Plakophilin-2 Haploinsufficiency Causes Calcium Handling Deficits and Modulates the Cardiac Response Towards Stress.

Human variants in plakophilin-2 (PKP2) associate with most cases of familial arrhythmogenic cardiomyopathy (ACM). Recent studies show that PKP2 not only maintains intercellular coupling, but also regulates transcription of genes involved in Ca2+ cycling and cardiac rhythm. ACM penetrance is low and it remains uncertain, which genetic and environmental modifiers are crucial for developing the cardiomyopathy. In this study, heterozygous PKP2 knock-out mice (PKP2-Hz) were used to investigate the influence of exercise, pressure overload, and inflammation on a PKP2-related disease progression. In PKP2-Hz mice, protein levels of Ca2+-handling proteins were reduced compared to wildtype (WT). PKP2-Hz hearts exposed to voluntary exercise training showed right ventricular lateral connexin43 expression, right ventricular conduction slowing, and a higher susceptibility towards arrhythmias. Pressure overload increased levels of fibrosis in PKP2-Hz hearts, without affecting the susceptibility towards arrhythmias. Experimental autoimmune myocarditis caused more severe subepicardial fibrosis, cell death, and inflammatory infiltrates in PKP2-Hz hearts than in WT. To conclude, PKP2 haploinsufficiency in the murine heart modulates the cardiac response to environmental modifiers via different mechanisms. Exercise upon PKP2 deficiency induces a pro-arrhythmic cardiac remodeling, likely based on impaired Ca2+ cycling and electrical conduction, versus structural remodeling. Pathophysiological stimuli mainly exaggerate the fibrotic and inflammatory response.

Connexins and pannexins in the immune system and lymphatic organs.

Connexin43 and pannexin1 are found in immune cells. While gap junctional communication has been demonstrated between immune cells, hemichannels have been implicated in many cellular functions. Among the functions involved as being connexin dependent and pannexin dependent are cell migration, phagocytosis, antigen presentation, T-cell reactivity and B-cell responses. Surprisingly, many of these connexin-related and pannexin-related functions are not recapitulated in in vivo models. This is leading to a reevaluation of the role of these proteins in immune function.

Connexin43 is dispensable for phagocytosis.

Macrophages that lack connexin43 (Cx43), a gap junction protein, have been reported to exhibit dramatic deficiencies in phagocytosis. In this study, we revisit these findings using well-characterized macrophage populations. Cx43 knockout (Cx43(-/-)) mice die soon after birth, making the harvest of macrophages from adult Cx43(-/-) mice problematic. To overcome this obstacle, we used several strategies: mice heterozygous for the deletion of Cx43 were crossed to produce Cx43(+/+) (wild type [WT]) and Cx43(-/-) fetuses. Cells isolated from 12- to 14-d fetal livers were used to reconstitute irradiated recipient animals. After reconstitution, thioglycollate-elicited macrophages were collected by peritoneal lavage and bone marrow was harvested. Bone marrow cells and, alternatively, fetal liver cells were cultured in media containing M-CSF for 7-10 d, resulting in populations of cells that were >95% macrophages based on flow cytometry. Phagocytic uptake was detected using flow cytometric and microscopic techniques. Quantification of phagocytic uptake of IgG-opsonized sheep erythrocytes, zymosan particles, and Listeria monocytogenes failed to show any significant difference between WT and Cx43(-/-) macrophages. Furthermore, the use of particles labeled with pH-sensitive dyes showed equivalent acidification of phagosomes in both WT and Cx43(-/-) macrophages. Our findings suggest that modulation of Cx43 levels in cultured macrophages does not have a significant impact on phagocytosis.

Interactions between ankyrin-G, Plakophilin-2, and Connexin43 at the cardiac intercalated disc.

RATIONALE: The early description of the intercalated disc defined 3 structures, all of them involved in cell-cell communication: desmosomes, gap junctions, and adherens junctions. Current evidence demonstrates that molecules not involved in providing a physical continuum between cells also populate the intercalated disc. Key among them is the voltage-gated sodium channel complex. An important component of this complex is the cytoskeletal adaptor protein Ankyrin-G (AnkG). OBJECTIVE: To test the hypothesis that AnkG partners with desmosome and gap junction molecules and exerts a functional effect on intercellular communication in the heart. METHODS AND RESULTS: We used a combination of microscopy, immunochemistry, patch-clamp, and optical mapping to assess the interactions between AnkG, Plakophilin-2, and Connexin43. Coimmunoprecipitation studies from rat heart lysate demonstrated associations between the 3 molecules. With the use of siRNA technology, we demonstrated that loss of AnkG expression caused significant changes in subcellular distribution and/or abundance of PKP2 and Connexin43 as well as a decrease in intercellular adhesion strength and electric coupling. Regulation of AnkG and of Na(v)1.5 by Plakophilin-2 was also demonstrated. Finally, optical mapping experiments in AnkG-silenced cells demonstrated a shift in the minimal frequency at which rate-dependence activation block was observed. CONCLUSIONS: These experiments support the hypothesis that AnkG is a key functional component of the intercalated disc at the intersection of 3 complexes often considered independent: the voltage-gated sodium channel, gap junctions, and the cardiac desmosome. Possible implications to the pathophysiology of inherited arrhythmias (such as arrhythmogenic right ventricular cardiomyopathy) are discussed.

Regulation of cardiac inward rectifier potassium current (I(K1)) by synapse-associated protein-97.

Synapse-associated protein-97 (SAP97) is a membrane-associated guanylate kinase scaffolding protein expressed in cardiomyocytes. SAP97 has been shown to associate and modulate voltage-gated potassium (Kv) channel function. In contrast to Kv channels, little information is available on interactions involving SAP97 and inward rectifier potassium (Kir2.x) channels that underlie the classical inward rectifier current, I(K1). To investigate the functional effects of silencing SAP97 on I(K1) in adult rat ventricular myocytes, SAP97 was silenced using an adenoviral short hairpin RNA vector. Western blot analysis showed that SAP97 was silenced by approximately 85% on day 3 post-infection. Immunostaining showed that Kir2.1 and Kir2.2 co-localize with SAP97. Co-immunoprecipitation (co-IP) results demonstrated that Kir2.x channels associate with SAP97. Voltage clamp experiments showed that silencing SAP97 reduced I(K1) whole cell density by approximately 55%. I(K1) density at -100 mV was -1.45 +/- 0.15 pA/picofarads (n = 6) in SAP97-silenced cells as compared with -3.03 +/- 0.37 pA/picofarads (n = 5) in control cells. Unitary conductance properties of I(K1) were unaffected by SAP97 silencing. The major mechanism for the reduction of I(K1) density appears to be a decrease in Kir2.x channel abundance. Furthermore, SAP97 silencing impaired I(K1) regulation by beta(1)-adrenergic receptor (beta1-AR) stimulation. In control, isoproterenol reduced I(K1) amplitude by approximately 75%, an effect that was blunted following SAP97 silencing. Our co-IP data show that beta1-AR associates with SAP97 and Kir2.1 and also that Kir2.1 co-IPs with protein kinase A and beta1-AR. SAP97 immunolocalizes with protein kinase A and beta1-AR in the cardiac myocytes. Our results suggest that in cardiac myocytes SAP97 regulates surface expression of channels underlying I(K1), as well as assembles a signaling complex involved in beta1-AR regulation of I(K1).

Novel pharmacophores of connexin43 based on the "RXP" series of Cx43-binding peptides.

Gap junction pharmacology is a nascent field. Previous studies have identified molecules that enhance intercellular communication, and may offer potential for innovative antiarrhythmic therapy. However, their specific molecular target(s) and mechanism(s) of action remain unknown. Previously, we identified a 34-aa peptide (RXP-E) that binds the carboxyl terminal domain of Cx43 (Cx43CT) and prevents cardiac gap junction closure and action potential propagation block. These results supported the feasibility of a peptide-based pharmacology to Cx43, but the structure of the core active element in RXP-E, an essential step for pharmacological development, remained undefined. Here, we used a combination of molecular modeling, surface plasmon resonance, nuclear magnetic resonance and patch-clamp strategies to define, for the first time, a unique ensemble of pharmacophores that bind Cx43CT and prevent closure of Cx43 channels. Two particular molecules are best representatives of this family: a cyclized heptapeptide (called CyRP-71) and a linear octapeptide of sequence RRNYRRNY. These 2 small compounds offer the first structural platform for the design of Cx43-interacting gap junction openers. Moreover, the structure of these compounds offers an imprint of a region of Cx43CT that is fundamental to gap junction channel function.

Loss of plakophilin-2 expression leads to decreased sodium current and slower conduction velocity in cultured cardiac myocytes.

RATIONALE: Plakophilin-2 (PKP2) is an essential component of the cardiac desmosome. Recent data show that it interacts with other molecules of the intercalated disc. Separate studies show preferential localization of the voltage-gated sodium channel (Na(V)1.5) to this region. OBJECTIVE: To establish the association of PKP2 with sodium channels and its role on action potential propagation. METHODS AND RESULTS: Biochemical, patch clamp, and optical mapping experiments demonstrate that PKP2 associates with Na(V)1.5, and that knockdown of PKP2 expression alters the properties of the sodium current, and the velocity of action potential propagation in cultured cardiomyocytes. CONCLUSIONS: These results emphasize the importance of intermolecular interactions between proteins relevant to mechanical junctions, and those involved in electric synchrony. Possible relevance to the pathogenesis of arrhythmogenic right ventricular cardiomyopathy is discussed.

RXP-E: a connexin43-binding peptide that prevents action potential propagation block.

Gap junctions provide a low-resistance pathway for cardiac electric propagation. The role of GJ regulation in arrhythmia is unclear, partly because of limited availability of pharmacological tools. Recently, we showed that a peptide called "RXP-E" binds to the carboxyl terminal of connexin43 and prevents chemically induced uncoupling in connexin43-expressing N2a cells. Here, pull-down experiments show RXP-E binding to adult cardiac connexin43. Patch-clamp studies revealed that RXP-E prevented heptanol-induced and acidification-induced uncoupling in pairs of neonatal rat ventricular myocytes. Separately, RXP-E was concatenated to a cytoplasmic transduction peptide (CTP) for cytoplasmic translocation (CTP-RXP-E). The effect of RXP-E on action potential propagation was assessed by high-resolution optical mapping in monolayers of neonatal rat ventricular myocytes, containing approximately 20% of randomly distributed myofibroblasts. In contrast to control experiments, when heptanol (2 mmol/L) was added to the superfusate of monolayers loaded with CTP-RXP-E, action potential propagation was maintained, albeit at a slower velocity. Similarly, intracellular acidification (pH(i) 6.2) caused a loss of action potential propagation in control monolayers; however, propagation was maintained in CTP-RXP-E-treated cells, although at a slower rate. Patch-clamp experiments revealed that RXP-E did not prevent heptanol-induced block of sodium currents, nor did it alter voltage dependence or amplitude of Kir2.1/Kir2.3 currents. RXP-E is the first synthetic molecule known to: (1) bind cardiac connexin43; (2) prevent heptanol and acidification-induced uncoupling of cardiac gap junctions; and (3) preserve action potential propagation among cardiac myocytes. RXP-E can be used to characterize the role of gap junctions in the function of multicellular systems, including the heart.

Connexin43 remodeling caused by inhibition of plakophilin-2 expression in cardiac cells.

Desmosomes and gap junctions are distinct structural components of the cardiac intercalated disc. Here, we asked whether the presence of plakophilin (PKP)2, a component of the desmosome, is essential for the proper function and distribution of the gap junction protein connexin (Cx)43. We used RNA silencing technology to decrease the expression of PKP2 in cardiac cells (ventricular myocytes, as well as epicardium-derived cells) obtained from neonatal rat hearts. We evaluated the content, distribution, and function of Cx43 gap junctions. Our results show that loss of PKP2 expression led to a decrease in total Cx43 content, a significant redistribution of Cx43 to the intracellular space, and a decrease in dye coupling between cells. Separate experiments showed that Cx43 and PKP2 can coexist in the same macromolecular complex. Our results support the notion of a molecular crosstalk between desmosomal and gap junction proteins. The results are discussed in the context of arrhythmogenic right ventricular cardiomyopathy, an inherited disease involving mutations in desmosomal proteins, including PKP2.

Adenoviral expression of IKs contributes to wavebreak and fibrillatory conduction in neonatal rat ventricular cardiomyocyte monolayers.

Previous studies have shown that the gating kinetics of the slow component of the delayed rectifier K(+) current (I(Ks)) contribute to postrepolarization refractoriness in isolated cardiomyocytes. However, the impact of such kinetics on arrhythmogenesis remains unknown. We surmised that expression of I(Ks) in rat cardiomyocyte monolayers contributes to wavebreak formation and facilitates fibrillatory conduction by promoting postrepolarization refractoriness. Optical mapping was performed in 44 rat ventricular myocyte monolayers infected with an adenovirus carrying the genomic sequences of KvLQT1 and minK (molecular correlates of I(Ks)) and 41 littermate controls infected with a GFP adenovirus. Repetitive bipolar stimulation was applied at increasing frequencies, starting at 1 Hz until loss of 1:1 capture or initiation of reentry. Action potential duration (APD) was significantly shorter in I(Ks)-infected monolayers than in controls at 1 to 3 Hz (P<0.05), whereas differences at higher pacing frequencies did not reach statistical significance. Stable rotors occurred in both groups, with significantly higher rotation frequencies, lower conduction velocities, and shorter action potentials in the I(Ks) group. Wavelengths in the latter were significantly shorter than in controls at all rotation frequencies. Wavebreaks leading to fibrillatory conduction occurred in 45% of the I(Ks) reentry episodes but in none of the controls. Moreover, the density of wavebreaks increased with time as long as a stable source sustained the fibrillatory activity. These results provide the first demonstration that I(Ks)-mediated postrepolarization refractoriness can promote wavebreak formation and fibrillatory conduction during pacing and sustained reentry and may have important implications in tachyarrhythmias.

Electrotonic myofibroblast-to-myocyte coupling increases propensity to reentrant arrhythmias in two-dimensional cardiac monolayers.

In pathological conditions such as ischemic cardiomyopathy and heart failure, differentiation of fibroblasts into myofibroblasts may result in myocyte-fibroblast electrical coupling via gap junctions. We hypothesized that myofibroblast proliferation and increased heterocellular coupling significantly alter two-dimensional cardiac wave propagation and reentry dynamics. Co-cultures of myocytes and myofibroblasts from neonatal rat ventricles were optically mapped using a voltage-sensitive dye during pacing and sustained reentry. The myofibroblast/myocyte ratio was changed systematically, and junctional coupling of the myofibroblasts was reduced or increased using silencing RNAi or adenoviral overexpression of Cx43, respectively. Numerical simulations in two-dimensional models were used to quantify the effects of heterocellular coupling on conduction velocity (CV) and reentry dynamics. In both simulations and experiments, reentry frequency and CV diminished with larger myofibroblast/myocyte area ratios; complexity of propagation increased, resulting in wave fractionation and reentry multiplication. The relationship between CV and coupling was biphasic: an initial decrease in CV was followed by an increase as heterocellular coupling increased. Low heterocellular coupling resulted in fragmented and wavy wavefronts; at high coupling wavefronts became smoother. Heterocellular coupling alters conduction velocity, reentry stability, and complexity of wave propagation. The results provide novel insight into the mechanisms whereby electrical myocyte-myofibroblast interactions modify wave propagation and the propensity to reentrant arrhythmias.

Identification of a novel peptide that interferes with the chemical regulation of connexin43.

The carboxyl-terminal domain of connexin43 (Cx43CT) is involved in various intra- and intermolecular interactions that regulate gap junctions. Here, we used phage display to identify novel peptidic sequences that bind Cx43CT and modify Cx43 regulation. We found that Cx43CT binds preferentially to peptides containing a sequence RXP, where X represents any amino acid and R and P correspond to the amino acids arginine and proline, respectively. A biased "RXP library" led to the identification of a peptide (dubbed "RXP-E") that bound Cx43CT with high affinity. Nuclear magnetic resonance data showed RXP-E-induced shifts in the resonance peaks of residues 343 to 346 and 376 to 379 of Cx43CT. Patch-clamp studies revealed that RXP-E partially prevented octanol-induced and acidification-induced uncoupling in Cx43-expressing cells. Moreover, RXP-E increased mean open time of Cx43 channels. The full effect of RXP-E was dependent on the integrity of the CT domain. These data suggest that RXP-based peptides could serve as tools to help determine the role of Cx43 as a regulator of function in conditions such as ischemia-induced arrhythmias.

Evidence for the presence of a free C-terminal fragment of cx43 in cultured cells.

Migration of the gap junction protein connexin 43 (Cx43) in SDS-PAGE yields 2 to 4 distinct bands, detectable in the 40-47 kDa range. Here, we show that antibodies against the carboxy-terminal domain of Cx43 recognized an additional 20-kDa product. This protein was detected in some culture cell lysates. The presence of the 20-kDa band was not prevented by the use of protease inhibitors (Complete(R) and phenylmethylsulfonyl fluoride (PMSF), 1-5 mM). The band was absent from cells treated with Cx43-specific RNAi, and from those derived from Cx43-deficient mice, indicating that this Cx43-immunoreactive protein is a product of the Cx43 gene. Treatment of CHO cells with cyclosporin A caused a reduction in the amount of full-length Cx43 and a concomitant increase in the amount of the 20-kDa band. Overall, our data show that a fraction of the Cx43-immunoreactive protein pool within a given cell may correspond to a C-terminal fragment of the protein.

Kir2.3 isoform confers pH sensitivity to heteromeric Kir2.1/Kir2.3 channels in HEK293 cells.

BACKGROUND: Data on pH regulation of the cardiac potassium current I(K1) suggest species-dependent differences in the molecular composition of the underlying Kir2 channel proteins. OBJECTIVE: The purpose of this study was to test the hypothesis that the presence of the Kir2.3 isoform in heterotetrameric channels modifies channel sensitivity to pH. METHODS: Voltage clamp was performed on HEK293 cells stably expressing guinea pig Kir2.1 and/or Kir2.3 isoforms and on sheep cardiac ventricular myocytes at varying extracellular pH (pH(o)) and in the presence of CO(2) to determine the sensitivity of macroscopic currents to pH. Single-channel activity was recorded from the HEK293 stables to determine the mechanisms of the changes in whole cell current. RESULTS: Biophysical characteristics of whole-cell and single-channel currents in Kir2.1/Kir2.3 double stables displayed properties attributable to isoform heteromerization. Whole-cell Kir2.1/Kir2.3 currents rectified in a manner reminiscent of Kir2.1 but were significantly inhibited by extracellular acidification in the physiologic range (pK(a) approximately 7.4). Whole-cell currents were more sensitive to a combined extracellular and intracellular acidification produced by CO(2). At pH(o) = 6.0, unitary conductances of heteromeric channels were reduced. Ovine cardiac ventricular cell I(K1) was pH(o) and CO(2) sensitive, consistent with the expression of Kir2.1 and Kir2.3 in this species. CONCLUSION: Kir2.1 and Kir2.3 isoforms form heteromeric channels in HEK293. The presence of Kir2.3 subunit(s) in heteromeric channels confers pH sensitivity to the channels. The single and double stable cells presented in this study are useful models for studying physiologic regulation of heteromeric Kir2 channels in mammalian cells.

Molecular composition of the intercalated disc in a spontaneous canine animal model of arrhythmogenic right ventricular dysplasia/cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by ventricular arrhythmias, sudden death, and fatty or fibrofatty replacement of right ventricular myocytes. Recent studies have noted an association between human ARVD/C and molecular remodeling of intercalated disc structures. However, progress has been constrained by limitations inherent to human studies. OBJECTIVE: We studied the molecular composition of the intercalated disc structure in a naturally occurring animal model of ARVD/C (Boxer dogs). METHODS: We studied hearts from 12 Boxers with confirmed ARVD/C and 2 controls. Ventricular sections from 4 animals were examined by immunofluorescent microscopy. Frozen tissue samples were used for Western blot analysis. Proteins investigated were N-cadherin, plakophilin 2, desmoplakin, plakoglobin, desmin, and connexin 43 (Cx43). RESULTS: In control dogs, all proteins tested by immunofluorescence analysis yielded intense localized signals at sites of end-to-end cell apposition. In contrast, myocardial tissues from ARVD/C-afflicted Boxers showed preservation of N-cadherin staining but loss of detectable signal for Cx43 at the intercalated disc location. Western blots indicated that the Cx43 protein was still present in the samples. Gene sequencing analysis showed no mutations in desmoplakin, plakoglobin, Cx43, or plakophilin 2. CONCLUSION: Mutation(s) responsible for ARVD/C in Boxers lead, directly or indirectly, to severe modifications of mechanical and electrical cell-cell interactions. Furthermore, significant reduction in gap junction formation may promote a substrate for malignant ventricular arrhythmias. This model may help to advance our understanding of the molecular basis, pathophysiology, and potential therapeutic approach to patients with ARVD/C.

High incidence of cardiac malformations in connexin40-deficient mice.

Gap junctions are intercellular channels formed by oligomerization of a protein called connexin (Cx). The heart expresses at least three connexin isotypes: Cx40, Cx43, and Cx45. A possible role for Cx40 in cardiac morphogenesis remains to be determined. We have characterized the anatomy and histology of fetal and newborn hearts obtained from crossing Cx40-deficient mice of mixed genetic background (C57BL/6x129Sv). Hearts were serial-sectioned (5 microm) along the coronal plane, stained with hematoxylin-eosin, and visualized by conventional light microscopy. Cardiac malformations in mice lacking Cx40 in one allele (Cx40+/-) included bifid atrial appendage, ventricular septal defect, tetralogy of Fallot (TOF), and an aortic arch abnormality. In Cx40-/- mice resulting from crossing of Cx40+/- mice, the most common cardiac malformations were double-outlet right ventricle (DORV), TOF, and endocardial cushion defects. Overall incidence of cardiac malformations was 6/33 (18%) in Cx40+/- mice and 4/12 (33%) in Cx40-/- mice. No cardiac malformations were observed in 15 wild-type mice studied. In addition, we examined 39 hearts from offspring of Cx40-/- matings. Frequency of cardiac malformations was even higher in this group (44%). Over one third of the hearts (14 of 39) showed conotruncal malformations corresponding to either DORV or TOF. Endocardial cushion defects were found in 3 out of 39 hearts. Our results suggest that Cx40 participates in cardiac morphogenesis, likely in association with other (unknown) products whose expression may vary with the genetic background of the mice.

Modifications in the biophysical properties of connexin43 channels by a peptide of the cytoplasmic loop region.

Connexin43 (Cx43) channels reside in at least 3 states: closed, open, or residual. It is hypothesized that the residual state results from the interaction of an intracellular "gating element" with structures at the vestibule of the pore. Recently, we showed in vitro that there is an intramolecular interaction of the carboxyl-terminal domain (referred to as "CT") with a region in the cytoplasmic loop of Cx43 (amino acids 119 to 144; referred to as "L2"). Here, we assessed whether the L2 region was able to interact with the gating particle in a functional channel. Cx43 channels were recorded in the presence of a peptide corresponding to the L2 region, delivered via the patch pipette. This manipulation did not modify unitary conductance, but decreased the frequency of transitions into the residual state, prolonged open time, and altered the voltage dependence of the channel in a manner analogous to that observed after truncation of the CT domain. The latter correlated with the ability of the peptide to bind to the CT domain, as determined by mirror resonance spectroscopy. Overall, we propose that the L2 acts as a "receptor" that interacts with a flexible intracellular gating element during channel gating. The full text of this article is available online at http://circres.ahajournals.org.

Role of the carboxyl terminal of connexin43 in transjunctional fast voltage gating.

Previous studies show that chemical regulation of connexin43 (Cx43) gap junction channels depends on the integrity of the carboxyl terminal (CT) domain. Experiments using Xenopus oocytes show that truncation of the CT domain alters the time course for current inactivation; however, correlation with the behavior of single Cx43 channels has been lacking. Furthermore, whereas chemical gating is associated with a "ball-and-chain" mechanism, there is no evidence whether transjunctional voltage regulation for Cx43 follows a similar model. We provide data on the properties of transjunctional currents from voltage-clamped pairs of mammalian tumor cells expressing either wild-type Cx43 or a mutant of Cx43 lacking the carboxyl terminal domain (Cx43M257). Cx43 transjunctional currents showed bi-exponential decay and a residual steady-state conductance of approximately 35% maximum. Transjunctional currents recorded from Cx43M257 channels displayed a single, slower exponential decay. Long transjunctional voltage pulses caused virtual disappearance of the residual current at steady state. Single channel data revealed disappearance of the residual state, increase in the mean open time, and slowing of the transition times between open and closed states. Coexpression of CxM257 with Cx43CT in a separate fragment restored the lower conductance state. We propose that Cx43CT is an effector of fast voltage gating. Truncation of Cx43CT limits channel transitions to those occurring across the higher energy barrier that separates open and closed states. We further propose that a ball-and-chain interaction provides the fast component of voltage-dependent gating between CT domain and a receptor affiliated with the pore.

Unique Kir2.x properties determine regional and species differences in the cardiac inward rectifier K+ current.

The inwardly rectifying potassium (Kir) 2.x channels mediate the cardiac inward rectifier potassium current (I(K1)). In addition to differences in current density, atrial and ventricular I(K1) have differences in outward current profiles and in extracellular potassium ([K+]o) dependence. The whole-cell patch-clamp technique was used to study these properties in heterologously expressed Kir2.x channels and atrial and ventricular I(K1) in guinea pig and sheep hearts. Kir2.x channels showed distinct rectification profiles: Kir2.1 and Kir2.2 rectified completely at potentials more depolarized than -30 mV (I approximately 0 pA). In contrast, rectification was incomplete for Kir2.3 channels. In guinea pig atria, which expressed mainly Kir2.1, I(K1) rectified completely. In sheep atria, which predominantly expressed Kir2.3 channels, I(K1) did not rectify completely. Single-channel analysis of sheep Kir2.3 channels showed a mean unitary conductance of 13.1+/-0.1 pS in 15 cells, which corresponded with I(K1) in sheep atria (9.9+/-0.1 pS in 32 cells). Outward Kir2.1 currents were increased in 10 mmol/L [K+]o, whereas Kir2.3 currents did not increase. Correspondingly, guinea pig (but not sheep) atrial I(K1) showed an increase in outward currents in 10 mmol/L [K+]o. Although the ventricles of both species expressed Kir2.1 and Kir2.3, outward I(K1) currents rectified completely and increased in high [K+]o-displaying Kir2.1-like properties. Likewise, outward current properties of heterologously expressed Kir2.1-Kir2.3 complexes in normal and 10 mmol/L [K+]o were similar to Kir2.1 but not Kir2.3. Thus, unique properties of individual Kir2.x isoforms, as well as heteromeric Kir2.x complexes, determine regional and species differences of I(K1) in the heart.

Structural bases for the chemical regulation of Connexin43 channels.

Connexins proteins associate with a variety of catalytic and non-catalytic molecules. Also, different domains of connexin can bind to each other, providing a mechanism for channel regulation. Here, we review some of these associations, placing particular emphasis on the intramolecular interactions that regulate Connexin43 (Cx43). We also describe some novel methods that allow for the characterization of protein-protein interactions such as those observed in the cardiac gap junction protein Connexin43. Overall, intra- and inter-molecular interactions may regulate gap junctions to filter the passage of molecular messages between cells at the appropriate time and between the appropriate cells. As a potential area for future investigations, we also speculate as to whether some of the inter-molecular interactions involving connexins lead to modifications in the function of the associated protein, rather than on the function of connexin itself.