Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel.

We report here a characterization of two families of calcium-activated K(+) channel beta-subunits, beta2 and beta3, which are encoded by distinct genes that map to 3q26.2-27. A single beta2 family member and four alternatively spliced variants of beta3 were investigated. These subunits have predicted molecular masses of 27. 1-31.6 kDa, share approximately 30-44% amino acid identity with beta1, and exhibit distinct but overlapping expression patterns. Coexpression of the beta2 or beta3a-c subunits with a BK alpha-subunit altered the functional properties of the current expressed by the alpha-subunit alone. The beta2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the beta3a-c subunits resulted in only partial inactivation of the current, and the beta3b subunit conferred an apparent inward rectification. Furthermore, unlike the beta1 and beta2 subunits, none of the beta3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these beta-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents.

Aromatic residues affecting permeation and gating in dSlo BK channels.

Structural determinants of permeation in large unit conductance calcium-activated potassium channels (BK channels) were investigated. Y293 and F294 in the P-region of dSlo were substituted by tryptophans. Compared to wild-type channels, Y293W channels displayed reduced inward unitary currents while F294W channels exhibited normal inward current amplitudes but flickery kinetics. Both mutations produced changes in current/voltage relations under bi-ionic conditions. Sensitivity to block by external tetraethylammonium (TEA) was affected in both channels, and the voltage dependence of TEA block was increased in F294W channels. Both mutations also affected gating by shifting the half-maximal activation voltage of macroscopic conductance/voltage relations to more positive potentials, and eliminating a slow component of deactivation. The double mutant did not produce ionic currents. These data are consistent with a model in which Y293 contributes to a potassium-binding site close to the outer mouth of the dSlo pore, while F294 contributes to an energy barrier near this site.

Inward rectifier potassium channels. Cloning, expression and structure-function studies.

A PCR-based cloning strategy was used to identify novel subunits of the two-transmembrane domain inward rectifier potassium channel family from rat brain, heart, and skeletal muscle. When expressed in Xenopus oocytes, two of these clones (Kir4.1 and Kir2.3) gave rise to inwardly rectifying potassium currents. Two-electrode voltage clamp commands to potentials negative to EK evoked inward potassium-selective currents which rapidly reached a peak amplitude and then relaxed to a steady-state level. Differences in the extent of current relaxation, the degree of rectification, and the voltage-dependent block by external cesium were detected. Two other members of this family (Kir5.1 and Kir3.4) did not produce macroscopic currents, when expressed by themselves, yet both subunits modified the currents when coexpressed with other specific members of the Kir family. Expression of chimeric subunits between Kir4.1 and either Kir5.1 or Kir3.4 suggested that the transmembrane domains determine the specificity of subunit heteropolymerization, while the C-terminal domains contribute to alterations in activation kinetics and rectification. Expression of covalently linked subunits demonstrated that the relative subunit positions, as well as stoichiometry, affect heteromeric channel activity.

Wanderlust kinetics and variable Ca(2+)-sensitivity of dSlo [correction of Drosophila], a large conductance CA(2+)-activated K+ channel, expressed in oocytes.

Cloned large conductance Ca2+-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminated a putative cyclic AMP-dependent protein kinase phosphorylation site exhibited large, slow fluctuations in open probability with time. These fluctuations, termed "wanderlust kinetics," occurred with a time course of tens of seconds to minutes and had kinetic properties inconsistent with simple gating models. Wanderlust kinetics was still observed in the presence of 5mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution was increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics did not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics could be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrated up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels.

Wanderlust kinetics and variable Ca(2+)-sensitivity of Drosophila, a large conductance Ca(2+)-activated K+ channel, expressed in oocytes.

Cloned large conductance Ca(2+)-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminated a putative cyclic AMP-dependent protein kinase phosphorylation site exhibited large, slow fluctuations in open probability with time. These fluctuations, termed "wanderlust kinetics," occurred with a time course of tens of seconds to minutes and had kinetic properties inconsistent with simple gating models. Wanderlust kinetics was still observed in the presence of 5 mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution was increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics did not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics could be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrated up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels.

Functional differences among alternatively spliced variants of Slowpoke, a Drosophila calcium-activated potassium channel.

The Slowpoke locus of Drosophila melanogaster encodes a family of alternatively spliced mRNAs which encode large conductance calcium-activated potassium channels. Variability residues in blocks of amino acids designated boxes A, C, E, G, and I. Oocytes were injected with cRNAs that had been chosen for direct functional comparison of single box differences. Single channel records from inside-out patches of oocyte membranes expressing A1 or A3 forms, E1 or E2 forms, and G2-G5 forms were analyzed and compared. The main functional difference between A1 and A3 was in unitary conductance, whereas the main difference in properties between E1 and E2 was in calcium sensitivity. Activation kinetics were different between G3 and G5, but not consistently in different A and E box backgrounds. The results indicate that alternative splicing of a common RNA precursor contributes to the functional diversity of the expressed channel. Our findings suggest that the variable region of the Slowpoke channel subunit comprises modular, yet interactive functional domains which influence the essential features of unit conductance, calcium sensitivity, and gating.

Reconstitution of expressed KCa channels from Xenopus oocytes to lipid bilayers.

Reconstitution of large conductance calcium-activated potassium (KCa) channels from native cell membranes into planar lipid bilayers provides a powerful method to study single channel properties, including ion conduction, pharmacology, and gating. Recently, KCa channels derived from the Drosophila Slowpoke (Slo) gene have been cloned and heterologously expressed in Xenopus oocytes. In this report, we describe the reconstitution of cloned and expressed Slo KCa channels from Xenopus oocyte membranes into lipid bilayers. The reconstituted channels demonstrate functional properties characteristic of native KCa channels. They possess a mean unitary conductance of approximately 260 pS in symmetrical potassium (250 mM), and they are voltage- and calcium-sensitive. At 50 microM Ca2+, their half-activation potential was near -20 mV; and their affinity for calcium is in the micromolar range. Reconstituted Slo KCa channels were insensitive to external charybdotoxin (40-500 nM) and sensitive to micromolar concentrations of external tetraethylammonium (KD = 158 microM, at 0 mV) and internal Ba2+ (KD = 76 microM, at 40 mV). In addition, they were blocked by internally applied "ball" inactivating peptide (KD = 480 microM, at 40 mV). These results demonstrate that cloned KCa channels expressed in Xenopus oocytes can be readily incorporated into lipid bilayers where detailed mechanistic studies can be performed under controlled internal and external experimental conditions.

Tetraethylammonium block of Slowpoke calcium-activated potassium channels expressed in Xenopus oocytes: evidence for tetrameric channel formation.

Unitary currents were recorded from inside-out membrane patches pulled from Xenopus oocytes that had been injected with RNA transcribed from a cDNA encoding the Drosophila maxi-K channel (Slowpoke). Site-directed mutagenesis was used to make cDNAs encoding channel subunits with single amino acid substitutions (Y308V and C309P). The extracellular side of the patch was exposed to tetraethylammonium (TEA) in the pipette solution; unitary currents in the presence of TEA were compared with currents in the absence of TEA to compute the inhibition. Amplitude distributions were fit by beta functions to estimate the blocking and unblocking rate constants. For wild-type channels, TEA blocked with an apparent Kd of 80 microM at 0 mV and sensed 0.18 of the membrane electric field; the voltage dependence lay entirely in the blocking rate constant. TEA blocked currents through C309P channels with a similar affinity to wild-type at 0 mV, but this was not voltage-dependent. Currents through Y308V channels were very insensitive to any block by TEA; the apparent Kd at 0 mV was 26 mM and the blockade sensed 0.18 of the electric field. Oocytes injected with a mixture of RNAs encoding wild-type and Y308V channels showed unitary currents of four discrete amplitudes in the presence of 3 mM TEA; at 40 mV these corresponded to inhibitions of approximately 80%, 55%, 25% and 10%.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and expression of a family of inward rectifier potassium channels.

Five new members of the two-transmembrane domain potassium channel family have been identified from rat brain, heart and skeletal muscle. The channel mRNAs are differentially expressed and found in both the central nervous system and periphery. Expression of two of these channels in Xenopus oocytes gave rise to inwardly rectifying potassium currents which were voltage-dependently blocked by barium and cesium. Voltage command pulses negative to Ek evoked inward currents which rapidly reached a peak amplitude and relaxed to a steady-state level. The quantity of current relaxation differed in the two channels and was increased at more negative potentials. The degree of current rectification was also different for the two channels. The results demonstrate the existence of a large and widely expressed family of inward rectifier potassium channel subunits with distinct tissue distributions and functional properties.

Single amino acid substitution affects desensitization of the 5-hydroxytryptamine type 3 receptor expressed in Xenopus oocytes.

5-Hydroxytryptamine type 3 receptors were expressed in Xenopus oocytes from a cloned cDNA. The peak inward current evoked by 5-hydroxytryptamine (30 microM) was linearly related to the holding potential (-100 to +20 mV) and reversed near 0 mV. The inward current (at -60 mV) declined during the continued presence of 5-hydroxytryptamine with a half-time of about 2 s; this desensitization was 20 times slower in calcium-free solution. Desensitization was markedly different in channels in which Leu286 was changed by site-directed mutagenesis; this residue is thought to lie near the middle of the M2 segment. Desensitization was faster with Phe, Tyr, or Ala in this position and slower with Thr. Phe and Thr substitutions in the equivalent position of the nicotinic acetylcholine receptor have similar effects on desensitization, suggesting that the underlying conformational change might be common to ligand-gated channels.

Calcium-activated potassium channels expressed from cloned complementary DNAs.

Calcium-activated potassium channels were expressed in Xenopus oocytes by injection of RNA transcribed in vitro from complementary DNAs derived from the slo locus of Drosophila melanogaster. Many cDNAs were found that encode closely related proteins of about 1200 aa. The predicted sequences of these proteins differ by the substitution of blocks of amino acids at five identified positions within the putative intracellular region between residues 327 and 797. Excised inside-out membrane patches showed potassium channel openings only with micromolar calcium present at the cytoplasmic side; activity increased steeply both with depolarization and with increasing calcium concentration. The single-channel conductance was 126 pS with symmetrical potassium concentrations. The mean open time of the channels was clearly different for channels having different substituent blocks of amino acids. The results suggest that alternative splicing gives rise to a large family of functionally diverse, calcium-activated potassium channels.

[Cryosurgery in the treatment of condylomatosis]. / La criochirurgia nel trattamento della condilomatosi.

The authors report their personal experience of the treatment of condylomas in both male and female patients using cryosurgery. The temperature of -80 degrees C which can be obtained using hyperdry nitrogen monoxide equipment allow radical treatment to be performed without pain in an outpatient setting without general or loco-regional anesthesia. The characteristics of this treatment allow patients to be regularly monitored, thus eliminating all signs of recidivation of those lesions which were too small to be seen at the start of treatment. The patient is considered cured after an interval of approximately 30-40 days after the disappearance of all condylomas.

Effects of Mg2+ on basal and beta-adrenergic-stimulated delayed rectifier potassium current in frog atrial myocytes.

1. The effects of internal Mg2+ ions on the delayed rectifier potassium current (IK) of bull-frog atrial myocytes were studied using the whole-cell configuration of the patch-clamp technique with a perfusable patch electrode. 2. Initial variations in IK amplitude were dependent on [Mg2+]i. With [Mg2+] greater than 1 mM, the amplitude of IK usually decreased after initiating the whole-cell recording configuration (run-down); with [Mg2+]i less than 1 mM, IK usually increased (run-up). Mg2+ blocked IK with an apparent half-maximal effect of 0.6 mM [Mg2+]i. 3. The basal free [Mg2+]i, indicated by the amplitude of IK before run-up or run-down, was estimated from the relationship between [Mg2+]i and IK to be 0.8 mM. 4. The amplitude of both the activation curve and the instantaneous voltage-current relationship was decreased by increasing [Mg2+]i. Under these conditions, the voltage dependence of IK was not affected. 5. The rate of activation of the current at +40 mV was slowed by increasing [Mg2+]i with little effect on the rate of deactivation at -50 mV. This is in contrast to the effects of isoprenaline, which speeded activation and slowed deactivation. 6. Isoprenaline increased IK on average by about 2.5 pA/pF, whether IK had previously run down or not, and regardless of [Mg2+]i. The reversibility of isoprenaline was partially inhibited at [Mg2+]i less than 1 mM. 7. It is concluded that Mg2+ affects IK via several mechanisms that might include a Mg(2+)-dependent phosphatase.

Regulation of Ca2+ current in frog ventricular cardiomyocytes by 5'-guanylylimidodiphosphate and acetylcholine.

1. Calcium currents (ICa) were measured in frog ventricular myocytes using the whole-cell patch clamp technique and a perfused pipette. The effect of internal perfusion with the hydrolysis-resistant GTP analogue, GppNHp (5'guanylylimidodiphosphate), on basal ICa and ICa stimulated with forskolin or isoprenaline was examined to gain insight into the role of G proteins in ICa regulation. 2. Without added guanine nucleotides, isoprenaline stimulated ICa approximately 14-fold with an EC50 of 0.09 microM. Forskolin stimulated ICa approximately 10-fold with an EC50 of 0.30 microM. 3. Internal 30 microM-GppNHp produced an approximately 80% decrease in ICa elevated by 0.3 microM-isoprenaline or 3 microM-forskolin. The inhibition of isoprenaline stimulation was due to a decrease in the maximal stimulation from approximately 14-fold to approximately 14-fold without a significant change in the EC50. In contrast, the reduction in forskolin stimulation was due to a 22-fold increase in the EC50 to 11.4 microM, with little change in maximal stimulation. 4. The inhibition of stimulated ICa by GppNHp is likely to be mediated by a G protein, because the effects of GppNHp are irreversible, and are blocked by excess GTP. ICa is affected similarly by GppNHp and by ACh. This suggests that GppNHp activates the same G protein that is normally activated by ACh, but activation by GppNHp occurs in the absence of agonist occupation of the muscarinic receptor. 5. The increase in the EC50 for forskolin produced by internal GppNHp was reversed by exposure to isoprenaline, which itself did not affect ICa amplitude. On average, exposure to isoprenaline in the presence of GppNHp caused an irreversible 81-fold decrease in the EC50 for forskolin to 0.14 microM. Stimulation of ICa by forskolin after internal GppNHp and exposure to isoprenaline was completely blocked by the protein kinase A inhibitor PKI(5-22). 6. These effects do not involve the phospholipase C system, because they are not mimicked by phorbol esters or internal inositol 1,4,5-trisphosphate (IP3) and are not blocked by bromophenacyl bromide or neomycin. 7. Direct effects of G proteins on ICa were not evident, because internal perfusion with PKI(5-22) completely inhibited isoprenaline- or forskolin-stimulated increases in ICa, and neither ACh nor internal GppNHp (30-500 microM) affected basal ICa or ICa elevated by internally perfused cyclic AMP. 8. These results suggest that the predominant site of action of the inhibitory G protein activated by either GppNHp or ACh is adenylyl cyclase. Furthermore, the internally perfused frog cardiomyocytes may provide a useful approach for probing the detailed interactions of G proteins, forskolin, and adenylyl cyclase in an intact cell.

[Our experience with early gastric cancer]. / La nostra esperienza in tema di early gastric cancer.

Gastric cancer has always required surgical therapy since in the majority of cases at the moment of treatment symptoms are already at an advances stage. Over the past years many advances have been made in the early diagnosis of many forms of neoplasia, but the rate of progress has been much slower with regard to gastric cancer. Only the preventive and regular use of gastroscopy will allow the disease to be diagnosed at a non-advanced stage. The term early gastric cancer is used to describe a carcinoma which only infiltrates the mucosa, or the mucosa and submucosa, irrespective of lymph node or other metastases. The present study was based on a retrospective analysis of cases of stomach cancer observed in the Surgical Department of the University of Perugia from January 1963 to December 1988. A total of 1,263 patients were affected by cancer of the stomach during the above period. One hundred and twenty-three cases were not included because of incomplete data or insufficient follow-up. A total of 1,140 patients were therefore included in the study; of these only 99 cases were affected by early gastric cancer. Age, sex, earlier gastric diseases, life styles, familial occurrence of disease, and symptomatology were among the different parameters evaluated. In addition, the site of disease, diagnostic methods, pre- and post-operative staging, intramural diffusion of the disease and surgical treatment were taken into account. In older cases the 5-year survival rate was calculated, whereas in more recent cases statistical methods, based on accumulated data, were used to estimate survival rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the delayed rectifier potassium current in frog cardiomyocytes by beta-adrenergic agonists and magnesium.

1. The regulation of IK and ICa were studied in single cells isolated from bull-frog atrium using the whole-cell configuration of the patch clamp and a perfused patch pipette. 2. IK was increased approximately 50-100% and ICa was increased approximately 6-10 times by 1 microM-isoprenaline, 5 microM-forskolin, or internal perfusion with 30 microM-cyclic AMP. The effects of cyclic AMP and isoprenaline were not additive. The shape of the concentration-response curves and the EC50 values for the effects of cyclic AMP on ICa and on IK were very similar (2.3 microM for IK and 1.7 microM for ICa). 3. Elevation of intracellular cyclic AMP had a similar effect on IK regardless of whether ICa was blocked with Cd2+ or not. Increasing ICa with dihydropyridine Ca2+ channel agonists had no effect on IK amplitude. 4. Isoprenaline or cyclic AMP caused an increase in the fully-activated IK and also shifted the activation curves to more negative potentials in most cells. The shift in the activation curve was reversible and was also observed when ICa was blocked with Cd2+. The rate of activation of IK was increased and the rate of deactivation of IK was slowed by isoprenaline. 5. After breaking the membrane patch and initiating whole-cell recording, IK ran down with time in about 50% of the cells examined when the intracellular solution contained 1 mM [Mg2+]. In contrast, when the solution contained 0.3 mM [Mg2+], rundown was almost never observed. Internal perfusion with increasing concentrations of [Mg2+] caused reversible decreases in the maximum amplitude of IK and shifted the IK activation curve slightly to more negative potentials, but had negligible effects upon the shape or the curvature of the fully activated current-voltage relationship.

Identification and developmental expression of a novel embryonic myosin heavy-chain gene in chicken.

The developmental expression of an embryonic chicken myosin heavy-chain (MHC) gene homologous to the genomic clone pCM4.1 was examined by S1 analysis. Transcripts homologous to pCM4.1 are first detected at day 12 in ovo, and are maximally expressed between days 15-17 in ovo. No pCM4.1 transcripts are detected at earlier stages of embryogenesis or at high levels in posthatch stages. This unique pattern of expression has led to the proposal that pCM4.1 represents a previously uncharacterized MHC gene, which is confined in its expression to late embryogenesis. Genomic hybridization data, in addition to a comparison between the DNA and amino acid sequences of pCM4.1 and other characterized chicken MHC 3' end clones, provide further evidence for this proposal. We also present observations made during the sequence analysis of pCM4.1 that may be relevant to our understanding of the 3'-end processing of homologous primary transcripts, and of the mechanism controlling developmental MHC isoform transitions.

[Wiscott-Aldrich syndrome. Description of a case]. / Sindrome di Wiscott-Aldrich descrizione di un caso

The Wiscott-Aldrich syndrome with the classical symptomatology (eczema, thrombocytopenia and susceptibility to infections) has been described in a 3-month-old child. The family history, together with the evidence of an immunological deficiency involving both thymus-dependent lymphocyte and immunoglobulin-antibody systems have given important clues to diagnosis. Impairment of immunoglobulin homoeostasis and its relevance to the assessment of heterozygotes is discussed.