bilateral addition of TPeA + ions confirmed a common site of interaction within the pore that possessed identical K m( V j) values for cis–trans concentrations of TPeA + ions as indicated by the modeled I–V relations and rapid channel block that precluded unitary current measurements. The TAA + block of K + currents and bilateral TPeA + interactions did not alter V j-gating of Cx40 gap junctions. N-octyl-tributylammonium and -triethylammonium also blocked rCx40 channels with higher affinity and faster kinetics than TBA + or TPeA +, indicative of a hydrophobic site within the pore near the site of block.

A dominant negative mutation of neuronal connexin 36 that blocks intercellular permeability

Rat connexin 36 (Cx36) was mutated by substituting serine for cysteine at residue 231 (C231S) and the mutant’s effect on the subcellular localization of wild-type Cx36 and the intercellular permeability that it confers was determined in human HeLa and rat PC12 cells. Cells transfected with the mutant or wild-type Cx36 cDNA expressed the expected 36 kDa protein and Cx36 immunoreactivity. Co-immunoprecipitation experiments with monkey COS-7 cells transiently transfected with both mutant and wild-type Cx36 cDNAs demonstrated that the mutant protein bound to the wild-type. Double immunofluorescence microscopy of stably transfected HeLa cells demonstrated that mutant Cx36 blocked the transport of the wild-type Cx36 to the cell membrane, primarily by trapping it in the endoplasmic reticulum around the nucleus. Coexpression of the mutant Cx36 with the wild-type protein abolished the ability of the latter to permit dye transfer in both HeLa and PC12 cells. The findings are the first demonstration of a mutation of Cx36 that inhibits wild-type Cx36 function in mammalian cells.