TPA Increases Conductance but Decreases Permeability in Neonatal Rat Cardiomyocyte Gap Junction Channels

Short-term (10 min) effects of 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), the protein kinase C (PKC) activator, on cardiac macroscopic (g_j) and single channel (γ_j) gap junctional conductances were studied in pairs of neonatal rat cardiomyocytes. Under dual whole-cell (WC) or perforated patch (PP) voltageclamp, g_j increased by 15.5 ± 7.2% (mean ± SD, n = 9) and by 46.3 ± 17.0% (n = 5), respectively. The latter difference is not related to intracellular calcium concentration, because raising the Ca²⁺ concentration in the electrode solution did not change the TPA-induced increase in g_j observed under WC conditions. The inactive phorbol ester, 4α-phorbol 12,13-didecanoate (αPDD), did not affect g_j. Single cardiac gap junction channel events, resolved in the presence of heptanol, indicated two γ_j sizes of 20 and 40-45 pS. Under control conditions, the larger events were most frequently observed. Whereas αPDD did not change this distribution, TPA shifted the γ_j distribution to the lower sizes. Diffusion of Lucifer Yellow (LY) and 6-carboxyfluorescein (6-CF), gap junction permeant tracers, was studied on small clusters of cardiomyocytes. Under control conditions, LY labeled 19.4 ± 7.2 cells (mean ± SD, n = 18) and 6-CF labeled 8.4 ± 2.2 cells (n = 20). Whereas αPDD did not change the extent of dye transfer, TPA restricted the diffusion of LY to 2.8 ± 1.3 cells (n = 11) and of 6-CF to 2.4 ± 1.4 cells (n = 20). This suggests that permeability and single channel conductance of connexin 43 channels are parallely related. Altogether, these results point to the opposite modulation of electrical and metabolic coupling of cardiac cells evoked by TPA.     

Regulation of the calcium slow channel by cyclic GMP dependent protein kinase in chick heart cells

In order to assess the interaction between the cAMP-dependent and the cGMP-dependent phosphorylation pathways on the slow Ca²⁺ current (I_Ca(L)), whole-cell voltage-clamp experiments were conducted on embryonic chick heart cells. Addition of 8Br-cGMP to the bath solution reduced the basal (unstimulated) I_Ca(L). Intracellular application of the catalytic subunit of PK-A (PK-A(cat); 1.5 M) via the patch pipette rapidly potentiated I_Ca(L) by 215±16% (n=4); subsequent addition of 1 mM 8Br-cGMP to the bath reduced the amplitude of I_Ca(L) towards the initial control values (123±29%). Intracellular application of PK-G (25 nM pre-activated by 10^–7 M cGMP), rapidly inhibited the basal I_Ca(L) by 64±6% (n=8). Heat-denatured PK-G was ineffective. Subsequent additions of relatively high concentrations of 8Br-cAMP (1 mM) or isoproterenol (ISO, 1–10 M) did not significantly remove the PK-G blockade of I_Ca(L). The results of the present study suggest that: (a) 8Br-cGMP can inhibit the basal or stimulated (by PK-A(cat)) I_Ca(L) in embryonic chick myocardial cells. (b) PK-G applied intracellularly inhibits the basal I_Ca(L).     

Predictions of non-linear responses to selection for forage yield under competition

Summary The expected response of a population of competing genotypes to selection of high yielding individuals is expressed in terms of a regression model of a type similar to that used to describe genotype-environment interactions (Perkins and Jinks 1968). It is shown that response to selection on the basis of the yielding ability of genotypes (direct effects) can also cause changes in the magnitude of their effects on their neighbours (indirect effects) and on the interactions of direct and indirect effects. Thus, if y_ij = + g_i a_j + _jg_i + s_ij, where y_ij is the expected yield of the ith genotype in competition with the jth, g_i is the direct effect of the ith genotype, a_j the indirect effect of the jth, and _j the regression onto direct effects of interactions involving the jth associate, it follows that response is R_(g) = g(1+b_(a/g) + b_/gg), where g is the change in average direct effect of the population brought about by selection, and b represents a regression coefficient. If b_(/g) is important, then response to selection will be non-linear over a range of genotypes, and where it is negative, then selection will become progressively less effective as it proceeds until a plateau is reached, beyond which it will be detrimental.Estimates of the above parameters were made from nine sets of data from diallel arrangements of binary mixtures. Although a degree of uncertainly was induced in these estimates by the need to reparametrize the model describing competition within and between binary components to cater only for inter-genotypic effects, the consistency of the prediction of non-linearity of response induces some confidence in the results. Predicted optima are as low as 8 pc abovethe mean inthe case of closely related material. Other deficiencies and implications of this selection model are discussed.     

Heptanol-induced decrease in cardiac gap junctional conductance is mediated by a decrease in the fluidity of membranous cholesterol-rich domains

To assess whether alterations in membrane fluidity of neonatal rat heart cells modulate gap junctional conductance (g _j ), we compared the effects of 2mm 1-heptanol and 20 μm 2-(methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)-octanoate (A₂C) in a combined fluorescence anisotropy and electrophysiological study. Both substances decreased fluorescence steady-state anisotropy (r_ss), as assessed with the fluorescent probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) by 9.6±1.1% (mean ±sem,n=5) and 9.8±0.6% (n=5), respectively, i.e., both substances increased bulk membrane fluidity. Double whole-cell voltage-clamp experiments showed that 2mm heptanol uncoupled cell pairs completely (n=6), whereas 20 μm A₂C, which increased bulk membrane fluidity to the same extent, did not affect coupling at all (n=5). Since gap junction channels are embedded in relatively cholesterol-rich domains of the membrane, we specifically assessed the fluidity of the cholesterol-rich domains with dehydroergosterol (DHE). Using DHE, heptanol increased r_ss by 14.9±3.0% (n=5), i.e., decreased cholesterol domain fluidity, whereas A₂C had no effect on r_ss (−0.4±6.7%,n=5). Following an increase of cellular “cholesterol” content (by loading the cells with DHE), 2mm heptanol did not uncouple cell pairs completely:g _j decreased by 80±20% (range 41–95%,n=5). The decrease ing _j was most probably due to a decrease in the open probability of the gap junction channels, because the unitary conductances of the channels were not changed nor was the number of channels comprising the gap junction. The sensitivity of non-junctional membrane channels to heptanol was unaltered in cholesterol-enriched myocytes. These results indicate that the fluidity of cholesterol-rich domains is of importance to gap junctional coupling, and that heptanol decreasesg _j by decreasing the fluidity of cholesterol-rich domains, rather than by increasing the bulk membrane fluidity.     

Multiple-channel conductance states and voltage regulation of embryonic chick cardiac gap junctions

Summary We used the double whole-cell voltage-clamp technique on ventricle cell pairs isolated from 7-day chick heart to measure the conductance of their gap junctions (G _j) and junctional channels ( _j) with a steady-state voltage difference (V _j) applied across the junction. Currents were recorded from single gap junction channels (i _j) as symmetrical rectangular signals of equal size and opposite sign in the two cells, and _j was measured from i _j/V _j. We observed channel openings at six reproducible conductance levels with means of 42.6, 80.7, 119.6, 157.7, 200.4 and 240.3 pS. More than half of all openings were to the 80-and 160-pS conductance levels. The probability that a high conductance event (e.g., 160 or 240 pS) results from the random simultaneous opening of several 40-pS channels is small, based on their frequency of occurrence and on the prevalence of shifts between small and large conductance states with no intervening 40-pS steps. Our results are consistent with three models of embryonic cardiac gap junction channel configuration: a homogeneous population of 40-pS channels that can open cooperatively in groups of up to six; a single population of large channels with a maximal conductance near 240 pS and five smaller substates; or several different channel types, each with its own conductance. G _j was determined from the junctional current (I _j) elicited by rectangular pulses of applied transjunctional voltage as I _j/V _j. It was highest near 0 V _j and was progressively reduced by application of V _j between 20 and 80 mV or –20 and –80 mV. In response to increases in V _j, G _j decayed in a voltage-and timedependent fashion. After a 6-sec holding period at 0 V _j, the initial conductance (G _init) measured immediately after the onset of an 80-mV step in V _j was nearly the same as that measured by a 10-mV prepulse. However, during 6-sec pulses of V _j>±20 mV, G _j declined over several seconds from G _init to a steady-state value (G _ss). At potentials greater than ±20 mV the current decay could be fit with biexponential curves with the slow decay time constant ( ₂) 5–20 times longer than ₁. For the response to a step to 80 mV V _j, for example, ₁=127 msec and ₂=2.6 sec. The rate of current decay in response to smaller positive or negative steps in V _j was slower, the magnitude of the decline was smaller, and the ratio ₂/ ₁ decreased. The relationship between G _init and V _j was approximately linear between 0 and 80 mV or –80 mV. whereas the relationship between G _ss and V _j was nonlinear beyond ±20 mV. Upon returning to 0 V _j, G _j recovered with a biexponential time course, reaching its maximal value after several seconds; recovery time constants after a step in V _j from 80 to 0 mV were 225 msec and 1.9 sec. In the resting state, at low junctional voltage, high conductance channel activity (160–240 pS) is favored. Voltage-dependent decline of G _j results in part from a shift from high to lower conductance states.We thank Ms. B.J. Duke for technical assistance and for preparation of the cell cultures and Drs. L.J. DeFelice and D. Eaton for stimulating and helpful discussions of the results.     

Procedures for selecting composites based on prediction methods

Summary Procedures for selecting among parental varieties to be used in the synthesis of composites are discussed. In addition to the criterion based on the mean and variance of composites of the same size (k) proposed by Cordoso (1976), we suggest the index I_j=w₁v_j+w_{2 j} or I_j=(2/k) I_j for a preliminary selection among parental varieties. We show that by increasing k (size of the composite) I_j tends to g_j, the general combining ability effect. Such a criterion is particularly important when n, the number of parental varieties, is large, so that the number of possible composites (N_c=2ⁿ–n–1) becomes too large to be handled when using the common prediction procedures. Yield data from a 9 × 9 variety diallel cross were used for illustration.     

The role of amino terminus of mouse Cx50 in determining transjunctional voltage-dependent gating and unitary conductance

Amino-terminus and carboxyl-terminus of connexins have been proposed to be responsible for the transjunctional voltage-dependent gating (V_j-gating) and the unitary gap junction channel conductance (γ_j). To better understand the molecular structure(s) determining the V_j-gating properties and the γ_j of Cx50, we have replaced part of the amino-terminus of mCx50 by the corresponding domain of mCx36 to engineer a chimera Cx50-Cx36N, and attached GFP at the carboxyl-terminus of mCx50 to construct Cx50-GFP. The dual whole-cell patch-clamp technique was used to test the resulting gap junction channel properties in N2A cells. The Cx50-Cx36N gap junction channel lowered the sensitivity of steady-state junctional conductance to V_j (G_j/V_j relationship), slowed V_j-gating kinetics, and reduced γ_j as compared to Cx50 channel. Cx50-GFP gap junction channel showed similar V_j-gating properties and γ_j to Cx50 channel. We further characterized a mutation, Cx50N9R, where the Asn (N) at the ninth position of Cx50 was replaced by the corresponding Arg (R) at Cx36. The G_j/V_j relationship of Cx50N9R channel was significantly changed; most strikingly, the macroscopic residual conductance (G_min) was near zero. Moreover, the single Cx50N9R channel only displayed one open state (γ_j = 132 ± 4 pS), and no substate could be detected. Our data suggest that the NT of Cx50 is critical for both the V_j-gating and the γ_j, and the introduction of a positively charged Arg at the ninth position reduced the G_min with a correlated disappearance of the substate at the single channel level.     

pH i -Dependent membrane conductance of proximal tubule cells in culture (OK): Differential effects on K+- and Na+-conductive channels

Summary Confluent monolayers of the established opossum kidney cell line were exposed to NH₄Cl pulses (20 mmol/liter) during continuous intracellular measurements of pH, membrane potential (PD _m ) and membrane resistance (R _m) in bicarbonate-free Ringer. The removal of extracellular NH₄Cl leads to an intracellular acidification from a control value of 7.33±0.08 to 6.47±0.03 (n=7). This inhibits the absolute K conductance (g _K+), reflected by a decrease of K⁺ transference number from 71±3% (n=28) to 26±6% (n=5), a 2.6±0.2-fold rise ofR _m, and a depolarization by 24.2±1.5 mV (n=52). In contrast, intracellular acidification during a block ofg _K+ by 3 mmol/liter BaCl₂ enhances the total membrane conductance, being shown byR _m decrease to 68±7% of control and cell membrane depolarization by 9.8±2.8 mV (n=17). Conversely, intracellular alkalinization under barium elevatesR _m and hyperpolarizes PD _m . The replacement of extracellular sodium by choline in the presence of BaCl₂ significantly hyperpolarizes PD _m and increasesR _m, indicating the presence of a sodium conductance. This conductance is not inhibited by 10^–4 mol/liter amiloride (n=7). Patch-clamp studies at the apical membrane (excised inside-out configuration) revealed two Na⁺-conductive channels with 18.8±1.4 pS (n=10) and 146 pS single-channel conductance. Both channels are inwardly rectifying and highly selective towards Cl^–. The low-conductive channel is 4.8 times more permeable for Na⁺ than for K⁺. Its open probability rises at depolarizing potentials and is dependent on the pH of the membrane inside (higher at pH 6.5 than at pH 7.8).     

Isomeric yohimbine alkaloids block calcium-activated K+ channels in medullary thick ascending limb cells of rabbit kidney

Summary The alpha₂-adrenergic antagonist yohimbine (YOH) and the closely related isomers corynanthine (COR) and rauwolscine (RAU) caused brief interruptions in current characteristic of a fast blocker Ca²⁺-activated K⁺ channels in cultured medullary thick ascending limb (MTAL) cells. The apparent dissociation constants (K _app), for COR, YOH, and RAU, respectively, at the intracellular face of the channel in the presence of 200mm K⁺ are 45±1, 98±2, and 310±33 m. TheK _app for COR on the extracellular side also in the presence of 200mm. K⁺ was much greater at 1.6±0.17mm. Increasing K⁺ on the same side as the blocker relieves the blocking reaction. TheK _app for the alkaloids varies with K⁺ in a manner quantitatively consistent with K⁺ and the alkaloids competing for a common binding site. Finally, blocking by the charged form of these alkaloids is voltage dependent with changes inK _app of 86±7 and 94±6 m pere-fold change in voltage for blockers applied either from the inside or outside. The alkaloids block at an electrical distance similar to tetraethylammonium, suggesting that the site within the channel pore of these molecules may be similar.     

Kinetics of the work of the ion channels controlled by nicotinic cholinoreceptors in the membrane of mollusk neurons

Currents entering through single channels with conductivity 10 pS were produced on the membrane of an isolated neuron of the fresh-water molluskPlanorbarius corneus in the presence of suberyldicholine (5 µM) by the patch-clamp technique (cell-attached configuration). The times of stay of the channels in the open and closed states, as well as the durations of pulse bursts and clusters, were measured. The distributions of the time intervals obtained experimentally were approximated for open states by one exponential function: t_o=27±3 msec (n=21), and for closed states by a sum of three exponentials: t_c1=9.5±1.0 msec (n=21); t_c2=171±33 msec (n=19); t_c3=5.2±1.0 sec (n=21). The burst durations are characterized by the presence of two exponential functions in the distribution: t_b2=20±14 msec (n=10), t_b2=203±23 msec (n=10), and the clusters by three exponential functions: t_k1=33±11 msec (n=8), t_k2=274±84 msec (n=8), and t_k3=1.5±0.5 sec (n=9). Thus, for work of a chemoactivated channel associated with nicotinic-type cholinoreceptors in a mollusk neuron we can suggest a kinetic scheme with one open and three nonconducting states: C O D₁A₂ D₂A₂. The two "long-lived" closed states of the channel may be associated with desensitization of the integral response of the neurons to the application of suberyldicholine. Values were obtained for the rate constants of these proposed reactions. It is suggested that this model may be useful in analyzing the action of cholinomimetics and blockers on the molluskan neuronal membrane.I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 23, No. 5, pp. 588–595, September–October, 1991.     

Root and stem xylem embolism,stomatal conductance,and leaf turgor in Acer grandidentatum populations along a soil moisture gradient

The objective of this study was to determine how adjustment in stomatal conductance (g ^s) and turgor loss point (_tlp) between riparian (wet) and neighboring slope (dry) populations of Acer grandidentum Nutt. was associated with the susceptibility of root versus stem xylem to embolism. Over two summers of study (1993–1994), the slope site had substantially lower xylem pressures (_px) and g ^s than the riparian site, particularly during the drought year of 1994. The _tlp was also lower at the slope (-2.9±0.1 MPa; all errors 95% confidence limits) than at riparian sites (-1.9±0.2 MPa); but it did not drop in response to the 1994 drought. Stem xylem did not differ in vulnerability to embolism between sites. Although slope-site stems lost a greater percentage of hydraulic conductance to embolism than riparian stems during the 1994 drought (46±11% versus 27±3%), they still maintained a safety margin of at least 1.7 MPa between midday _px and the critical pressure triggering catastrophic xylem embolism (_pxCT). Root xylem was more susceptible to embolism than stem xylem, and there were significant differences between sites: riparian roots were completely cavitated at -1.75 MPa, compared with -2.75 MPa for slope roots. Vulnerability to embolism was related to pore sizes in intervessel pit membranes and bore no simple relationship to vessel diameter. Safety margins from _pxCT averaged less than 0.6 MPa in roots at both the riparian and slope sites. Minimal safety margins at the slope site during the drought of 1994 may have led to the almost complete closure of stomata (g _s=9±2 versus 79±15 mmol m^-2 s^-1 at riparian site) and made any further osmotic adjustment of _tlp non-adaptive. Embolism in roots was at least partially reversed after fall rains. Although catastrophic embolism in roots may limit the minimum for gas exchange, partial (and reversible) root embolism may be adaptive in limiting water use as soil water is exhausted.     

Activity coefficients of intracellular Na+ and K+ during development of frog oocytes

Summary The chemical activities, (a), of Na⁺ and K⁺ were determined in large mature and in small immature frog oocytes, using open-tipped micropipettes and ionselective microelectrodes. The average chemical concentrations,c, of Na⁺ and K⁺ were determined by spectrophotometry and by electron probe X-ray microanalysis. The apparent activity coefficient (^app) was calculated for each ion as the ratio,a/c.With development, (a _Na/a _K) decreased four to fivefold and (c _Na/c _K) increased six to sevenfold. In the large mature oocytes, _Na ^app was measured to be 0.08±0.02 and _K ^app lay within the range 1.15±0.03 to 1.29±0.04, constituting the smallest value for Na⁺ and largest value for K⁺, respectively, thus far reported. This intracellular value of _K ^app was substantially greater than the activity coefficient of K⁺ in the external medium (0.76). The data suggest that the inequality of _Na ^app and _K ^app in this and probably other cells reflects the development of subcellular compartmentalization of ions. Possible intracellular sites of ionic compartmentalization are considered.     

Regulation of Ion Fluxes,Cell Volume and Gap Junctional Coupling by cGMP in GFSHR-17 Granulosa Cells

Gap junctional communication between granulosa cells seems to play a crucial role for follicular growth and atresia. Application of the double whole-cell patch-clamp- and ratiometric fura-2-techniques allowed a simultaneous measurement of gap junctional conductance (G _j) and cytoplasmic concentration of free Ca²⁺ ([Ca²⁺]_i) in a rat granulosa cell line GFSHR-17. The voltage-dependent gating of G _j varied for different cell pairs. One population exhibited a bell-shape dependence of G _j on transjunctional voltage, which was strikingly similar to that of Cx43/Cx43 homotypic gap junction channels expressed in pairs of oocytes of Xenopus laevis. Within 15–20 min, gap junctional uncoupling occurred spontaneously, which was preceded by a sustained increase of [Ca²⁺]_i and accompanied by shrinkage of cellular volume. These responses to the whole-cell configuration were avoided by absence of extracellular Ca²⁺, blockage of K⁺ efflux, or addition of 8-bromoguanosine 3,5-cyclic monophosphate (8-Br-cGMP) to the pipette solution. Even in the absence of extracellular Ca²⁺ or blockage of K⁺ efflux, formation of whole-cell configuration generated a Ca²⁺ spike that could be suppressed by the presence of 8-Br-cGMP. We propose that intracellular cGMP regulates Ca²⁺ release from intracellular Ca²⁺ stores, which activates sustained Ca²⁺ influx, K⁺ efflux and cellular shrinkage. We discuss whether gap junctional conductance is directly affected by cGMP or by cellular shrinkage and whether gap junctional coupling and/or cell shrinkage is involved in the regulation of apoptotic/necrotic processes in granulosa cells.     

Signal transduction pathways of muscarinic receptors in circular smooth muscle from the rabbit caecum

The effects of muscarinic acetylcholine receptor stimulation on phosphoinositides breakdown and adenylate cyclase activity were examined in the circular smooth muscle of the rabbit caecum. InMyo-[³H]inositol-labeled circular smooth muscle cells, carbachol caused a concentration-dependent increase in [³H]inositol phosphates ([³H]IPs) accumulation (EC₅₀ of 3±1 M). The M₁-selective antagonist pirenzepine (PRZ), the M₂-selective AF-DX 116 (11-2[[2-[(diethyl-amino)methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6Hpyrido[2,3-b][1,4]benzodiazepin-6-one) and the M₃-selective para-fluoro-hexahydrosiladifenidol (p-F-HHSiD) inhibited the carbachol-induced [³]inositol phosphates accumulation with the following order of potency: p-F-HHSiD>PRZ>AF-DX 116. In saponin-permeabilized circular smooth muscle cells, carbachol and GTP[S] elicited a concentration-dependent increase in [³H]inositol phosphates accumulation. The concentration-response curve for GTP[S] was shifted to the left when cells were incubated with 1 M carbachol. The [³H]inositol phosphates accumulation elicited by simultaneous addition of 0.1 M GTP[S] and 1 M carbachol to permeabilized cells was significantly decreased (78.28±18.23% inhibition) when cells were preincubated for 5 min with 0.1 mM GDP[S]. In nonpermeabilized cells, pertussis toxin did not alter the carbachol-induced increase in [³H]inositol phosphates accumulation. On the other hand, the 0.1 mM carbachol-induced inhibition of forskolin-stimulated adenylate cyclase activity in circular smooth muscle homogenates was significantly reversed by atropine and AF-DX 116, whereas PRZ and p-F-HHSiD were ineffective (muscarinic antagonists were used at 1 M final concentration). Moreover, the carbachol-induced inhibition of the cyclic AMP accumulation elicited by 10 M isoproterenol was abolished by pertussis toxin pretreatment of isolated circular smooth muscle cells. In conclusion, our data suggest that in circular smooth muscle of rabbit caecum, the muscarinic receptor stimulation of [³H]inositol phsophates accumulation is mediated by M₃ subtype receptors coupled to a pertussis toxin-insensitive G protein, whereas inhibition of adenylate cyclase activity is mediated by M₂ subtype receptors coupled to a pertussis toxin-sensitive GTP-binding protein G_i.     

Properties of the sarcoplasmic reticulum Ca2+-pump in coronary artery skinned smooth muscle

Pig coronary artery cultured smooth muscle cells were skinned using saponin. In the presence of an ATP-regenerating system and oxalate, the skinned cells showed an ATP-dependent azide insensitive Ca²⁺-uptake which increased linearly with time for >1 h. The Ca²⁺-uptake occurred with K_m values of 0.20±0.03 M for Ca²⁺ and 400±34 M for MgATP^2–. Thapsigargin and cyclopiazonic acid inhibited this uptake with IC₅₀ values of 0.13±0.02 and 0.56±0.04 M, respectively. These properties of SR Ca²⁺-pump are similar to those reported for membrane fractions isolated from fresh smooth muscle of coronary artery and other arteries. However, optimum pH of the uptake in the skinned cells (6.2) was lower than that reported previously using isolated membranes (6.4–6.8).Abbreviations SR sarcoplasmic reticulum - ER endoplasmic reticulum - PM plasma membrane - CPA cyclopiazonic acid - DTT dithiothreitol     

Switched single-electrode voltage-clamp amplifiers allow precise measurement of gap junction conductance

Measurement of gapjunction conductance(g_j) with patch-clampamplifiers can, due to series resistance problems, be subject toconsiderable errors when large currents are measured. Formulas developed to correct for these errors unfortunately depend on exactestimates of series resistance, which are not always easy to obtain.Discontinuous single-electrode voltage-clamp amplifiers (DSEVCs) wereshown to overcome series resistance problems in single whole cellrecording. With the use of two synchronized DSEVCs, the simulatedg_j in a modelcircuit can be measured with a maximum error of <5% in all recordingsituations investigated (series resistance, 5-47 M; membraneresistance, 20-1,000 M; g_j, 1-100nS). At a very lowg_j of 100 pS, theerror sometimes exceeded 5% (maximum of 15%), but the error wasalways <5% when membrane resistance was >100 M. The precisionof the measurements is independent of series resistance, membraneresistance, and g_j. Consequently,it is possible to calculateg_j directly from Ohm's law, i.e., without using correction formulas. Our results suggest that DSEVCs should be used to measureg_j if largecurrents must be recorded, i.e., if cells are well coupled or ifmembrane resistance is low.     

Atrial fibrillation-associated Connexin40 mutants make hemichannels and synergistically form gap junction channels with novel properties

Mutations of Cx40 (GJA5) have been identified in people with lone chronic atrial fibrillation including G38D and M163V which were found in the same patient. We used dual whole cell patch clamp procedures to examine the transjunctional voltage (V_j) gating and channel conductance properties of these two rare mutants. Each mutant exhibited slight alterations of V_j gating properties and increased the gap junction channel conductance (γ_j) by 20–30 pS. While co-expression of the two mutations had similar effects on V_j gating, it synergistically increased γ_j by 50%. Unlike WTCx40 or M163V, G38D induced activity of a dominant 271 pS hemichannel.     

β-Adrenoreceptors of multiple affinities in a clonal capillary endothelial cell line and its functional implication

-Adrenoreceptor has been studied in a clonal capillary endothelial cell line established from the vascular bed of the bovine adrenal medulla. [³H]Dihydroalprenolol ([³H]DHA) binding to the isolated plasma membranes from these cells has demonstrated the presence of -adrenoreceptors with two different affinities. the dissociation constants (K_d) have been found to be 0.27±0.09×10^–9 M and 2.96±0.31×10^–9 M, respectively with the corresponding B_max of 5.1±0.05 and 70.0±0.2 pmol/mg protein, respectively. Inhibition of [³H]DHA binding to the -receptor by atenolol (a ₁-antagonist) and ICI 118,551 (a ₁-antagonist) has suggested that the IC_50cor (=K_i) for atenolol and ICI 118,551 for high affinity site are 0.08±0.03×10^–12 M and 0.25±0.08×10^–12 M, respectively. This, therefore, indicates that both atenolol and ICI 118,551 are able to displace the bound ligand effectively but the ₁-selective antagonist atenolol is 3 times more potent than its ₂ counterpart, ICI 118,551. Displacement of [³H]DHA binding to the endothelial cell plasma membrane by the agonists isoproterenol, epinephrine and norephinephrine has established a relative order of K_i for these agents as isoproterenol (0.56±0.19×10^–9 M)–9 M)>-norepinephrine (0.71±0.24×10^–9 M) for the high affinity site. The corresponding values for the low affinity site, however, are 4.62±0.64×10^–9 M, 6.21±0.86×10^–9 M and 5.90±0.82×10^–9 M, respectively for the same agonists. Increased intracellular cAMP accompanied with cellular proliferation in the presence of isoproterenol has suggested not only the coupling of -adrenoreceptors to the adenylate cyclase system but also its involvement in endothelial cell proliferation.Abbreviations DHA Dihydroalprenolol - cAMP 3:5 cyclic adenosine monophosphate - DTT dithiothreitol - MEM minimal essential medium - 8Br-cAMP 8-bromo-adenosine 3:5 cyclic monophosphate     

Seasonal changes in sediment phosphorus forms in relation to sedimentation and benthic bacterial biomass in Lake Erken

Surficial sediment and sedimenting material were sampled during spring and summer 1991 in Lake Erken. Sediment was analyzed for redox potential, P concentrations and bacterial biomass. Sedimentation and chlorophyll a concentrations of sedimenting matter were determined. Additionally, different phosphorus forms in surficial sediment were quantified using sequential fractionation. The resulting dataset was used to study the effects of sedimentation events following phytoplankton blooms and benthic bacterial biomass on the size of the various phosphorus pools in the sediment.Sedimentation of spring diatoms caused a rapid increase in the NH₄Cl- and NaOH-extractable P (NH₄Cl–P and NaOH–rP) in the sediment. During sedimentation, NaOH–rP and NH₄Cl–P increased within 3 days from 422 ± 17 g g^–1 DW to 537 ± 8.0 g g^–1 DW and from 113 ± 13 g g^–1 DW to 186 ± 26 g g^–1 DW, respectively. The NaOH–nrP (non-reactive P) fraction made up about 17% of Tot-P in sediment samples, whereas NaOH–rP and HCl–P made up 25% each. All P forms showed considerable seasonal variation. Significant relationships were found between bacterial biomass and the NaOH–nrP and NH₄Cl–P fractions in the sediment, respectively. Also regressions of NaOH–nrP and NH₄Cl–P versus the chlorophyll a concentration of sedimenting matter were highly significant. These regressions lend support to the conjecture that NaOH–nrP is a conservative measure of bacterial poly-P.     

Specificity of the connexin W3/4 locus for functional gap junction formation

The N-terminal (NT) domain of the connexins forms an essential transjunctional voltage (V_j) sensor and pore-forming domain that when truncated, tagged, or mutated often leads to formation of a nonfunctional channel. The NT domain is relatively conserved among the connexins though the α- and δ-group connexins possess a G2 residue not found in the β- and γ-group connexins. Deletion of the connexin40 G2 residue (Cx40G2Δ) affected the V_j gating, increased the single channel conductance (γ_j), and decreased the relative K⁺/Cl^? permeability (P_K/P_Cl) ratio of the Cx40 gap junction channel. The conserved α/β-group connexin D2/3 and W3/4 loci are postulated to anchor the NT domain within the pore via hydrophilic and hydrophobic interactions with adjacent connexin T5 and M34 residues. Cx40D3N and D3R mutations produced limited function with progressive reductions in V_j gating and noisy low γ_j gap junction channels that reduced the γ_j of wild-type Cx40 channels from 150 pS to < 50 pS when coexpressed. Surprisingly, hydrophobic Cx40 W4F and W4Y substitution mutations were not compatible with function despite their ability to form gap junction plaques. These data are consistent with minor and major contributions of the G2 and D3 residues to the Cx40 channel pore structure, but not with the postulated hydrophobic W4 intermolecular interactions. Our results indicate an absolute requirement for an amphipathic W3/4 residue that is conserved among all α/β/δ/γ-group connexins. We alternatively hypothesize that the connexin D2/3-W3/4 locus interacts with the highly conserved FIFR M1 motif to stabilize the NT domain within the pore.