Subconductance states of Cx30 gap junction channels: data from transfected HeLa cells versus data from a mathematical model

Human HeLa cells expressing mouse connexin30 were used to study the electrical properties of gap junction channel substates. Experiments were performed on cell pairs using a dual voltage-clamp method. Single-channel currents revealed discrete levels attributable to a main state, a residual state, and five substates interposed, suggesting the operation of six subgates provided by the six connexins of a gap junction hemichannel. Substate conductances, gamma(j,substate), were unevenly distributed between the main-state and the residual-state conductance (gamma(j,main state) = 141 pS, gamma(j,residual state) = 21 pS). Activation of the first subgate reduced the channel conductance by approximately 30%, and activation of subsequent subgates resulted in conductance decrements of 10-15% each. Current transitions between the states were fast (<2 ms). Substate events were usually demarcated by transitions from and back to the main state; transitions among substates were rare. Hence, subgates are recruited simultaneously rather than sequentially. The incidence of substate events was larger at larger gradients of V(j). Frequency and duration of substate events increased with increasing number of synchronously activated subgates. Our mathematical model, which describes the operation of gap junction channels, was expanded to include channel substates. Based on the established V(j)-sensitivity of gamma(j,main state) and gamma(j,residual state), the simulation yielded unique functions gamma(j,substate) = f(V(j)) for each substate. Hence, the spacing of subconductance levels between the channel main state and residual state were uneven and characteristic for each V(j).

Pitfalls when examining gap junction hemichannels: interference from volume-regulated anion channels

Human HeLa cells transfected with mouse connexin45 were used to explore the experimental conditions suitable to measure currents carried by gap junction hemichannels. Experiments were performed with a voltage-clamp technique and whole-cell recording. Lowering [Ca(2+)](o) from 2 mM to 20 nM evoked an extra current, I (m), putatively carried by Cx45 hemichannels. However, the variability of I (m) (size, voltage sensitivity, kinetics) suggested the involvement of other channels. The finding that growth medium in the incubator increased the osmolarity with time implied that volume-regulated anion channels (VRAC) may participate. This assumption was reinforced by the following observations. On the one hand, keeping [Ca(2+)](o) normal while the osmolarity of the extracellular solution was reduced from 310 to 290 mOsm yielded a current characteristic of VRAC; I (VRAC) activated/deactivated at negative/positive voltage, giving rise to the conductance functions g (VRAC,inst)=f(V (m)) (inst: instantaneous; V (m): membrane potential) and g (VRAC,ss)=f(V (m)) (ss: steady state). Moreover, it was reversibly inhibited by mibefradil, a Cl(-)channel blocker (binding constant K (d)=38 microM, Hill coefficient n=12), but not by the gap junction channel blocker 18alpha-glycyrrhetinic acid. On the other hand, minimizing the osmotic imbalance while [Ca(2+)](o) was reduced led to a current typical for Cx45 hemichannels; I (hc) activated/deactivated at positive/negative voltage. Furthermore, it was reversibly inhibited by 18alpha-glycyrrhetinic acid or palmitoleic acid, but not by mibefradil. Computations based on g (VRAC,ss)=f(V (m)) and g (hc,ss)=f(V (m)) indicated that the concomitant operation of both currents results in a bell-shaped conductance-voltage relationship. The functional implications of the data presented are discussed. Conceivably, VRAC and hemichannels are involved in a common signaling pathway.

Gap junction channels and cardiac impulse propagation

The role of gap junction channels on cardiac impulse propagation is complex. This review focuses on the differential expression of connexins in the heart and the biophysical properties of gap junction channels under normal and disease conditions. Structural determinants of impulse propagation have been gained from biochemical and immunocytochemical studies performed on tissue extracts and intact cardiac tissue. These have defined the distinctive connexin coexpression patterns and relative levels in different cardiac tissues. Functional determinants of impulse propagation have emerged from electrophysiological experiments carried out on cell pairs. The static properties (channel number and conductance) limit the current flow between adjacent cardiomyocytes and thus set the basic conduction velocity. The dynamic properties (voltage-sensitive gating and kinetics of channels) are responsible for a modulation of the conduction velocity during propagated action potentials. The effect is moderate and depends on the type of Cx and channel. For homomeric-homotypic channels, the influence is small to medium; for homomeric-heterotypic channels, it is medium to strong. Since no data are currently available on heteromeric channels, their influence on impulse propagation is speculative. The modulation by gap junction channels is most prominent in tissues at the boundaries between cardiac tissues such as sinoatrial node-atrial muscle, atrioventricular node-His bundle, His bundle-bundle branch and Purkinje fibers-ventricular muscle. The data predict facilitation of orthodromic propagation.

Closing the gap between coil and balloon in the neurointerventional armamentarium? Initial clinical experience with a nitinol vascular occlusion plug

INTRODUCTION: The use of vascular plug devices for the occlusion of high-flow lesions is a relatively new and successful procedure in peripheral and cardiopulmonary interventions. We report on the use and efficiency of the Amplatzer vascular plug in a small clinical series and discuss its potential for occlusion of large vessels and high-flow lesions in neurointerventions. METHODS: Between 2005 and 2007 four patients (mean age 38.5 years, range 16-62 years) were treated with the device, in three patients to achieve parent artery occlusion of the internal carotid artery, in one patient to occlude a high-flow arteriovenous fistula of the neck. The application, time to occlusion, and angiographic and clinical results and the follow-up were evaluated. RESULTS: Navigation, positioning and detachment of the device were satisfactory in all cases. No flow-related migration of the plug was seen. The cessation of flow was delayed by a mean of 10.5 min after deployment of the first device. In the procedures involving vessel sacrifice, two devices had to be deployed to achieve total occlusion. No patient experienced new neurological deficits; the 3-month follow-up revealed stable results. CONCLUSION: The Amplatzer vascular plug can be adapted for the treatment of high-flow lesions and parent artery occlusions in the head and neck. In this small series the use of the devices was uncomplicated and safe. The rigid and large delivery device and the delayed cessation of flow currently limit the device's use in neurointerventions.

Long-term effects of heart transplantation: the gap between physical performance and emotional well-being

The purpose of our study was to assess physical and emotional factors in heart transplant patients. A prospective design was used to compare patients' physical symptoms, emotional complaints, and restrictions at admission to the waiting list, immediately after, and 1 and 5 years after heart transplantation. Thirty-three patients were included (30 male, 3 female) in the study. Their mean age at admission was 48 +/- 10.2 years. Of these, 23 suffered from cardiomyopathy, 8 from coronary heart disease, and 2 from valvular insufficiency. At admission, the patients suffered from symptoms of cardiac insufficiency, and were restricted in sports, gardening, hobbies, sexual life, job, food-intake, and mobility. More than three-fourths rated their physical and emotional status as moderate to poor. Emotionally, they suffered from irritability, restlessness, depression, psychic lability, lowered drive, lack of social contact, low self-esteem, and anxiety. At the end of rehabilitation (4-8 weeks after the operation), all physical and emotional complaints, as well as restrictions had significantly decreased (p < 0.0001 to p < 0.001), except for trembling, numbness of hands/feet, and food-intake. One year postoperatively, patients reported even fewer physical complaints (p < 0.01). Three-fourths rated their physical and emotional status good or excellent. Five years postoperatively--in contrast to physical status, restrictions, and physical complaints--the emotional complaints had increased significantly (p < 0.0001). Patients reported excellent physical performance up to 5 years postoperatively. On the other hand, the study revealed that their emotional well-being had significantly deteriorated from 1 to 5 years postoperatively. Attention should, therefore, not only be paid to the good physical health of the survivors, but also to the worsening of their emotional status.

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

Transmission electron microscopy has provided most of what is known about the ultrastructural organization of tissues, cells, and organelles. Due to tremendous advances in crystallography and magnetic resonance imaging, almost any protein can now be modeled at atomic resolution. To fully understand the workings of biological "nanomachines" it is necessary to obtain images of intact macromolecular assemblies in situ. Although the resolution power of electron microscopes is on the atomic scale, in biological samples artifacts introduced by aldehyde fixation, dehydration and staining, but also section thickness reduces it to some nanometers. Cryofixation by high pressure freezing circumvents many of the artifacts since it allows vitrifying biological samples of about 200 mum in thickness and immobilizes complex macromolecular assemblies in their native state in situ. To exploit the perfect structural preservation of frozen hydrated sections, sophisticated instruments are needed, e.g., high voltage electron microscopes equipped with precise goniometers that work at low temperature and digital cameras of high sensitivity and pixel number. With them, it is possible to generate high resolution tomograms, i.e., 3D views of subcellular structures. This review describes theory and applications of the high pressure cryofixation methodology and compares its results with those of conventional procedures. Moreover, recent findings will be discussed showing that molecular models of proteins can be fitted into depicted organellar ultrastructure of images of frozen hydrated sections. High pressure freezing of tissue is the base which may lead to precise models of macromolecular assemblies in situ, and thus to a better understanding of the function of complex cellular structures.