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Calcium-activated nonselective cationic currents have been known for 30 years, but their physiological implications have remained unresolved until the recent cloning of the TRPM4 ion channel. Since then, TRPM4 has been identified as a key modulator of numerous calcium-dependent mechanisms such as the immune response, insulin secretion, cerebral artery constriction, respiratory rhythm, and cardiac conduction.

Dendritic cell (DC) maturation and migration are events critical for the initiation of immune responses. After encountering pathogens, DCs upregulate the expression of costimulatory molecules and subsequently migrate to secondary lymphoid organs. Calcium (Ca(2+)) entry governs the functions of many hematopoietic cell types, but the role of Ca(2+) entry in DC biology remains unclear. Here we report that the Ca(2+)-activated nonselective cation channel TRPM4 was expressed in and controlled the Ca(2+) homeostasis of mouse DCs. The absence of TRPM4, which elicited Ca(2+) overload, did not influence DC maturation but did considerably impair chemokine-dependent DC migration. Our results establish TRPM4-regulated Ca(2+) homeostasis as crucial for DC mobility but not maturation and emphasize that DC maturation and migration are independently regulated.

Introduction: It has been speculated that pacemaker current (I(f)) in human atria could play a role in causing ectopic atrial automaticity. Ivabradine is a novel selective and specific I(f) inhibitor in the sinus node that reduces heart rate without any negative inotropic effect. The aim of the study was to explore possible effects of ivabradine on I(f) in atrial myocytes., Methods and Results: Using patch-clamp technique, we studied effects of ivabradine on I(f) present in atrial myocytes isolated from human right appendages of patients undergoing cardiac surgery. The identification of HCN isoforms was obtained by means of multiplex single-cell RT-PCR. Ivabradine induced a marked concentration and use-dependent I(f) inhibition with an IC50 at steady state of 2.9 microM. Time constant of block development (Tau(on)) decreases with the increase in the ivabradine concentration. Use-dependent inhibition induced by ivabradine (3 microM) was not modified in the presence of cAMP (10 microM) in the pipette solution. Multiplex single-cell RT-PCR indicates that the major HCN gene subtype detected in atria was HCN2. HCN4 is detected weakly and HCN1 is not significantly detected., Conclusions: Ivabradine inhibits I(f) current in the nonpacemaker cell with characteristics similar to those described previously in rabbit sinus node cells, but revealed a lesser sensitivity for I(f) recorded in human atrial cell than hHCN4 subunits considered as the major contributors to native f-channels in human sinoatrial node. A potential protection of atrial arrhythmias by ivabradine is discussed.

Cardiac hypertrophy is an adaptive process that occurs in response to increased physical stress on the heart. Hypertrophy, which may be induced by hypertension among other factors, is characterized by an increase in left ventricular mass and an associated increase in force production capacity. However, as sustained cardiac hypertrophy may lead to heart failure and sudden death, an understanding of the molecular processes involved in both the onset and consequences of hypertrophy is of significant importance. Calcium is a key player in the process underlying the development of cardiac hypertrophy. Recently, several Transient Receptor Potential proteins (TRPs), including calcium-permeable and calcium-regulated ion channels, have been shown to be related to various aspects of cardiac hypertrophy. TRPs are implicated in the development of cardiac hypertrophy (TRPC1, TRPC3, TRPC6), the electrophysiological perturbations associated with hypertrophy (TRPM4) and the progression to heart failure (TRPC7). This review describes the major characteristics of cardiac hypertrophy and focuses on the roles of TRPs in the physiological processes underlying hypertrophy.

This chapter describes methods to investigate mammalian cardiac channel properties at the single-channel level. Cell isolation is performed from adult heart by enzymatic digestion using the Langendorff apparatus. Isolation proceeding is suitable for rabbit, rat, and mouse hearts. Also, isolation of human atrial cardiomyocytes is described. In freshly isolated cells or cells maintained in primary culture, the single-channel variants of the patch-clamp technique (cell-attached, inside-out, and outside-out) are used to investigate channel properties. Biophysical properties such as conductance and ionic selectivity are determined. Also, regulations by extracellular and intracellular mechanisms are investigated. To illustrate the study, the author provides an example by the characterization of a calcium-activated nonselective cation channel (TRPM4).

The spontaneous activity of cardiac tissue originates in specialized pacemaker cells in the sino-atrial node that generate autonomous rhythmic electrical impulses. A number of regions in the brain are also able to generate spontaneous rhythmic activity to control and regulate important physiological functions. The generation of pacemaker potentials relies on a complex interplay between different types of currents carried by cation channels. Among these currents, the hyperpolarization-activated current (termed I(f), cardiac pacemaker "funny" current, and I(h) in neurons) is the major component contributing to the initiation of cardiac and neuronal excitability and to the modulation of this excitability by neurotransmitters and hormones. I(f) is an inward current activated by hyperpolarization of the membrane potential and by intracellular cyclic nucleotides such as cAMP. The identification at the end of the 1990s of a family of mammalian genes that encode for four Hyperpolarization-activated Cyclic Nucleotide-gated channels, HCN1-4, has made analysis of the location of these channels and the study of their biophysical properties an obtainable goal. As a result, specific agents have been developed for their ability to selectively reduce heart rate by lowering cardiac pacemaker activity where f-channels are their main natural target. These drugs include alinidine, zatebradine, cilobradine, ZD-7288 and ivabradine. Recent data indicate that pharmacological tools such as W7 and genistein, which have been used to identify some intracellular pathways involved in ionic channel modulation, also have the ability to inhibit I(f) directly. This opens new perspectives for the future development of other specific rhythm-lowering agents.

Cardiac hypertrophy is associated with electrophysiological modifications, including modification of action potential shape that can give rise to arrhythmias. We report here a higher detection of a calcium-activated nonselective cation current in cardiomyocytes of spontaneously hypertensive rats (SHRs), a model of hypertension and heart hypertrophy when compared with Wistar-Kyoto (WKY) rat, its normotensive equivalent. Freshly isolated cells from the left ventricles of 3- to 6-month-old WKY rats or SHRs were used for patch-clamp recordings. In inside-out patches, the channel presented a linear conductance of 25+/-0.5 pS, did not discriminate Na(+) over K(+), and was not permeable to Ca(2+). Open probability was increased by depolarization and a rise in [Ca(2+)](i) (dissociation constant=10+/-5.4 micromol/L) but reduced by 0.5 mmol/L [ATP](i), 10 micromol/L glibenclamide, or flufenamic acid (IC(50)=5.5+/-1.7 micromol/L). Thus, it owns the fingerprint of the TRPM4 current. Although rarely detected in WKY cardiomyocytes, the current was present in >50% of patches from SHR cardiomyocytes. Moreover, by performing RT-PCR from ventricular samples, we observed that TRPM4 mRNA detection was higher in SHRs than in WKY rats. We propose that a TRPM4 current is expressed in ventricular cardiomyocytes from SHRs. According to its properties, this channel may contribute to the transient inward current implicated in delayed-after-depolarizations observed during [Ca(2+)] overload of cardiomyocytes.

A voltage-activated proton current in human cardiac fibroblasts, measured using the whole-cell recording configuration of the patch-clamp technique, is reported. Increasing the pH of the bathing solution shifted the current activation threshold to more negative potentials and increased both the current amplitude and its rate of activation. Changing the pH gradient by one unit caused a 51mV shift in the reversal potential of the current, demonstrating a high selectivity for protons of the channel carrying the current. Extracellularly applied Zn(2+) reversibly inhibited the current. Activation of the current contributes to the resting membrane conductance under conditions of intracellular acidosis. It is proposed that this current in cardiac fibroblasts is involved in the regulation of the intracellular pH and the membrane potential under physiological conditions as well as in response to pathological conditions such as ischemia.

Introduction: Among a range of chloride channels, outwardly rectifying Cl- channels have been reported in the heart of various species. Although the anionic current carried by this channel has been subjected to intense electrophysiological investigations, paradoxically no examination of single-channel currents has been reported for human cardiomyocytes., Methods and Results: Using the cell-attached and cell-free configurations of the patch-clamp technique, we have characterized the properties of an outwardly rectifying chloride current (ORCC) at the unitary level in freshly isolated human atrial cardiomyocytes. In excised inside-out patches, the channel presented a nonlinear I/V relationship with a conductance of 76.5 +/- 14.7 pS in the positive voltage range and 8.1 +/- 2 pS in the negative voltage range, indicating an outward rectification. Preincubation with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) significantly increased the number of spontaneously active channels observed. The channel was Cl- selective (Cl- to Na+ permeability ratio, PCl/PNa= 18) with the permeability sequence I- > Br- > Cl- > F- > gluconate. It was blocked by the classical Cl- channels blockers glibenclamide, NPPB, SITS, and DIDS. Channel activity was not dependent upon internal calcium concentration. In the cell-attached configuration, ORCC channel activation was observed under perfusion of a hypotonic solution., Conclusion: Human atrial myocytes express an outwardly rectifying Cl- channel that is sensitive to PKC activation. This channel shares biophysical and pharmacological properties with the swelling-activated chloride current implicated in cardiac pathologies such as myocardial ischemia and dilated cardiopathies.

A rare mRNA variant of the human lymphocyte-specific protein tyrosine kinase LCK gene that retains intron B and excludes exon 7 (B+7-) due to alternative splicing of the canonical LCK transcripts was identified and characterized. LCK B+7- mRNA is detected in all tested peripheral blood T lymphocytes total RNA samples but is apparently sequestered in the nucleus. The presence of intron B sequence does not disrupt the reading frame and results in the insertion of 58 aminoacids, containing a proline-rich region just upstream of p56lck SH3 domain. This putative isoform encodes an unstable 516 aminoacids protein (LckB+7-) which can be expressed in transfected COS-7 cells. Furthermore in Jurkat T cell extracts, a recombinant intron B plus SH3 p56lck domain fails to interact with some TCR-induced tyrosine phosphorylated polypeptides and known p56lck partners such as Sam68 and c-Cbl. The biological function of this rare messenger remains to be elucidated.

NTAL (non-T-cell activation linker, also called LAB) and LAT (linker for activation of T cells) are evolutionarily related transmembrane adaptor proteins that are phosphorylated upon immunoreceptor engagement. Using quantitative reverse transcription-PCR, both NTAL and LAT were found to be expressed in B cells. However, LAT expression was limited to early B cells, whereas NTAL expression typified mature B cells. To delineate their roles in B-cell development and function, Ntal-deficient mice were generated and crossed with Lat-deficient mice. B cells developed in Lat(-/-) Ntal(-/-) double-deficient mice and in mice lacking either of the two adaptors with the same efficiency as in wild-type mice. Upon B-cell antigen receptor cross-linking, Ntal(-/-) B cells exhibited slightly increased Ca(2+) mobilization and proliferation. In addition, Ntal-deficient mice had increased levels of natural antibodies and slightly increased humoral response to a T-dependent antigen. Normal titers of serum-specific immunoglobulins were produced in response to a T-cell-independent antigen. Although NTAL is also expressed in plasma cells, its absence did not affect the hypergammaglobulinemia E and G1 that developed in mice with a mutation in tyrosine 136 of LAT. Therefore, NTAL does not play a role in B cells symmetric to the role played by LAT in T cells.

Chloride channels located on the basolateral membrane are known to be involved in chloride absorption in several parts of the renal tubule, and particularly in the thick ascending limb and distal convoluted tubule. The data available suggest that the ClC-K channels play the major role in this process. We provide here a description of the electrophysiological properties of these channels, still very incomplete at this stage, and we attempt to compare ClC-Ks to three chloride channels that we have identified in the basolateral membrane of microdissected fragments of the mouse renal tubule using the patch-clamp technique. Based on anion selectivity and dependence on external pH and calcium shown by the ClC-Ks, we propose candidate ClC-K1 and ClC-K2 in native tissue. We also discuss the possibility that chloride channels that do not belong to the ClC family may also be involved in the absorption of chloride across the cortical thick ascending limb., (Copyright (c) 2005 S. Karger AG, Basel.)

Cardiac arrhythmias, which occur in a wide variety of conditions where intracellular calcium is increased, have been attributed to the activation of a transient inward current (Iti). Iti is the result of three different [Ca]i-sensitive currents: the Na(+)-Ca2+ exchange current, a Ca(2+)-activated chloride current and a Ca(2+)-activated non-selective cationic current. Using the cell-free configuration of the patch-clamp technique, we have characterized the properties of a Ca(2+)-activated non-selective cation channel (NSC(Ca)) in freshly dissociated human atrial cardiomyocytes. In excised inside-out patches, the channel presented a linear I-V relationship with a conductance of 19 +/- 0.4 pS. It discriminated poorly among monovalent cations (Na+ and K+) and was slightly permeable to Ca2+ ions. The channel's open probability was increased by depolarization and a rise in internal calcium, for which the Kd for [Ca2+]i was 20.8 microM. Channel activity was reduced in the presence of 0.5 mM ATP or 10 microM glibenclamide on the cytoplasmic side to 22.1 +/- 16.8 and 28.5 +/- 8.6%, respectively, of control. It was also inhibited by 0.1 mM flufenamic acid. The channel shares several properties with TRPM4b and TRPM5, two members of the 'TRP melastatin' subfamily. In conclusion, the NSC(Ca) channel is a serious candidate to support the delayed after-depolarizations observed in [Ca2+] overload and thus may be implicated in the genesis of arrhythmias.

Introduction: Cardiac hypertrophy is associated with changes in electrophysiologic properties due to ionic channel modifications and increases in protein kinase C (PKC) activity and diacylglycerol (DAG) content. These changes may contribute to an increased propensity for arrhythmia. Similar electrophysiologic modifications have been reported in adult rat cardiomyocytes undergoing dedifferentiation in primary culture., Methods and Results: Single-channel measurements on such cells identified the appearance of a Ca(2+)-activated nonselective cation channel (NSC(Ca)) during the dedifferentiation process. The current study investigated the sensitivity of this channel to PKC and DAG analogues. In the cell-attached configuration, channel conductance was 20.2 pS under physiologic conditions. Perfusion with the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG, 0.1 mM) or the PKC activator phorbol 12-myristate 13-acetate (PMA, 0.5 microM) increased the channel normalized open probability (nPo), whereas in the presence of the PKC inhibitor calphostin C (1 microM), only OAG retained this effect. In the inside-out configuration, perfusion of both DAG analogues OAG (0.1 mM) and 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG, 10 microM) on the inside of the membrane increased nPo. These results indicate that DAG regulates the NSC(Ca) channel via both the PKC pathway and by a direct interaction., Conclusion: DAG content, PKC activity, and channel expression increased during hypertrophy. This indicates that the NSC(Ca) channel exhibits high activity in this condition and, therefore, is a candidate for the genesis of arrhythmias in ventricular cardiomyocytes. In addition, regulation of the channel by DAG and PKC contributes to current understanding of the physiologic role of this channel, which shares properties with the cloned TRPM4b channel.

Invariant chain (Ii)-deficient mice exhibit profound B cell defects that have remained poorly understood, because they could not be simply explained by impaired Ag presentation. We found that Ii deficiency induced cell autonomous defects of two distinct B cell lineages. The life span of mature follicular (FO) B cells was reduced, accounting for their markedly decreased frequency, whereas, in contrast, marginal zone (MZ) B cells accumulated. Other Ii-expressing lineages such as B1 B cells and dendritic cells were unaffected. Surprisingly, the life span of FO B cells was fully corrected in Ii/I-Abeta doubly deficient mice, revealing that Ii-free I-Abeta chains alter FO B cell survival. In contrast, the accumulation of MZ B cells was controlled by a separate mechanism independent of I-Abeta. Interestingly, in Ii-deficient mice lacking FO B cells, the MZ B cells invaded the FO zone, suggesting that intact follicules contribute to the retention of B cells in the MZ. These findings reveal unexpected consequences of Ii deficiency on the development and organization of B cell follicles.

The marginal zone of the spleen is a precisely ordered region that contains specialized subsets of B lymphocytes and macrophages. Disruption of the negative signaling inositol phosphatase, SH2-containing inositol-5-phosphatase 1 (SHIP), results in the loss of marginal zone B cells (MZBs) with reorganization of marginal zone macrophages (MZMOs) to the red pulp of the spleen. This primary macrophage defect, as revealed by selectively depleting SHIP in myeloid cells shows that MZMOs are specifically required for the retention of MZBs. The MZMO phenotype was reverted in SHIP/Bruton's tyrosine kinase (Btk) double knockout mice, thus identifying the Btk activating pathway as an essential component being regulated by SHIP. Furthermore, we identified a direct interaction between the MARCO scavenger receptor on MZMOs and MZBs. Activation or disruption of this interaction results in MZB migration to the follicle. The migration of the MZMOs was further studied after the response to Staphylococcus aureus, which induced MZMOs to move into the red pulp while MZBs migrated into the follicular zone. The marginal zone is therefore a dynamic structure in which retention and trafficking of B cells requires specific macrophage-B cell interactions.

The lymphoid organs contain specialized microanatomic structures composed of lymphoid, myeloid and stromal cells that are vital to the generation of an effective adaptive immune response. Although the existence of these specialized structures has been known for over a century, the developmental signals that generate them and the specific roles of these structures in the immune response have remained largely elusive. Because of their position adjacent to the marginal sinuses, marginal zone B (MZB) cells are amongst the first population of cells seen by blood born antigens and are presumed to have a critical role in host defense against bacterial pathogens. Here we demonstrate that a deficiency of the tyrosine kinase (Pyk-2) results in a cell autonomous defect of MZB cell production. In response to repetitive polysaccharide antigens (T-independent type II (TI-II)) Pyk-2-deficient mice displayed marked suppression of IgM, IgG3 and IgG2a production. Furthermore, complement receptor engagement proved necessary for the specific targeting of polysaccharide antigens to MZB cells. These results suggest how innate immune responses mediated through complement coupling are translated into an adaptive response by MZB cells, and provide a potential mechanism for the T cell independence of humoral responses to polysaccharide antigens.

The correlation between synaptogenesis and onset of spontaneous action potentials was assessed in rat brainstem cells up to 29 days in primary culture. Cells exhibited different stages of maturation followed by electron microscopy and patch clamp recordings. Terminal boutons with no preferential orientation of presynaptic vesicles appeared after 2 days in culture. After 5 days, preferential orientation of presynaptic vesicles and thickening of postsynaptic membranes were observed. The spontaneous discharge of action potentials, single or bursting, was observed after 7 days in vitro. This was followed by the expression of a 128-pS K(+) channel starting at 13 days in vitro. A 69-pS K(+) channel was also present throughout the duration of the cultures. These results suggest that spontaneous discharge of action potentials does not occur before synapses are formed and K(+) channel types develop differentially in brainstem neurons in vitro.

Fc gammaRIIB is an inhibitory receptor that terminates activation signals initiated by antigen cross-linking of the BCR through the recruitment of SHIP. Fc gammaRIIB can also signal independently of BCR coligation to directly mediate an apoptotic response, requiring only an intact transmembrane domain. Failure to recruit SHIP, either by deletion of SHIP or mutation of Fc gammaRIIB, results in enhanced Fc gammaRIIB-triggered apoptosis. Thus, in the germinal center, where ICs are retained by FDCs, Fc gammaRIIB may be an active determinant in the negative selection of B cells whose BCRs have reduced affinity for antigen as a result of somatic hypermutation. Selection of B cells may represent the sum of opposing signals generated by the interaction of ICs with the BCR and Fc gammaRIIB through pathways modulated by SHIP.

The entry of B lymphocytes into secondary lymphoid organs is a critical step in the development of an immune response, providing a site for repertoire shaping, antigen-induced activation and selection. These events are controlled by signals generated through the B cell antigen receptor (BCR) and are associated with changes in the migration properties of B cells in response to chemokine gradients. The chemokine stromal cell-derived factor (SDF)-1alpha is thought to be one of the driving forces during those processes, as it is produced inside secondary lymphoid organs and induces B lymphocyte migration that arrests upon BCR engagement. The signaling pathway that mediates this arrest was genetically dissected using B cells deficient in specific BCR-coupled signaling components. BCR-induced inhibition of SDF-1alpha chemotaxis was dependent on Syk, BLNK, Btk, and phospholipase C (Plc)gamma2 but independent of Ca2+ mobilization, suggesting that the target of BCR stimulation was a protein kinase C (PKC)-dependent substrate. This target was identified as the SDF-1alpha receptor, CXCR4, which undergoes PKC- dependent internalization upon BCR stimulation. Mutation of the internalization motif SSXXIL in the COOH terminus of CXCR4 resulted in B cells that constitutively expressed this receptor upon BCR engagement. These studies suggest that one pathway by which BCR stimulation results in inhibition of SDF-1alpha migration is through PKC-dependent downregulation of CXCR4.

The cystic fibrosis transmembrane conductance regulator forms an anion-selective channel. We previously showed that charge selectivity, the ability to discriminate between anions and cations, occurs near the cytoplasmic end of the channel. The molecular determinants of charge selectivity, however, are unknown. We investigated the role of Arg352, a residue flanking the predicted cytoplasmic end of the M6 segment, in the mechanism of charge selectivity. We determined the Cl- to Na+ permeability ratio (PCl/PNa) from the reversal potential measured in a 10-fold NaCl gradient. For the wild type, PCl/PNa was 36 (range of 28-51). For the R352H mutant, PCl/PNa was dependent on cytoplasmic pH. At pH 5.4, the PCl/PNa was 33 (range of 27-41), similar to that of the wild type, but at pH 7.2, where the histidine should be largely uncharged, PCl/PNa was 3 (range of 2.9-3.1). For the R352C and R352Q mutants, PCl/PNa was 7 (range of 6-8) and 4 (range of 3.5-4.4), respectively. Furthermore, Na+ which does not carry a significant fraction of the current through the wild type is measurably conducted through R352Q. Thus, the charge of the side chain at position 352 is a strong determinant of charge selectivity. In the wild type, the positive charge on Arg352 contributes to an electrostatic potential in the channel that forms a barrier to cation permeation. Mutation of Arg352 did not alter the halide selectivity sequence. Selectivity among halides must involve other residues.

Autoantibodies and immune complexes are major pathogenic factors in autoimmune injury, responsible for initiation of the inflammatory cascade and its resulting tissue damage. This activation results from the interaction of immunoglobulin (Ig)G Fc receptors containing an activation motif (ITAM) with immune complexes (ICs) and cytotoxic autoantibodies which initiates and propagates an inflammatory response. In vitro, this pathway can be interrupted by coligation to FcgammaRIIB, an IgG Fc receptor containing an inhibitory motif (ITIM). In this report, we describe the in vivo consequences of FcgammaRII deficiency in the inflammatory response using a mouse model of IC alveolitis. At subthreshold concentrations of ICs that fail to elicit inflammatory responses in wild-type mice, FcgammaRII-deficient mice developed robust inflammatory responses characterized by increased hemorrhage, edema, and neutrophil infiltration. Bronchoalveolar fluids from FcgammaRII-/- stimulated mice contain higher levels of tumor necrosis factor and chemotactic activity, suggesting that FcgammaRII deficiency lowers the threshold of IC stimulation of resident cells such as the alveolar macrophage. In contrast, complement- and complement receptor-deficient mice develop normal inflammatory responses to suprathreshold levels of ICs, while FcRgamma-/- mice are completely protected from inflammatory injury. An inhibitory role for FcgammaRII on macrophages is demonstrated by analysis of FcgammaRII-/- macrophages which show greater phagocytic and calcium flux responses upon FcgammaRIII engagement. These data reveal contrasting roles for the cellular receptors for IgG on inflammatory cells, providing a regulatory mechanism for setting thresholds for IC sensitivity based on the ratio of ITIM to ITAM FcgammaR expression. Exploiting the FcgammaRII inhibitory pathway could thus provide a new therapeutic approach for modulating antibody-triggered inflammation.

The Wiskott-Aldrich syndrome (WAS) is a rare immunodeficiency disease affecting mainly platelets and lymphocytes. Here, we show that the WAS gene product, WASp, is tyrosine phosphorylated upon aggregation of the high affinity IgE receptor (Fc epsilonRI) at the surface of RBL-2H3 rat tumor mast cells. Lyn and the Bruton's tyrosine kinase (Btk), two protein tyrosine kinases involved in Fc epsilonRI-signaling phosphorylate WASp and interact with WASp in vivo. Interestingly, expression of a GTPase defective mutant form of CDC42, that interacts with WASp, is accompanied by a substantial increase in WASp tyrosine phosphorylation. This study suggests that activated CDC42 recruits WASp to the plasma membrane where it becomes phosphorylated by Lyn and Btk. We conclude that WASp represents a connection between protein tyrosine kinase signaling pathways and CDC42 function in cytoskeleton and cell growth regulation in hematopoietic cells.

The cystic fibrosis transmembrane conductance regulator (CFTR) forms an anion-selective channel involved in epithelial chloride transport. Recent studies have provided new insights into the structural determinants of the channel's functional properties, such as anion selectivity, single-channel conductance, and gating. Using the scanning-cysteine-accessibility method we identified 7 residues in the M1 membrane-spanning segment and 11 residues in and flanking the M6 segment that are exposed on the water-accessible surface of the protein; many of these residues may line the ion-conducting pathway. The pattern of the accessible residues suggests that these segments have a largely alpha-helical secondary structure with one face exposed in the channel lumen. Our results suggest that the residues at the cytoplasmic end of the M6 segment loop back into the channel, narrowing the lumen, and thereby forming both the major resistance to ion movement and the charge-selectivity filter.

Cross-linking of the antigen receptor on lymphocytes by antigens or antibodies to the receptor results in activation of enzymes of the protein kinase C (PKC) family. Mice homozygous for a targeted disruption of the gene encoding the PKC-betaI and PKC-betaII isoforms develop an immunodeficiency characterized by impaired humoral immune responses and reduced cellular responses of B cells, which is similar to X-linked immunodeficiency in mice. Thus PKC-betaI and PKC-betaII play an important role in B cell activation and may be functionally linked to Bruton's tyrosine kinase in antigen receptor-mediated signal transduction.

Basolateral membranes of microdissected collagenase-treated fragments of renal tubules from the mouse were examined using the cell-attached and the cell-free variants of the patch-clamp technique. With a K(+)-rich solution in the pipette, a highly active, inwardly rectifying K+ channel was observed on intact cells of the cortical collecting tubule (CCT). The mean inward and outward conductances were 38.5 +/- 3.1 pS and 17.3 +/- 1.8 pS, respectively (n = 4). In contrast, cell-attached patches were usually inactive when a Na(+)-rich solution filled the patch pipette. However, another type of channel with a conductance of 20-30 pS exhibited a sparse activity in 4/20 CCT. In excised, inside-out patches, the most frequent channel in CCT had an ohmic unit conductance of 27.1 +/- 1.2 pS (n = 17), excluded anions (PCl/PNa = 0.09), discriminated little between NH4+, K+ and Na+ (PNH4/PNa = 1.5; PK/PNa = 0.9), and was much less permeable to Ca2+ and Ba2+ than to Na+ (PCa/PNa = 0.09; PBa/PNa approximately 0). The cation channel was moderately voltage-dependent, showing a decreased open probability (Po) at negative voltages. It was activated by internal calcium (threshold: 1 mumol/l-0.1 mmol/l calcium), and inhibited by the adenine nucleotides ATP, ADP and AMP with half-maximal inhibition of Po at 1.2 mumol/l AMP. As in other cell models, 3',5'-dichlorodiphenylamine-2-carboxylic acid blocked channel activity when added to the internal surface of the membrane patch. Extending our study to other parts of the renal tubule, we found that the basolateral membranes of the proximal (pars recta), distal convoluted, connecting and outer medullary collecting tubules, the thin descending limb and the medullary thick ascending limb all contained a similar Ca- and ATP-sensitive cation channel. The calcium sensitivity varied from one part to another.