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.

A Spanish founder mutation in the chloride channel gene, CLCNKB, as a cause of atypical Bartter syndrome in adult age

BACKGROUND: Mutations in the chloride channel gene, CLCNKB, usually cause classic Bartter syndrome (cBS) or a mixed Bartter-Gitelman phenotype in the first years of life. METHODS: We report an adult woman with atypical BS caused by a homozygous missense mutation, A204T, in the CLCNKB gene, which has previously been described as the apparently unique cause of cBS in Spain. RESULTS: The evaluation of this patient revealed an overlap of phenotypic features ranging from severe biochemical and systemic disturbances typical of cBS to scarce symptoms and diagnosis in the adult age typical of Gitelman syndrome. The tubular disease caused a dramatic effect on mental, growth and puberal development leading to low IQ, final short stature and abnormal ovarian function. Furthermore, low serum PTH concentrations with concomitant nephrocalcinosis and normocalcaemia were observed. Both ovarian function and serum PTH levels were normalized after treatment with cyclooxygenase inhibitors. CONCLUSIONS: The present report confirms a weak genotype-phenotype correlation in patients with CLCNKB mutations and supports the founder effect of the A204T mutation in Spain. In our country, the genetic diagnosis of adult patients with hereditary hypokalaemic tubulopathies should include a screening of A204T mutation in the CLCNKB gene.

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.

The supramolecular assemblies of voltage-dependent anion channels in the native membrane

Voltage-dependent anion channels (VDACs) are major constituents of the outer mitochondrial membrane (OMM). These primary transporters of nucleotides, ions and metabolites mediate a substantial portion of the OMM molecular traffic. To study the native supramolecular organization of the VDAC, we have isolated, characterized and imaged OMMs from potato tubers. SDS-PAGE and mass spectrometry of OMMs revealed the presence of the VDAC isoforms POM34 and POM36, as well as the translocase of the OMM complex. Tubular two-dimensional crystals of the VDAC spontaneously formed after incubation of OMMs for two to three months at 4 degrees C. Transmission electron microscopy revealed an oblique lattice and unit cells housing six circular depressions arranged in a hexagon. Atomic force microscopy of freshly isolated OMMs demonstrated (i) the existence of monomers to tetramers, hexamers and higher oligomers of the VDAC and (ii) its spatial arrangement within the oligomers in the native membrane. We discuss the importance of the observed oligomerization for modulation of the VDAC function, for the binding of hexokinase and creatine kinase to the OMM and for mitochondria-mediated apoptosis.

Comparison of the effects of cetylpyridinium chloride with an essential oil mouth rinse on dental plaque and gingivitis - a six-month randomized controlled clinical trial

OBJECTIVE: To compare the effects of an experimental mouth rinse containing 0.07% cetylpyridinium chloride (CPC) (Crest Pro-Health) with those provided by a commercially available mouth rinse containing essential oils (EOs) (Listerine) on dental plaque accumulation and prevention of gingivitis in an unsupervised 6-month randomized clinical trial. MATERIAL AND METHODS: This double-blind, 6-month, parallel group, positively controlled study involved 151 subjects balanced and randomly assigned to either positive control (EO) or experimental (CPC) mouth rinse treatment groups. At baseline, subjects received a dental prophylaxis procedure and began unsupervised rinsing twice a day with 20 ml of their assigned mouthwash for 30 s after brushing their teeth for 1 min. Subjects were assessed for gingivitis and gingival bleeding by the Gingival index (GI) of Löe ; Silness (1963) and plaque by the Silness ; Löe (1964) Plaque index at baseline and after 3 and 6 months of rinsing. At 3 and 6 months, oral soft tissue health was assessed. Microbiological samples were also taken for community profiling by the DNA checkerboard method. RESULTS: Results show that after 3 and 6 months of rinsing, there were no significant differences (p=0.05) between the experimental (CPC) and the positive control mouth rinse treatment groups for overall gingivitis status, gingival bleeding, and plaque accumulation. At 6 months, the covariant (baseline) adjusted mean GI and bleeding sites percentages for the CPC and the EO rinses were 0.52 and 0.53 and 8.7 and 9.3, respectively. Both mouth rinses were well tolerated by the subjects. Microbiological community profiles were similar for the two treatment groups. Statistically, a significant greater reduction in bleeding sites was observed for the CPC rinse versus the EO rinse. CONCLUSION: The essential findings of this study indicated that there was no statistically significant difference in the anti-plaque and anti-gingivitis benefits between the experimental CPC mouth rinse and the positive control EO mouth rinse over a 6-month period.

Increased expression of the auxiliary beta2-subunit of ventricular L-type Ca2+ channels leads to single-channel activity characteristic of heart failure

BACKGROUND: Increased activity of single ventricular L-type Ca(2+)-channels (L-VDCC) is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary beta-subunits as a possible explanation. METHODS AND RESULTS: By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC beta-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1) or beta(3) isoforms, beta(2a) and beta(2b) induce a high-activity channel behavior typical of failing myocytes. In accordance, beta(2)-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac Ca(V)1.2 also reveal increased single-channel activity and sarcolemmal beta(2) expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing ("Adaptive Phase"), reveal the opposite phenotype, viz: reduced single-channel activity accompanied by lowered beta(2) expression. Additional evidence for the cause-effect relationship between beta(2)-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive Ca(V)1.2 and inducible beta(2) cardiac overexpression. Here in non-failing hearts induction of beta(2)-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure. CONCLUSIONS: Our study presents evidence of the pathobiochemical relevance of beta(2)-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure.

Patients with biallelic mutations in the chloride channel gene CLCNKB: long-term management and outcome

BACKGROUND: Little information on the management and long-term follow-up of patients with biallelic mutations in the chloride channel gene CLCNKB is available. METHODS: Long-term follow-up was evaluated from 5.0 to 24 years (median, 14 years) after diagnosis in 13 patients with homozygous (n = 10) or compound heterozygous (n = 3) mutations. RESULTS: Medical treatment at last follow-up control included supplementation with potassium in 12 patients and sodium in 2 patients and medical treatment with indomethacin in 9 patients. At the end of follow-up, body height was 2.0 standard deviation score or less in 6 patients; 2 of these patients had growth hormone deficiency. Body weight (chloride channel gene CLCNKB tend to present with pathological proteinuria and impaired kidney function after a median follow-up of 14 years, and growth retardation is common and sometimes related to growth hormone deficiency in these patients.

CSTX-1, a toxin from the venom of the hunting spider Cupiennius salei, is a selective blocker of L-type calcium channels in mammalian neurons

The inhibitor cystine-knot motif identified in the structure of CSTX-1 from Cupiennius salei venom suggests that this toxin may act as a blocker of ion channels. Whole-cell patch-clamp experiments performed on cockroach neurons revealed that CSTX-1 produced a slow voltage-independent block of both mid/low- (M-LVA) and high-voltage-activated (HVA) insect Ca(v) channels. Since C. salei venom affects both insect as well as rodent species, we investigated whether Ca(v) channel currents of rat neurons are also inhibited by CSTX-1. CSTX-1 blocked rat neuronal L-type, but no other types of HVA Ca(v) channels, and failed to modulate LVA Ca(v) channel currents. Using neuroendocrine GH3 and GH4 cells, CSTX-1 produced a rapid voltage-independent block of L-type Ca(v) channel currents. The concentration-response curve was biphasic in GH4 neurons and the subnanomolar IC(50) values were at least 1000-fold lower than in GH3 cells. L-type Ca(v) channel currents of skeletal muscle myoballs and other voltage-gated ion currents of rat neurons, such as I(Na(v)) or I(K(v)) were not affected by CSTX-1. The high potency and selectivity of CSTX-1 for a subset of L-type channels in mammalian neurons may enable the toxin to be used as a molecular tool for the investigation of this family of Ca(v) channels.

Soft-decision equalization techniques for frequency selective MIMO channels

Multi-input multi-output (MIMO) technology is an emerging solution for high data rate wireless communications. We develop soft-decision based equalization techniques for frequency selective MIMO channels in the quest for low-complexity equalizers with BER performance competitive to that of ML sequence detection. We first propose soft decision equalization (SDE), and demonstrate that decision feedback equalization (DFE) based on soft-decisions, expressed via the posterior probabilities associated with feedback symbols, is able to outperform hard-decision DFE, with a low computational cost that is polynomial in the number of symbols to be recovered, and linear in the signal constellation size. Building upon the probabilistic data association (PDA) multiuser detector, we present two new MIMO equalization solutions to handle the distinctive channel memory. With their low complexity, simple implementations, and impressive near-optimum performance offered by iterative soft-decision processing, the proposed SDE methods are attractive candidates to deliver efficient reception solutions to practical high-capacity MIMO systems. Motivated by the need for low-complexity receiver processing, we further present an alternative low-complexity soft-decision equalization approach for frequency selective MIMO communication systems. With the help of iterative processing, two detection and estimation schemes based on second-order statistics are harmoniously put together to yield a two-part receiver structure: local multiuser detection (MUD) using soft-decision Probabilistic Data Association (PDA) detection, and dynamic noise-interference tracking using Kalman filtering. The proposed Kalman-PDA detector performs local MUD within a sub-block of the received data instead of over the entire data set, to reduce the computational load. At the same time, all the inter-ference affecting the local sub-block, including both multiple access and inter-symbol interference, is properly modeled as the state vector of a linear system, and dynamically tracked by Kalman filtering. Two types of Kalman filters are designed, both of which are able to track an finite impulse response (FIR) MIMO channel of any memory length. The overall algorithms enjoy low complexity that is only polynomial in the number of information-bearing bits to be detected, regardless of the data block size. Furthermore, we introduce two optional performance-enhancing techniques: cross- layer automatic repeat request (ARQ) for uncoded systems and code-aided method for coded systems. We take Kalman-PDA as an example, and show via simulations that both techniques can render error performance that is better than Kalman-PDA alone and competitive to sphere decoding. At last, we consider the case that channel state information (CSI) is not perfectly known to the receiver, and present an iterative channel estimation algorithm. Simulations show that the performance of SDE with channel estimation approaches that of SDE with perfect CSI.

Bislug flow in circular and noncircular channels and the role of interface stretching on energy dissipation

The area of microfluidics has increased in popularity with such fields as MEMS, microreactors, microscaleheat exchangers, etc. A comprehensive understanding of dissipation mechanisms for fluid flow in microchannels is required to accurately predict the behavior in these small systems. Tests were performed using a constant pressure potential created by two immiscible fluids juxtaposed in a microchannel. This study focused on the flow and dissipation mechanisms in round and square microchannels. There are four major dissipation mechanisms in slug flow; wall shear, dissipation at the contact line, menisci interaction and the stretching of the interface. A force balance between the internal driving potential, viscous drag and interface stretching was used to develop a model for the prediction of the velocity of a bislug in a microchannel. Interface stretching is a dissipation mechanism that has been included due to the unique system properties and becomes increasingly more important as the bislug decreases in length.