Receptor-regulated ion channels.

Recent advances in the study of receptor-regulated ion channels include the cloning of the genes encoding three types of potassium channel that are favorite targets of receptors for transmitters and hormones. Studies of these channels have also provided a strong indication that G-protein betagamma subunits may gate ion channels via direct protein-protein interactions. Similarities between channel regulation by natriuretic peptides and channel regulation by secreted peptide products of the Alzheimer's beta-amyloid precursor protein offer hints for the existence of a receptor for the latter. There are also other novel examples of channel regulation in excitable and nonexcitable cells, including liver cells and blood cells.

Two types of asymmetric divisions in the Drosophila sensory organ precursor cell lineage.

Asymmetric partitioning of cell-fate determinants during development requires coordinating the positioning of these determinants with orientation of the mitotic spindle. In the Drosophila peripheral nervous system, sensory organ progenitor cells (SOPs) undergo several rounds of division to produce five cells that give rise to a complete sensory organ. Here we have observed the asymmetric divisions that give rise to these cells in the developing pupae using green fluorescent protein fusion proteins. We find that spindle orientation and determinant localization are tightly coordinated at each division. Furthermore, we find that two types of asymmetric divisions exist within the sensory organ precursor cell lineage: the anterior-posterior pI cell-type division, where the spindle remains symmetric throughout mitosis, and the strikingly neuroblast-like apical-basal division of the pIIb cell, where the spindle exhibits a strong asymmetry at anaphase. In both these divisions, the spindle reorientates to position itself perpendicular to the region of the cortex containing the determinant. On the basis of these observations, we propose that two distinct mechanisms for controlling asymmetric cell divisions occur within the same lineage in the developing peripheral nervous system in Drosophila.

Sequoia, a tramtrack-related zinc finger protein, functions as a pan-neural regulator for dendrite and axon morphogenesis in Drosophila.

Morphological complexity of neurons contributes to their functional complexity. How neurons generate different dendritic patterns is not known. We identified the sequoia mutant from a previous screen for dendrite mutants. Here we report that Sequoia is a pan-neural nuclear protein containing two putative zinc fingers homologous to the DNA binding domain of Tramtrack. sequoia mutants affect the cell fate decision of a small subset of neurons but have global effects on axon and dendrite morphologies of most and possibly all neurons. In support of sequoia as a specific regulator of neuronal morphogenesis, microarray experiments indicate that sequoia may regulate downstream genes that are important for executing neurite development rather than altering a variety of molecules that specify cell fates.

Ultrastructural localization of rhodopsin in the vertebrate retina.

Early work by Dewey and collaborators has shown the distribution of rhodopsin in the frog retina. We have repeated these experiments on cow and mouse eyes using antibodies specific to rhodopsin alone. Bovine rhodopsin in emulphogene was purified on an hydroxyapatite column. The purity of this reagent was established by spectrophotometric criteria, by sodium dodecyl sulfate (SDS) gel electrophoresis, and by isoelectric focusing. This rhodopsin was used as an immunoadsorbent to isolate specific antibodies from the antisera of rabbits immunized with bovine rod outer segments solubilized in 2% digitonin. The antibody so prepared was shown by immunoelectrophoresis to be in the IgG class and did not cross-react with lipid extracts of bovine rod outer segments. Papain-digested univalent antibodies (Fab) coupled with peroxidase were used to label rhodopsin in formaldehyde-fixed bovine and murine retinas. In addition to the disk membranes, the plasma membrane of the outer segment, the connecting cilium, and part of the rod inner segment membrane were labeled. We observed staining on both sides of the rod outer segment plasma membrane and the disk membrane. Discrepancies were observed between results of immunolabeling experiments and observations of membrane particles seen in freeze-cleaved specimens. Our experiments indicate that the distribution of membrane particles in freeze cleaving experiments reflects the distribution of membrane proteins. Immunolabeling, on the other hand, can introduce several different types of artifact, unless controlled with extreme care.

Specification of subunit assembly by the hydrophilic amino-terminal domain of the Shaker potassium channel.

The functional heterogeneity of potassium channels in eukaryotic cells arises not only from the multiple potassium channel genes and splice variants but also from the combinatorial mixing of different potassium channel polypeptides to form heteromultimeric channels with distinct properties. One structural element that determines the compatibility of different potassium channel polypeptides in subunit assembly has now been localized to the hydrophilic amino-terminal domain. A Drosophila Shaker B (ShB) potassium channel truncated polypeptide that contains only the hydrophilic amino-terminal domain can form a homomultimer; the minimal requirement for the homophilic interaction has been localized to a fragment of 114 amino acids. Substitution of the amino-terminal domain of a distantly related mammalian potassium channel polypeptide (DRK1) with that of ShB permits the chimeric DRK1 polypeptide to coassemble with ShB.

Role of ER export signals in controlling surface potassium channel numbers.

Little is known about the identity of endoplasmic reticulum (ER) export signals and how they are used to regulate the number of proteins on the cell surface. Here, we describe two ER export signals that profoundly altered the steady-state distribution of potassium channels and were required for channel localization to the plasma membrane. When transferred to other potassium channels or a G protein-coupled receptor, these ER export signals increased the number of functional proteins on the cell surface. Thus, ER export of membrane proteins is not necessarily limited by folding or assembly, but may be under the control of specific export signals.

Cloning of genomic and complementary DNA from Shaker, a putative potassium channel gene from Drosophila.

On the basis of electrophysiological analysis of Shaker mutants, the Shaker locus of Drosophila melanogaster has been proposed to encode a structural component of a voltage-dependent potassium channel, the A channel. Unlike sodium channels, acetylcholine receptors, and calcium channels, K+ channels have not been purified biochemically. To facilitate biochemical studies of a K+ channel, genomic DNA from the Shaker locus has been cloned. Rearrangements in five Shaker mutants have been mapped to a 60-kilobase segment of the genome. Four complementary DNA clones have been analyzed. These clones indicate that the Shaker gene contains multiple exons distributed over at least 65 kilobases of genomic DNA in the region where the mutations mapped. Furthermore, the gene may produce several classes of alternatively spliced transcripts. Two of the complementary DNA clones have been sequenced and their sequences support the hypothesis that Shaker encodes a component of a K+ channel.

Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila.

Potassium currents are crucial for the repolarization of electrically excitable membranes, a role that makes potassium channels a target for physiological modifications that alter synaptic efficacy. The Shaker locus of Drosophila is thought to encode a K+ channel. The sequence of two complementary DNA clones from the Shaker locus is reported here. The sequence predicts an integral membrane protein of 70,200 daltons containing seven potential membrane-spanning sequences. In addition, the predicted protein is homologous to the vertebrate sodium channel in a region previously proposed to be involved in the voltage-dependent activation of the Na+ channel. These results support the hypothesis that Shaker encodes a structural component of a voltage-dependent K+ channel and suggest a conserved mechanism for voltage activation.

Molecular basis for interactions of G protein betagamma subunits with effectors.

Both the alpha and betagamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) communicate signals from receptors to effectors. Gbetagamma subunits can regulate a diverse array of effectors, including ion channels and enzymes. Galpha subunits bound to guanine diphosphate (Galpha-GDP) inhibit signal transduction through Gbetagamma subunits, suggesting a common interface on Gbetagamma subunits for Galpha binding and effector interaction. The molecular basis for interaction of Gbetagamma with effectors was characterized by mutational analysis of Gbeta residues that make contact with Galpha-GDP. Analysis of the ability of these mutants to regulate the activity of calcium and potassium channels, adenylyl cyclase 2, phospholipase C-beta2, and beta-adrenergic receptor kinase revealed the Gbeta residues required for activation of each effector and provides evidence for partially overlapping domains on Gbeta for regulation of these effectors. This organization of interaction regions on Gbeta for different effectors and Galpha explains why subunit dissociation is crucial for signal transmission through Gbetagamma subunits.

Four cDNA clones from the Shaker locus of Drosophila induce kinetically distinct A-type potassium currents in Xenopus oocytes.

The Shaker gene encodes the A-channel of larval and pupal muscle, or one or more of its subunits. Alternative splicing produces messages for several different proteins; two mRNA species have previously been shown to induce the expression of A-currents in Xenopus oocytes. Two additional mRNAs have now been tested and found to produce A-currents in oocytes. The four currents differ in kinetics of inactivation, indicating that the Shaker products may contribute to kinetic diversity in A-channels of the fly and that sequences in both the amino- and carboxy-terminal regions are important for inactivation.

The S4-S5 loop contributes to the ion-selective pore of potassium channels.

Mutagenesis experiments on voltage-gated K+ channels have suggested that the ion-selective pore is comprised mostly of H5 segments. To see whether regions outside of the H5 segment might also contribute to the pore structure, we have studied the effect of single amino acid substitutions in the segment that connects the S4 and S5 putative transmembrane segments (S4-S5 loop) on various permeation properties of Shaker K+ channels. Mutations in the S4-S5 loop alter the Rb+ selectivity, the single-channel K+ and Rb+ conductances, and the sensitivity to open channel block produced by intracellular tetraethylammonium ion, Ba2+, and Mg2+. The block of Shaker K+ channels by intracellular Mg2+ is surprising, but is reminiscent of the internal Mg2+ blockade of inward rectifier K+ channels. The results suggest that the S4-S5 loop constitutes part of the ion-selective pore. Thus, the S4-S5 loop and the H5 segment are likely to contribute to the long pore characteristic of voltage-gated K+ channels.

Hydrophobic substitution mutations in the S4 sequence alter voltage-dependent gating in Shaker K+ channels.

Voltage-activated Na+, Ca2+, and K+ channels contain a common motif, the S4 sequence, characterized by a basic residue at every third position interspersed mainly with hydrophobic residues. The S4 sequence is proposed to function as the voltage sensor and to move in response to membrane depolarization, triggering conformational changes that open the channel. This hypothesis has been tested in previous studies which revealed that mutations of the S4 basic residues often shift the curve of voltage dependence of activation along the voltage axis. We find that comparable or larger shifts are caused by conservative substitutions of hydrophobic residues in the S4 sequence of the Shaker K+ channel. We suggest that the S4 structure plays an essential role in determining the relative stabilities of the closed and open states of the channel.

Molecular basis for K(ATP) assembly: transmembrane interactions mediate association of a K+ channel with an ABC transporter.

K(ATP) channels are large heteromultimeric complexes containing four subunits from the inwardly rectifying K+ channel family (Kir6.2) and four regulatory sulphonylurea receptor subunits from the ATP-binding cassette (ABC) transporter family (SUR1 and SUR2A/B). The molecular basis for interactions between these two unrelated protein families is poorly understood. Using novel trafficking-based interaction assays, coimmunoprecipitation, and current measurements, we show that the first transmembrane segment (M1) and the N terminus of Kir6.2 are involved in K(ATP) assembly and gating. Additionally, the transmembrane domains, but not the nucleotide-binding domains, of SUR1 are required for interaction with Kir6.2. The identification of specific transmembrane interactions involved in K(ATP) assembly may provide a clue as to how ABC proteins that transport hydrophobic substrates evolved to regulate other membrane proteins.

A trafficking checkpoint controls GABA(B) receptor heterodimerization.

Surface expression of GABA(B) receptors requires heterodimerization of GB1 and GB2 subunits, but little is known about mechanisms that ensure efficient heterodimer assembly. We found that expression of the GB1 subunit on the cell surface is prevented through a C-terminal retention motif RXR(R); this sequence is reminiscent of the ER retention/retrieval motif RKR identified in subunits of the ATP-sensitive K+ channel. Interaction of GB1 and GB2 through their C-terminal coiled-coil alpha helices masks the retention signal in GB1, allowing the plasma membrane expression of the assembled complexes. Because individual GABA(B) receptor subunits and improperly assembled receptor complexes are not functional even if expressed on the cell surface, we conclude that a trafficking checkpoint ensures efficient assembly of functional GABA(B) receptors.

Determination of the subunit stoichiometry of an inwardly rectifying potassium channel.

Inwardly rectifying K+ channels are distantly related to their voltage-gated counterparts and possess a structural motif of only two putative transmembrane segments in each subunit. They are formed by the assembly of an unknown number of subunits. We have examined the subunit stoichiometry of a strongly rectifying K+ channel, IRK1, by linking together the coding sequence of three or four subunits and distinguishing channels with different numbers of subunits carrying a double mutation that alters inward rectification and single-channel properties. We find that IRK1 channels, like voltage-gated K+ channels, are tetrameric channels. Interestingly, the high sensitivity to Mg2+ and polyamines, cations that produce inward rectification by blocking the channel pore from the cytoplasmic side is largely retained in a channel containing only one wild-type subunit and three subunits bearing mutations that abolish high affinity Mg2+ and polyamine block.

Mutations that affect the length, fasciculation, or ventral orientation of specific sensory axons in the Drosophila embryo.

In wild-type Drosophila embryos, five lateral chordotonal (lch) axons in each abdominal hemisegment originate from a midlaterally positioned cluster of neurons and grow, fasciculate, and orient ventrally as they connect with targets in the CNS. We have identified 22 recessive lethal mutations in 12 complementation groups, 8 of which are novel, that differentially affect lch axon growth, fasciculation, or ventral orientation. Mutations in 3 loci result in shorter, but fasciculated and ventrally directed axon bundles. Mutations in 4 complementation groups cause lch axon defasciculation. Mutations in 7 complementation groups cause some lch axon bundles to grow dorsally along a trajectory 180 degrees from normal.

Control of rectification and permeation by residues in two distinct domains in an inward rectifier K+ channel.

Inwardly rectifying K+ channels conduct more inward than outward current as a result of voltage-dependent block of the channel pore by intracellular Mg2+ and polyamines. We investigated the molecular mechanism and structural determinants of inward rectification and ion permeation in a strongly rectifying channel, IRK1. Block by Mg2+ and polyamines is found not to conform to one-to-one binding, suggesting that a channel pore can accommodate more than one blocking particle. A negatively charged amino acid in the hydrophilic C-terminal domain is found to be critical for both inward rectification and ion permeation. This residue and a negatively charged residue in the putative second transmembrane segment (M2) contribute independently to high affinity binding of Mg2+ and polyamines. Mutation of this residue also induces Mg(2+)- and polyamine-independent inward rectification and dramatically alters single-channel behavior. We propose that the hydrophilic C-terminal domain comprises part of the channel pore and that involvement of both hydrophilic and hydrophobic domains in pore lining may provide a molecular basis for the multi-ion, long-pore nature of inwardly rectifying K+ channels.

tramtrack acts downstream of numb to specify distinct daughter cell fates during asymmetric cell divisions in the Drosophila PNS.

Asymmetric cell divisions allow a sensory organ precursor (SOP) cell to generate a neuron and its support cells in the Drosophila PNS. We demonstrate a role of tramtrack (ttk), previously identified as a zinc finger-containing putative transcription factor, in the determination of different daughter cell fates. Both loss of function and overexpression of ttk affect the fates of the SOP progeny. Whereas loss of ttk function transforms support cells to neurons, ttk overexpression results in the reverse transformation. ttk is expressed in support cells but not in neurons. It has been shown that numb, a membrane-associated protein asymmetrically distributed during the SOP division, confers different daughter cell fates. Loss of ttk or numb function results in reciprocal cell fate transformation. Epistatic studies suggest that ttk acts downstream of numb. We propose that ttk executes the command dictated by asymmetrically localized numb to specify distinct daughter cell fates during multiple asymmetric divisions.

Control of daughter cell fates during asymmetric division: interaction of Numb and Notch.

During development of the Drosophila peripheral nervous system, a sensory organ precursor (SOP) cell undergoes rounds of asymmetric divisions to generate four distinct cells of a sensory organ. Numb, a membrane-associated protein, is asymmetrically segregated into one daughter cell during SOP division and acts as an inherited determinant of cell fate. Here, we show that Notch, a transmembrane receptor mediated cell-cell communication, functions as a binary switch in cell fate specification during asymmetric divisions of the SOP and its daughter cells in embryogenesis. Moreover, numb negatively regulates Notch, probably through direct protein-protein interaction that requires the phosphotyrosine-binding (PTB) domain of Numb and either the RAM23 region or the very C-terminal end of Notch. Notch then positively regulates a transcription factor encoded by tramtrack (ttk). This leads to Ttk expression in the daughter cell that does not inherit Numb. Thus, the inherited determinant Numb bestows a bias in the machinery for cell-cell communication to allow the specification of distinct daughter cell fates.

A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels.

Proper ion channel function often requires specific combinations of pore-forming alpha and regulatory beta subunits, but little is known about the mechanisms that regulate the surface expression of different channel combinations. Our studies of ATP-sensitive K+ channel (K(ATP)) trafficking reveal an essential quality control function for a trafficking motif present in each of the alpha (Kir6.1/2) and beta (SUR1) subunits of the K(ATP) complex. We show that this novel motif for endoplasmic reticulum (ER) retention/retrieval is required at multiple stages of K(ATP) assembly to restrict surface expression to fully assembled and correctly regulated octameric channels. We conclude that exposure of a three amino acid motif (RKR) can explain how assembly of an ion channel complex is coupled to intracellular trafficking.