Molecular mechanism of use-dependent calcium channel block by phenylalkylamines: Role of���inactivation

The role of channel inactivation in the molecular mechanism of calcium (Ca2+) channel block by phenylalkylamines (PAA) was analyzed by designing mutant Ca2+ channels that carry the high affinity determinants of the PAA receptor site [Hockerman, G. H., Johnson, B. D., Scheuer, T., and Catterall, W. A. (1995) J. Biol. Chem. 270, 22119���22122] but inactivate at different rates. Use-dependent block by PAAs was studied after expressing the mutant Ca2+ channels in Xenopus oocytes. Substitution of single putative pore-orientated amino acids in segment IIIS6 by alanine (F-1499-A, F-1500-A, F-1510-A, I-1514-A, and F-1515-A) gradually slowed channel inactivation and simultaneously reduced inhibition of barium currents (IBa) by (���)D600 upon depolarization by 100 ms steps at 0.1 Hz. This apparent reduction in drug sensitivity was only evident if test pulses were applied at a low frequency of 0.1 Hz and almost disappeared at the frequency of 1 Hz. (���)D600 slowed IBa recovery after maintained membrane depolarization (1���3 sec) to a comparable extent in all channel constructs. A drug-induced delay in the onset of IBa recovery from inactivation suggests that PAAs promote the transition to a deep inactivated channel conformation. These findings indicate that apparent PAA sensitivity of Ca2+ channels is not only defined by drug interaction with its receptor site but also crucially dependent on intrinsic gating properties of the channel molecule. A molecular model for PAA-Ca2+ channel interaction that accounts for the relationship between drug induced inactivation and channel block by PAA is proposed.

Basolateral membrane targeting of a renal-epithelial inwardly rectifying potassium channel from the cortical collecting duct, CCD-IRK3, in���MDCK���cells

We recently cloned an inward-rectifying K channel (Kir) cDNA, CCD-IRK3 (mKir 2.3), from a cortical collecting duct (CCD) cell line. Although this recombinant channel shares many functional properties with the ���small-conductance��� basolateral membrane Kir channel in the CCD, its precise subcellular localization has been difficult to elucidate by conventional immunocytochemistry. To circumvent this problem, we studied the targeting of several different epitope-tagged CCD-IRK3 in a polarized renal epithelial cell line. Either the 11-amino acid span of the vesicular stomatitis virus (VSV) G glycoprotein (P5D4 epitope) or a 6-amino acid epitope of the bovine papilloma virus capsid protein (AU1) was genetically engineered on the extreme N terminus of CCD-IRK3. As determined by patch-clamp and two-microelectrode voltage-clamp analyses in Xenopus oocytes, neither tag affected channel function; no differences in cation selectivity, barium block, single channel conductance, or open probability could be distinguished between the wild-type and the tagged constructs. MDCK cells were transfected with tagged CCD-IRK3, and several stable clonal cell lines were generated by neomycin-resistance selection. Immunoprecipitation studies with anti-P5D4 or anti-AU1 antibodies readily detected the predicted-size 50-kDa protein in the transfected cells lines but not in wild-type or vector-only (PcB6) transfected MDCK cells. As visualized by indirect immunofluorescence and confocal microscopy, both the tagged CCD-IRK3 forms were exclusively detected on the basolateral membrane. To assure that the VSV G tag was not responsible for the targeting, the P5D4 epitope modified by a site-directed mutagenesis (Y2F) to remove a potential basolateral targeting signal contained in this tag. VSV(Y2F) was also detected exclusively on the basolateral membrane, confirming bona fide IRK3 basolateral expression. These observations, with our functional studies, suggest that CCD-IRK3 may encode the small-conductance CCD basolateral K channel.

Permeant ion regulation of N-methyl-d-aspartate receptor channel block by Mg2+

Block of the channel of N-methyl-d-aspartate (NMDA) receptors by external Mg2+ (Mgo2+) has broad implications for the many physiological and pathological processes that depend on NMDA receptor activation. An essential property of channel block by Mgo2+ is its powerful voltage dependence. A widely cited explanation for the strength of the voltage dependence of block is that the Mgo2+-binding site is located deep in the channel of NMDA receptors; Mgo2+ then would sense most of the membrane potential field during block. However, recent electrophysiological and mutagenesis studies suggest that the blocking site cannot be deep enough to account for the voltage dependence of Mgo2+ block. Here we describe the basis for this discrepancy: the magnitude and voltage dependence of channel block by Mgo2+ are strongly regulated by external and internal permeant monovalent cations. Our data support a model in which access to the channel by Mgo2+ is prevented when permeant ion-binding sites at the external entrance to the channel are occupied. Mgo2+ can block the channel only when the permeant ion-binding sites are unoccupied and then can either unblock back to the external solution or permeate the channel. Unblock to the external solution is prevented if external permeant ions bind while Mg2+ blocks the channel, although permeation is still permitted. The model provides an explanation for the strength of the voltage dependence of Mgo2+ block and quantifies the interdependence of permanent and blocking ion binding to NMDA receptors.

The active oligomeric state of the minimalistic influenza virus M2 ion channel is a���tetramer

The influenza A virus M2 integral membrane protein is an ion channel that permits protons to enter virus particles during uncoating of virions in endosomes and also modulates the pH of the trans-Golgi network in virus-infected cells. The M2 protein is a homo-oligomer of 97 residues, and analysis by chemical cross-linking and SDS/PAGE indicates M2 forms a tetramer. However, a higher order molecular form is sometimes observed and, thus, it is necessary to determine the active form of the molecule. This was done by studying the currents of oocytes that expressed mixtures of the wild-type M2 protein (epitope tagged) and the mutant protein M2-V27S, which is resistant to the inhibitor amantadine. The composition of mixed oligomers of the two proteins expressed at the plasma membrane of individual oocytes was quantified after antibody capture of the cell surface expressed molecules and it was found that the subunits mixed freely. When the ratio of wild-type to mutant protein subunits was 0.85:0.15, the amantadine sensitivity was reduced to 50% and for a ratio of 0.71:0.29 to 20%. These results are consistent with the amantadine-resistant mutant being dominant and the oligomeric state being a tetramer.

Channel formation by antiapoptotic protein���Bcl-2

Bcl-2 is the prototypical member of a large family of apoptosis-regulating proteins, consisting of blockers and promoters of cell death. The three-dimensional structure of a Bcl-2 homologue, Bcl-XL, suggests striking similarity to the pore-forming domains of diphtheria toxin and the bacterial colicins, prompting exploration of whether Bcl-2 is capable of forming pores in lipid membranes. Using chloride efflux from KCl-loaded unilamellar lipid vesicles as an assay, purified recombinant Bcl-2 protein exhibited pore-forming activity with properties similar to those of the bacterial toxins, diphtheria toxin, and colicins, i.e., dependence on low pH and acidic lipid membranes. In contrast, a mutant of Bcl-2 lacking the two core hydrophobic ��-helices (helices 5 and 6), predicted to be required for membrane insertion and channel formation, produced only nonspecific effects. In planar lipid bilayers, where detection of single channels is possible, Bcl-2 formed discrete ion-conducting, cation-selective channels, whereas the Bcl-2 (��h5, 6) mutant did not. The most frequent conductance observed (18 �� 2 pS in 0.5 M KCl at pH 7.4) is consistent with a four-helix bundle structure arising from Bcl-2 dimers. However, larger channel conductances (41 �� 2 pS and 90 �� 10 pS) also were detected with progressively lower occurrence, implying the step-wise formation of larger oligomers of Bcl-2 in membranes. These findings thus provide biophysical evidence that Bcl-2 forms channels in lipid membranes, suggesting a novel function for this antiapoptotic protein.

Voltage-controlled gating in a large conductance Ca2+-sensitive K+channel���(hslo)

Large conductance calcium- and voltage-sensitive K+ (MaxiK) channels share properties of voltage- and ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca2+ favor pore opening. We demonstrate the presence of voltage sensor rearrangements with voltage (gating currents) whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca2+ concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4���5 elementary charges.

Congestive heart failure in rats is associated with increased expression and targeting of aquaporin-2 water channel in collecting���duct

We tested whether severe congestive heart failure (CHF), a condition associated with excess free-water retention, is accompanied by altered regulation of the vasopressin-regulated water channel, aquaporin-2 (AQP2), in the renal collecting duct. CHF was induced by left coronary artery ligation. Compared with sham-operated animals, rats with CHF had severe heart failure with elevated left ventricular end-diastolic pressures (LVEDP): 26.9 �� 3.4 vs. 4.1 �� 0.3 mmHg, and reduced plasma sodium concentrations (142.2 �� 1.6 vs. 149.1 �� 1.1 mEq/liter). Quantitative immunoblotting of total kidney membrane fractions revealed a significant increase in AQP2 expression in animals with CHF (267 �� 53%, n = 12) relative to sham-operated controls (100 �� 13%, n = 14). In contrast, immunoblotting demonstrated a lack of an increase in expression of AQP1 and AQP3 water channel expression, indicating that the effect on AQP2 was selective. Furthermore, postinfarction animals without LVEDP elevation or plasma Na reduction showed no increase in AQP2 expression (121 �� 28% of sham levels, n = 6). Immunocytochemistry and immunoelectron microscopy demonstrated very abundant labeling of the apical plasma membrane and relatively little labeling of intracellular vesicles in collecting duct cells from rats with severe CHF, consistent with enhanced trafficking of AQP2 to the apical plasma membrane. The selective increase in AQP2 expression and enhanced plasma membrane targeting provide an explanation for the development of water retention and hyponatremia in severe CHF.

Scanning mutagenesis of the putative transmembrane segments of Kir2.1, an inward rectifier potassium���channel

Structural models of inward rectifier K+ channels incorporate four identical or homologous subunits, each of which has two hydrophobic segments (M1 and M2) which are predicted to span the membrane as �� helices. Since hydrophobic interactions between proteins and membrane lipids are thought to be generally of a nonspecific nature, we attempted to identify lipid-contacting residues in Kir2.1 as those which tolerate mutation to tryptophan, which has a large hydrophobic side chain. Tolerated mutations were defined as those which produced measurable inwardly rectifying currents in Xenopus oocytes. To distinguish between water-accessible positions and positions adjacent to membrane lipids or within the protein interior we also mutated residues in M1 and M2 individually to aspartate, since an amino acid with a charged side chain should not be tolerated at lipid-facing or interior positions, due to the energy cost of burying a charge in a hydrophobic environment. Surprisingly, 17 out of 20 and 17 out of 22 non-tryptophan residues in M1 and M2, respectively, tolerated being mutated to tryptophan. Moreover, aspartate was tolerated at 15 out of 22 and 15 out of 21 non-aspartate M1 and M2 positions respectively. Periodicity in the pattern of tolerated vs. nontolerated mutations consistent with �� helices or �� strands did not emerge convincingly from these data. We consider the possibility that parts of M1 and M2 may be in contact with water.

Calcium channel activation and self-biting in mice

The L type calcium channel agonist (��)Bay K 8644 has been reported to cause characteristic motor abnormalities in adult mice. The current study shows that administration of this drug can also cause the unusual phenomenon of self-injurious biting, particularly when given to young mice. Self-biting is provoked by injecting small quantities of (��)Bay K 8644 directly into the lateral ventricle of the brain, suggesting a central effect of the drug. Similar behaviors can be provoked by administration of another L type calcium channel agonist, FPL 64176. The self-biting provoked by (��)Bay K 8644 can be inhibited by pretreating the mice with dihydropyridine L type calcium channel antagonists such as nifedipine, nimodipine, or nitrendipine. However, self-biting is not inhibited by nondihydropyridine antagonists including diltiazem, flunarizine, or verapamil. The known actions of (��)Bay K 8644 as an L type calcium channel agonist, the reproduction of similar behavior with another L type calcium channel agonist, and the protection afforded by certain L type calcium channel antagonists implicate calcium channels in the mediation of the self-biting behavior. This phenomenon provides a model for studying the neurobiology of this unusual behavior.

Ablation of P/Q-type Ca2+ channel currents, altered synaptic transmission, and progressive ataxia in mice lacking the ��1A-subunit

The Ca2+ channel ��1A-subunit is a voltage-gated, pore-forming membrane protein positioned at the intersection of two important lines of research: one exploring the diversity of Ca2+ channels and their physiological roles, and the other pursuing mechanisms of ataxia, dystonia, epilepsy, and migraine. ��1A-Subunits are thought to support both P- and Q-type Ca2+ channel currents, but the most direct test, a null mutant, has not been described, nor is it known which changes in neurotransmission might arise from elimination of the predominant Ca2+ delivery system at excitatory nerve terminals. We generated ��1A-deficient mice (��1A���/���) and found that they developed a rapidly progressive neurological deficit with specific characteristics of ataxia and dystonia before dying ���3���4 weeks after birth. P-type currents in Purkinje neurons and P- and Q-type currents in cerebellar granule cells were eliminated completely whereas other Ca2+ channel types, including those involved in triggering transmitter release, also underwent concomitant changes in density. Synaptic transmission in ��1A���/��� hippocampal slices persisted despite the lack of P/Q-type channels but showed enhanced reliance on N-type and R-type Ca2+ entry. The ��1A���/��� mice provide a starting point for unraveling neuropathological mechanisms of human diseases generated by mutations in ��1A.

Phospholipase C-linked receptors regulate the ATP-sensitive potassium channel by means of phosphatidylinositol 4,5-bisphosphate metabolism

In the COS7 cells transfected with cDNAs of the Kir6.2, SUR2A, and M1 muscarinic receptors, we activated the ATP-sensitive potassium (KATP) channel with a K+ channel opener and recorded the whole-cell KATP current. The KATP current was reversibly inhibited by the stimulation of the M1 receptor, which is linked to phospholipase C (PLC) by the Gq protein. The receptor-mediated inhibition was observed even when protein kinase C (PKC) was inhibited by H-7 or by chelating intracellular Ca2+ with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N���,N���-tetraacetate (BAPTA) included in the pipette solution. However, the receptor-mediated inhibition was blocked by U-73122, a PLC inhibitor. M1-receptor stimulation failed to inhibit the KATP current activated by the injection of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) through the whole-cell patch pipette. The receptor-mediated inhibition became irreversible when the replenishment of PIP2 was blocked by wortmannin (an inhibitor of phosphatidylinositol kinases), or by including adenosine 5���-[��,�����imido]triphosphate (AMPPNP, a nonhydrolyzable ATP analogue) in the pipette solution. In inside-out patch experiments, the ATP sensitivity of the KATP channel was significantly higher when the M1 receptor in the patch membrane was stimulated by acetylcholine. The stimulatory effect of pinacidil was also attenuated under this condition. We postulate that stimulation of PLC-linked receptors inhibited the KATP channel by increasing the ATP sensitivity, not through PKC activation, but most probably through changing PIP2 levels.

Interaction of batrachotoxin with the local anesthetic receptor site in transmembrane segment IVS6 of the voltage-gated sodium channel

The voltage-gated sodium channel is the site of action of more than six classes of neurotoxins and drugs that alter its function by interaction with distinct, allosterically coupled receptor sites. Batrachotoxin (BTX) is a steroidal alkaloid that binds to neurotoxin receptor site 2 and causes persistent activation. BTX binding is inhibited allosterically by local anesthetics. We have investigated the interaction of BTX with amino acid residues I1760, F1764, and Y1771, which form part of local anesthetic receptor site in transmembrane segment IVS6 of type IIA sodium channels. Alanine substitution for F1764 (mutant F1764A) reduces tritiated BTX-A-20-��-benzoate binding affinity, causing a 60-fold increase in Kd. Alanine substitution for I1760, which is adjacent to F1764 in the predicted IVS6 transmembrane alpha helix, causes only a 4-fold increase in Kd. In contrast, mutant Y1771A shows no change in BTX binding affinity. For wild-type and mutant Y1771A, BTX shifted the voltage for half-maximal activation ���40 mV in the hyperpolarizing direction and increased the percentage of noninactivating sodium current to ���60%. In contrast, these BTX effects were eliminated completely for the F1764A mutant and were reduced substantially for mutant I1760A. Our data suggest that the BTX receptor site shares overlapping but nonidentical molecular determinants with the local anesthetic receptor site in transmembrane segment IVS6 as well as having unique molecular determinants in transmembrane segment IS6, as demonstrated in previous work. Evidently, BTX conforms to a domain���interface allosteric model of ligand binding and action, as previously proposed for calcium agonist and antagonist drugs acting on l-type calcium channels.

KATP channel inhibition by ATP requires distinct functional domains of the cytoplasmic C terminus of the pore-forming subunit

ATP-sensitive potassium (���KATP���) channels are rapidly inhibited by intracellular ATP. This inhibition plays a crucial role in the coupling of electrical activity to energy metabolism in a variety of cells. The KATP channel is formed from four each of a sulfonylurea receptor (SUR) regulatory subunit and an inwardly rectifying potassium (Kir6.2) pore-forming subunit. We used systematic chimeric and point mutagenesis, combined with patch-clamp recording, to investigate the molecular basis of ATP-dependent inhibition gating of mouse pancreatic �� cell KATP channels expressed in Xenopus oocytes. We identified distinct functional domains of the presumed cytoplasmic C-terminal segment of the Kir6.2 subunit that play an important role in this inhibition. Our results suggest that one domain is associated with inhibitory ATP binding and another with gate closure.

Perceptual learning reflects external noise filtering and internal noise reduction through channel reweighting

To investigate the nature of plasticity in the adult visual system, perceptual learning was measured in a peripheral orientation discrimination task with systematically varying amounts of external (environmental) noise. The signal contrasts required to achieve threshold were reduced by a factor or two or more after training at all levels of external noise. The strong quantitative regularities revealed by this novel paradigm ruled out changes in multiplicative internal noise, changes in transducer nonlinearites, and simple attentional tradeoffs. Instead, the regularities specify the mechanisms of perceptual learning at the behavioral level as a combination of external noise exclusion and stimulus enhancement via additive internal noise reduction. The findings also constrain the neural architecture of perceptual learning. Plasticity in the weights between basic visual channels and decision is sufficient to account for perceptual learning without requiring the retuning of visual mechanisms.

Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to Eag and cyclic nucleotide-gated���channels

We have isolated a novel cDNA, that appears to represent a new class of ion channels, by using the yeast two-hybrid system and the SH3 domain of the neural form of Src (N-src) as a bait. The encoded polypeptide, BCNG-1, is distantly related to cyclic nucleotide-gated channels and the voltage-gated channels, Eag and H-erg. BCNG-1 is expressed exclusively in the brain, as a glycosylated protein of ���132 kDa. Immunohistochemical analysis indicates that BCNG-1 is preferentially expressed in specific subsets of neurons in the neocortex, hippocampus, and cerebellum, in particular pyramidal neurons and basket cells. Within individual neurons, the BCNG-1 protein is localized to either the dendrites or the axon terminals depending on the cell type. Southern blot analysis shows that several other BCNG-related sequences are present in the mouse genome, indicating the emergence of an entire subfamily of ion channel coding genes. These findings suggest the existence of a new type of ion channel, which is potentially able to modulate membrane excitability in the brain and could respond to regulation by cyclic nucleotides.