Lester / Davidson Lab 1998 Annual Report

We are examining the physiological role of nicotinic acetylcholine receptors (AChR) in the nervous system by introducing mutations into the corresponding mouse genes that significantly alter the function of the receptors. We have chosen for our initial studies the a4 subunit which, in association with the b2 subunit, constitutes the most abundant form of AChR in the central nervous system. Mutations in the M2 region of the muscle AChR have been extensively studied in the Xenopus oocyte system in our lab. The Leu9'Ser mutation in the M2 transmembrane region, which lines the pore of the ion channel, increases the apparent sensitivity to ACh (decreases the EC₅₀) by about 10-fold for every mutated subunit in the mouse muscle receptor. Naturally occurring mutations of this type in the M2 region of the AChR of human muscle produce myasthenic syndromes known as Slow Channel Syndrome which are caused by a prolonged synaptic potential. Neuronal a4b2 receptors with a Leu9'Ser mutation in the a subunit have ACh sensitivity about 100-fold higher (100-fold lower EC₅₀) than wild type receptors when examined in the Xenopusoocyte system. The M2 transmembrane region was mapped to exon 5 of mouse a4 genomic DNA. We introduced a point mutation (Leu9'Ser) by site directed mutagenesis in a 9.5 kb fragment of genomic DNA containing exon 5. The targeting vector was then constructed by introducing a neomycin cassette

flanked by loxP sites immediately downstream from exon 5 to provide positive selection. A diptheria toxin (DT) cassette was introduced in the polylinker of the vector adjacent to the genomic DNA to provide negative selection for random insertion of the construct into the genome (when there is random insertion the diphteria toxin is expressed and the cells die). The construct was introduced into embryonic stem (ES) cells by electroporation and neomycin resistant clones were selected. We screened the neomycin resistant clones for homologous recombination which will incorporate the Leu9'Ser mutation into the mouse genome. Several clones were identified by Southern blotting, PCR, and sequencing. The positive ES cell clones will be injected into blastocysts to generate mice carrying the mutation. To avoid interference with transcription and RNA splicing the neo cassette will be deleted on breeding with a mouse line expressing cre recombinase. Progeny will be then analyzed for changes in physiology and behavior. This project is a collaboration with Dr. James Boulter (UCLA).

Hyperactive 5HT3 receptor knock-in mice

Hong Dang, Cesar Labarca, Purnima Deshpande

To study how ion channels function in the brains of intact animals, we are introducing a hyper-active 5HT3 serotonin receptor channel into the native genetic environment of mice by gene-targeting (knock-in). The choice of this particular ligand-gated ion channel was based on (1) the likelihood that this receptor is encoded by a single gene (there is no genetic evidence for other subunits, thus little chance for compensation from other subunits) and (2) the low calcium selectivity in vitro of the 5HT3 receptor, which indicates that it is unlikely to be cytotoxic.

Heterologous expression in Xenopus oocytes was used to examine changes of the receptor’s sensitivity to serotonin caused by various point mutations. A valine to serine mutation in the M2 channel-lining domain increased the sensitivity of the receptor to serotonin by 100 fold. Mixed injection of wild type and mutant mRNAs at 1:1 ratio into oocytes apparently gave rise to a range of heteromultimers that decreased the overall EC₅₀ by 10 fold.

The same mutation was made in a piece of genomic DNA containing the exon encoding the M2 region. A neomycin resistance gene (Neo) flanked by two loxP sequences was inserted in the nearby intron to provide a positive selection for homologous recombinants. A thymidine kinase gene (TK) was attached at one end of the genomic insert to serve as a negative selection for random integration events. This DNA was electroporated into mouse embryonic stem (ES) cells, and the correct recombinants, where one copy of the wild-type exon replaced by the mutated DNA, were selected by G418 and FIAU resistance. The removal of the Neo cassette from the genomic DNA was achieved through transient transfection (electroporation) with the Cre gene, whose product, CRE recombinase, deletes DNA sequences between two similarly oriented loxP units, leaving a footprint of 34 bps in the intron.

Several clones of ES cells containing the intended mutation have been identified through a combination of PCR, Southern blot, and sequencing analyses. These will be used to generate mice carrying the mutant gene. These animals may then be analyzed to identify specific alterations associated with the mutation.

This work is carried out in collaboration with the David Julius lab (UCSF).

Gene targeting inactivation of a glial inwardly rectifying K+ channel in mice

Paulo Kofuji, Purnima Deshpande and Cesar Labarca

Inwardly rectifying K⁺ (IRK) channels are highly expressed in glial cells and neuronal cells. The biophysical properties of these channels are compatible with a role in the passive uptake of K⁺ into glia after neuronal activity and have thus been implicated to serve a role in potassium homeostasis in brain. Glial K⁺ channels may also be crucial in modulating glial proliferation. The channel Kir 4.1 (BIR10) has been recently identified as the major IRK channel subtype expressed in glial cells (1). We have initiated gene targeting experiments to inactivate the functional expression of Kir 4.1 in mice brain. A lambda genomic library derived from mice (129/SveTac substrain) was screened using a Kir 4.1 cDNA probe and several individual genomic clones were isolated. Restriction mapping and sequencing analysis indicated that Kir 4.1 polypeptide is encoded by a single exon. A genomic fragment of 5.8 kb was then used in the construction of a replacement type targeting vector. A neomycin phosphotransferase cassette was introduced roughly in the middle of the coding exon and a thymidine kinase cassette was introduced in the flanking 5’ end. Electroporation of CJ7 embryonic stem cells with the targeting vector was performed and the cells were maintained in media supplemented with G418 and FIAU for 8 days. Three hundred and eighty four colonies were further expanded for Southern blot analysis. Four clones revealed the expected patterns on Southern blot analysis for homologous recombinants and were used for further analysis. Three of these clones were injected into blastocysts and surgically transferred into pseudopregnant recipients. Heterozygous animals for the targeted gene will be mated with each other to produce mice homozygous for the targeted gene Kir 4.1. The final stage in the process will be to study homozygous animals for the mutation induced by gene replacement. These animals will provide a unique opportunity to investigate many of standing questions related to glial function in physiological and pathophysiological states.

(1) Takumi et al., JBC 270: 16339-16346, 1995

Neurobiology of brain P2X4 receptor channels for extracellular ATP.

Baljit Singh Khakh

P2X receptors are ATP-gated cation channels for extracellular ATP. Molecular cloning has identified seven genes encoding distinct P2X receptor subunits (P2X1 to P2X7) and these are widespread in the brain. However, little is known about the functional properties, subunit composition or patho/physiological roles of P2X receptors in native neurons where they can mediate fast synaptic transmission and presynaptic modulation of neurotransmitter release. Assessing the function of individual P2X receptor subunits is critical to determine their contribution to neuronal physiology.

Currently, as with other ion channels, P2X receptors in the brain are being studied using in situ hybridization, immunocytochemistry, electrophysiology and the generation of knock-out mice. Ongoing work in our laboratory has in addition been using new approaches to study the full function of nicotinic and 5HT3 receptor channels. These methods include the in vitro design of desired hypersensitive mutant channels and their in vivo introduction into mice by gene targeting, a method termed ‘‘knock-in’’ expression as opposed to knock-out where the native genes are inactivated. Mutant mice are then studied in detail from whole organism behavior to studies of cellular physiology. The hope is that these methods will further our understanding of the functional expression, distribution and role of individual channel subunits in cellular physiology in a system where they are easier to detect. The hypersensitive channel approach and the transgenic mice may be predictive for phenotypes of P2X receptor inherited diseases and for the consequences of ongoing P2X receptor activation that is likely to occur when cells die and release their ATP. These techniques will be used to study the ubiquitous P2X4 receptor subunit; unraveling its physiological function in the CNS is of paramount importance. Using in vitro site-directed mutagenesis pore forming regions of P2X4 receptors shall be identified; mutant channels will then be functionally tested using heterologous expression systems. Mutations that cause increased sensitivity to ATP will be studied in particular detail. Once a desired hypersensitive channel is identified it will be expressed in vivo in mice using targeted exon replacement. Mutant and control mice will be studied in respect to their the behavior, development and cellular physiology. Of particular interest will be the occurrence and properties of ATP mediated synaptic transmission in key brain nuclei.

Effects of normal and mutant excitability proteins expressed at physiological levels in hippocampal neurons

Hendrickje Nadeau, Baljit Singh Khakh, David J. Anderson

Selectively reducing the excitability of glutamatergic neurons will (1) allow for the creation of animal models of human neurological disorders and (2) provide insight into possible treatments for conditions such as epilepsy, stroke, and excitotoxin poisoning. Because there are no pharmacologic agents that target subsets of glutamatergic cells, we are focusing on genetic approaches, combining inducible and cell-specific promoters with genes that either prevent the firing of action potentials in the cells in which they are expressed (presynaptic silencing) or, alternatively, prevent that cell from communicating with its neighbors (postsynaptic silencing). For our first experiments, we are investigating the kidney inward rectifier ROMK1 as a possible example of the former, and a GTPase deficient mutant of the vesicular release protein Rab3a for the latter.

Our goal is to construct an in vitro model that mimics as much as possible conditions that can be obtained in transgenic mice and in human gene therapy. Using the lentiviral (HIV)-based system of Trono et al. (1996), we have succeeded in stably transferring a low number of copies of the selected genes into cultured rat hippocampal neurons. The vectors are bicistronic, expressing green fluorescent protein along with the gene of interest to allow for easy identification of infected cells. We are performing patch-clamp and imaging experiments on the infected cultures, examining the inserted gene's effect on the infected cell and its synaptically-connected neighbors. The high infectivity and low toxicity of the virus allow us to adjust the percentage of infected cells in each dish. The virus stably integrates into a random site in the genome, making this a very good model of the situation in a transgenic mouse.

Future goals involve the creation of a tetracycline-inducible retroviral vector, the expression of other constitutively active potassium channels, and the production of transgenic mice.

Encoding properties of brain neurones silenced by gene transfer of a kidney potassium channel (Kir1.1)

Baljit S. Khakh, Daniel Sahlein, Hendrickje Nadeau, Sheri McKinney, David J. Anderson

A major determinant of cellular excitability is the interplay of voltage-dependent ion channels. Potassium channels hyperpolarize neurons and thus regulate neurotransmitter release, synaptic activity, action potential duration and resting membrane potentials. Kir1.1 (ROMK1) is a weakly inwardly rectifying potassium channel that was cloned from the kidney and does not seem to be widely expressed in brain neurons. Functional properties of Kir1.1, such as high potassium conductance at resting membrane potentials and lack of channel inactivation make this channel a candidate for use in silencing neurons by gene transfer using viruses (see abstract by Nadeau et al). As an initial test we are using cultured embryonic hippocampal neurons and assaying properties of cell excitability in neurons that are either (1) uninfected neurons, (2) infected with a virus for Kir1.1.+GFP, and (3) infected with a control virus for GFP alone. The cellular parameters that are of interest include differences in resting membrane potentials and the threshold currents for action potential firing (see Ehrengruber et al., 1997 for details). Ongoing experiments indicate that infection of hippocampal neurons with Kir1.1 results in membrane hyperpolarization of about 6 mV when compared to uninfected neurons.

In future work we will test whether infection of neurons with Kir1.1 can modulate both pre- and post- synaptic activity. We will test the validity of silencing neurons by gene transfer of Kir1.1 in various in vitro models of pathophysiologies whose origins are excessive neuronal excitation, eg. epileptiform activity in brain neurons and firing properties of peripheral pain sensors.

Investigating the Role of Proline in Ligand-Gated Ion Channel Receptor Gating

Pamela M. England, Hong Dang, Yinong Zhang, Dennis A. Dougherty*

A conserved proline residue is found in the first transmembrane domain (M1) of every subunit of the nicotinic acetylcholine receptor superfamily (nAChR) of ligand-gated ion channels. The location of this conserved proline residue, between the extracellular ligand-binding domain and the main channel-lining domain (M2) in the primary sequence, suggests an involvement in the transduction of ligand-binding signals to the gating of the ion channel. Indeed, replacing this residue in both the muscle and neuronal isoforms of the nAChR with amino acids other than proline results in non-functional channels, yet still containing an intact agonist binding site, expressed on the surface of cells. Using the nonsense codon suppression methodology, we have replaced the peptide backbone at this site with an ester backbone in both the nAChR and the serotonin 5HT3 receptors. The amide to ester backbone modification provides functional receptors even with a sidechain that is not proline-like. It appears that this M1 proline residue plays a role in gating and suggests the structural perturbations originating in the agonist binding site travel to the pore region via the M1 domain.

*Division of Chemistry and Chemical Engineering

Backbone mutations in transmembrane domains of the nicotinic acetylcholine receptor: implications for the mechanism of gating

Pamela M. England, Yinong Zhang, Dennis A. Dougherty*

An approach to identify backbone conformational changes underlying ion channel gating was developed and applied to the nicotinic acetylcholine receptor (nAChR). This approach utilizes the nonsense codon suppression method to replace specific backbone peptide bonds with an ester backbone at several sites in the nicotinic acetylcholine receptor (nAChR). The resulting structural modification probes conformational dynamics by disrupting backbone hydrogen bonds at the site of mutation. We mutated the peptide backbone to an ester backbone at two sites of interest in the protein. In the first transmembrane (M1) domain, a conserved proline residue (aPro221) was identified as a crucial gating element. The amide-to-ester mutation at this site provides receptors with near-normal sensitivity, although the natural amino acids tested other than Pro produce receptors that gate with an EC₅₀ ~ 100 fold higher than normal. These data suggest that a backbone hydrogen bond at this site interferes with normal gating. Notably, the M1 Pro, while conserved in each of the subunits, is essential only in the a subunits. This is in contrast to mutations in the middle of the M2 domains, the effects of which are essentially equivalent for all subunits. This suggests that the coupling pathway from the agonist binding site (on the extracellular portion of the a subunits) to the channel pore region (M2) includes the aM1 domain. In the aM2 domain, the amide-to-ester mutation was used to distinguish between two distinct models for gating. The ester mutation yielded functional receptors at 15 positions, three of which provided receptors with ~10-fold lower EC₅₀than wild type. These results support a model for gating that includes significant changes of backbone conformation within the M2 domain.

Division of Biology, California Institute of Technology, Pasadena, CA 91125

*†Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125

Mapping Functional Domains in the nAChR using SNIPP

Pamela M. England, Gabriel S. Brandt*, Dennis A. Dougherty*

The photoreactive unnatural amino acid (2-nitrophenyl) glycine [Npg, (1)] can be used to identify functional domains of proteins. Introduced site-specifically using the nonsense codon suppression technique, the residue provides peptide backbone cleavage upon irradiation with UV light. Previous work has established the utility of site-specific, nitrobenzyl-induced photochemical proteolysis (SNIPP)1 of proteins. We have continued to apply this method to the study of the nicotinic acetylcholine receptor (nAChR) expressed in Xenopus oocytes. An interesting feature of the nAChR is the conserved 13 amino acid disulfide loop. This "signature loop" is present in each of the subunits of the nAChR as well as all other members of the nAChR superfamily of ligand-gated ion channels. Using SNIPP, we are now addressing the question of a functional role for this loop in each of the nAChR subunits. Cleavage of the disulfide loop in the a-subunits results in ~90% reduction in whole-cell currents as well as a decrease in a-bungarotoxin binding. Similarly, cleavage of the b, g, and d signature loops results in ~50% reduction in whole cell current. (Figure 1) Thus, the Cys loops in all four subunits of the receptor are critical for function. Having established an important role for these loops in the nAChR, we are now working to delineate more precisely the nature of this role.

Reference:

1) England, P. M.; Lester, H. A.; Davidson N.; Dougherty, D. A. (1997)

Site-Specific, Photochemical Proteolysis Applied to Ion Channels In Vivo. Proc. Natl. Acad. Sci. USA 94, 11025-11030.

*Division of Chemistry and Chemical Engineering

Unnatural Amino Acids for In Vivo Site-Specific Labeling of Membrane Proteins

Justin P. Gallivan*, Dennis A. Dougherty*

The structure of an integral membrane protein is in part determined by its transmembrane topology. Knowledge of which regions of a protein are located within a cell, those which are not, and those which are embedded in the membrane, is not only essential for determining how a protein is folded, but also how it functions. Despite its importance, there are relatively few direct methods to determine the transmembrane topology of functional proteins expressed in living cells. To address this issue, we developed a new experimental method to determine the surface accessibility, and ultimately the transmembrane topology, of membrane proteins expressed in Xenopus oocytes. We used the in vivo nonsense suppression technique to incorporate biotinylated unnatural amino acids into functional ion channels expressed in Xenopus oocytes. Binding of 125I-streptavidin to biotinylated receptors was used as a means of determining the surface accessibility of individual amino acids, and thus the topology of the protein. In previous work, we incorporated biotinylated amino acids into sites in the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (nAChR) and assayed their accessibility to streptavidin, thus validating the method. Prresent work seeks to apply this method to other regions of the nicotinic receptor to obtain further chemical-scale insights into its structure.

*Division of Chemistry and Chemical Engineering

Couplings Between Distant Regions in the Nicotinic Acetylcholine Receptor: Acidic Residues Near the Agonist Binding Site and Hydrophobic Residues in the Pore

Wenge Zhong*, Haiyun Zhang, and Dennis A. Dougherty*

Conventional and nonsense suppression mutagenesis methods and double mutant cycles were employed to study functional interactions within the muscle nicotinic acetylcholine receptor expressed in Xenopus oocytes. We measured macroscopic dose response relations for receptors with natural and unnatural mutations at tyrosine 93 in the a subunit (aY93) and/or D (aspartic acid) to N (asparagine) mutations at 174 in the g and 180 in the d subunits. Comparison among the EC₅₀ values for these receptors leads to the conclusion that there is no hydrogen bond between aY93 and gD174 or dD180. However, these D-to-N mutations decrease the receptor’s sensitivity to acetylcholine (ACh). Single-channel analysis indicates that the D-to-N mutations affect gating of the channel primarily by a decrease in the opening rate, in addition to a moderate increase in the channel closing rate. The D-to-N mutations were also studied in combination with mutations from wild-type leucine to serine at the 9’ position of the M2 pore-lining region. The opposing effects on EC₅₀ values are not all multiplicative, suggesting remote energetic couplings between the acidic residues and the 9’ leucine residues, particularly in the b subunit. These acidic residues participate in the intramolecular transduction pathway that couples agonist binding to channel gating.

*Division of Chemistry and Chemical Engineering

Leucine Mutations at the 9’ Position of the Acetylcholine Receptor M2 Domain:Kinetic Bases of Increased Sensitivity to Agonist

Haiyun Zhang, Purnima Deshpande, Cesar Labarca, and Jun Li

In nicotinic acetylcholine receptors (nAChRs) a leucine residue at the 9’ position of the M2 transmembrane segment is highly conserved. Previous studies revealed that mutating this leucine to serine (Leu9’Ser) increased the apparent sensitivity to acetylcholine (ACh) by about 10-fold for each subunit mutated. To better understand the kinetic bases of this effect, we studied single-channel currents of nAChRs with varying numbers (denoted as m*_s) of Leu9’Ser mutant subunits expressed in Xenopus oocytes. The mutated nAChRs have increased spontaneous opening frequencies. The lifetime of the spontaneous openings, however, remains relatively constant (~ 0.3 ms) from m*_s = 1 to m*_s = 5. The ACh-induced openings become longer in mutant nAChRs. For example, while the wild-type receptors typically open for 1-3 ms, all mutant receptors (m*_s = 1 to m*_s = 5) display a new class of opening that lasts tens of milliseconds. These results suggest that 9’ mutations have produced significant changes in channel gating properties. In order to determine whether binding is also affected, we estimated the binding and gating rate constants using a maximum interval-likelihood algorithm. Our analyses indicated that the binding rate constants were also altered in the mutant receptors, despite the ~ 50 Å between the binding site and the channel lumen. Also, monoliganded receptors account for most of the conductance for m*_s = 4. Thus both gating and binding effects generate the ~ 3000-fold decrease in macroscopic EC₅₀ value between the wild-type receptor and the m*_s = 4 receptor. To examine the possible functional consequences of Leu9’Ser mutations in the context of synaptic transmission, we constructed responses analogous to postsynaptic currents by delivering brief ACh pulses to outside-out patches. The decay phase of the response lengthens with increasing m*_s, qualitatively as expected from the steady-state single-channel kinetic data. Thus conservation of the 9’ leucine in M2 is associated with appropriately brief synaptic responses.

From Ab Initio Quantum Mechanics to Molecular Neurobiology: A Cation-pi Binding Site in the Nicotinic Receptor

Wenge Zhong*, Justin P. Gallivan*, Yinong Zhang, Lintong Li, and Dennis A. Dougherty*

The nicotinic acetylcholine receptor (nAChR), the prototype ligand-gated ion channel, functions at the vertebrate neuromuscular junction and in the central nervous system. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation-p interactions may be involved in binding the quaternary ammonium group of the agonist, ACh. We show a compelling correlation between: a) ab initio quantum mechanical predictions of cation-pi binding abilities; and b) EC₅₀ values for ACh at the receptor for a series of tryptophan derivatives, which were incorporated into the receptor using the in vivo nonsense suppression method for unnatural amino acid incorporation. Such a correlation is seen at one and only one of the aromatic residues - Trp149 of the a subunit. This indicates that, on binding, the cationic, quaternary ammonium group of ACh makes van der Waals contact with the indole sidechain of aTrp149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position a149 produces a constitutively active receptor.

*Division of Chemistry and Chemical Engineering

Incorporation of Fluorescent Unnatural Amino Acids into the Nicotinic Acetylcholine Receptor

Marcus C. Sarofim*, Dennis A. Dougherty*

Fluorophores can function as versatile probes of protein dynamics and structure. We are using the nonsense codon suppression technique to introduce highly fluorescent amino acids into the nicotinic acetylcholine receptor (nAChR), expressed in Xenopus oocytes. Initial targets include the dynamics and lipid accessibility of the M1 transmembrane domain. We can investigate the possibility that this domain is part of the intramolecular signal transduction pathway between the binding site and the pore by monitoring the changes in emission characteristics of incorporated fluorophores that should result from large movements of the region. Two residues have been synthesized based on the small, environment sensitive fluorophore NBD (nitrobenzoxadiazole). Future studies may include mapping surface accessibility and using fluorescence resonance energy transfer (FRET) to measure distances in the receptor.

*Division of Chemistry and Chemical Engineering

Flash Decaging of Tyrosine Sidechains in an Ion Channel

Jeffrey C. Miller, Scott K. Silverman*, Pamela M. England, and Dennis A. Dougherty*

A nonsense codon suppression technique was employed to incorporate ortho-nitrobenzyl tyrosine, "caged tyrosine", in place of tyrosine at any of three positions (93, 127, or 198) in the ? subunit of the muscle nicotinic ACh receptor (nAChR) expressed in Xenopus oocytes. The ortho-nitrobenzyl group was then removed by 1 ms flashes at 300-350 nm to yield tyrosine itself while macroscopic currents were recorded during steady ACh exposure. Responses to multiple flashes showed (a) that each flash decages up to 17% of the tyrosines and (b) that two tyrosines must be decaged per receptor for a response. The conductance relaxations showed multiple kinetic components; rate constants (< 0.1 s^-1to 10³ s^-1) depended on pH and the site of incorporation; and relative amplitudes depended on the number of prior flashes. This method, which is potentially quite general, (a) provides a time-resolved assay for the behavior of a protein when a mutant sidechain is abruptly changed to the wild-type residue and (b) will also allow for selective decaging of sidechains that are candidates for covalent modification (such as phosphorylation) in specific proteins in intact cells.

*Division of Chemistry and Chemical Engineering

Studying phosphorylation kinetics of potassium channels with unnatural amino acid probes

Yanhe Tong, Eric Slimko

The activation of potassium channels can be modulated by phosphorylation. Recent work has demonstrated that voltage-gated potassium channel Kv1.3 can be phosphorylated at multiple sites by v-Src kinase. My studies have shown that the inward rectifier potassium channel Kir2.1 can be phosphorylated both by v-Src kinase and by PYK2 kinase. The TrkB receptor tyrosine kinase can also modulate both Kv1.3 and Kir2.1. Using the nonsense codon suppression method, unnatural amino acid ortho-nitrobenzyl tyrosine (Tyr(ONB)) has been successfully incorporated into putative tyrosine phosphorylation sites of Kv1.3 and Kir2.1 expressed in Xenopus oocytes. Electrophysiological studies showed that Kv1.3 and Kir2.1 channels truncated at phosphorylation sites, resulting from the inefficiency of unnatural amino acid incorporation, have no dominant-negative effects on wild-type channels. The Tyr(ONB) incorporated Kv1.3 and Kir2.1 channels have the same characteristics as wild-type channels, yet the phosphorylation at the Tyr(ONB) sites is protected from kinases by the ortho-nitrobenzyl group before irradiation. The modulation of the channels by v-Src kinase was studied after the tyrosine side chain was revealed by irradiation. The phosphorylation rate at some positions, such as Y111 and Y111-113 of Kv1.3 as well as Y242 of Kir2.1, was measured. The results show that the dynamic process of protein phosphorylation can be monitored by using the nonsense codon suppression method; this study will generate data not available with conventional mutagenesis.

Asymmetric Contributions of Subunit Pore Regions to Ion Selectivity in an Inward-Rectifier K⁺ Channel

Scott K. Silverman* and Dennis A. Dougherty*

We have investigated aspects of ion selectivity in K+ channels by functional expression of wild-type and mutant heteromultimeric G protein-coupled inward-rectifier K+ (GIRK) channels in Xenopus oocytes. Within the K+ channel pore (P) region signature sequence, a large number of point mutations in GIRK1 and GIRK4 subunits have been made at a key tyrosine residueóthe ìsignatureî tyrosine of the GYG. Studies of mutant GIRK1/GIRK4 heteromultimers reveal that the GIRK1 and GIRK4 subunits contribute asymmetrically to K+ selectivity. The signature tyrosine of GIRK1 can be mutated to many different residues while retaining selectivity; in contrast, the analogous position in GIRK4 must be tyrosine for maximal selectivity. Other residues of the P region also contribute to selectivity, and studies with GIRK1/GIRK4 chimeras reveal that an intact, heteromultimeric P region is necessary and sufficient for optimal K+ selectivity. We propose that the GIRK1 and GIRK4 P regions play roles similar to the two P regions of an emerging family of K+ channels whose subunits each have two P regions connected in tandem. We find different consequences between similar mutations in inward-rectifier and voltage-gated K+ channels, which suggests that the pore structures and selectivity mechanisms in the two classes of channel may not be identical. We confirm that GIRK4 subunits alone can form functional channels in oocytes, but we find that these channels are measurably permeable to Na+ and Ca2+.

*Division of Chemistry and Chemical Engineering

Functional interactions between integrins and GIRK channels

Jancy C. McPhee and Yan Dang

The sequence arginine-glycine-aspartate (RGD), located extracellularly between the first membrane-spanning region and the pore, is conserved among all identified G protein-activated inward rectifier K⁺ (GIRK) channels subunits. Many integrins recognize this RGD sequence on other proteins, usually in the extracellular matrix. We are asking whether GIRK activity might be regulated by direct interaction with integrin. Previously, we showed that mutation of the RGD site on the GIRK subunit, particularly on the GIRK4 subunit, decreases or abolishes the GIRK current when channels were expressed in Xenopus laevis oocytes. Recently, we demonstrated that a similar effect of the RGD mutation could be seen when the channel was expressed in Chinese Hamster Ovary (CHO) cells. These data show that mutation of the RGD site on the channel to RGE disrupts GIRK currents, consistent with the idea that the channel and integrin interact. We are currently testing the importance of the other residues of the RGD motif, notably the glycine, and whether other inward rectifiers may also interact with integrin. We are also examining whether externally-applied RGD-containing peptides disrupt naturally-occuring atrial cell GIRK currents and the details of the localization of GIRK and integrin in atrial cells using immunohistochemical labeling and confocal microscopy.

Using antibodies against each of the channel subunits or the b 1 subunit of integrin, we initiated a series of studies to co-immunoprecipitate the channel and endogenous integrin from detergent-solubilized oocytes. We showed that wildtype channels can be co-immunoprecipitated with integrin but mutant channels cannot. These studies show directly that GIRK and integrin bind to each other when the channel is expressed in heterologous cells and that mutation of the RGD site disrupts the binding.

If direct binding of GIRK to integrin allows channel activation, the underlying mechanism would go beyond present concepts of GIRK function. Several possible mechanisms, however, are suggested by contemporary findings on both channels and integrins, such as a role for the cytoskeleton, tyrosine kinases or phosphoinositide signaling. We will explore these possiblities and others as we try to elucidate the mechanism for the modulation of GIRK function by integrin.

Functional roles of aromatic residues in the ligand-binding domain of cyclic nucleotide-gated channels

Jun Li

The ligand-binding domains of cyclic nucleotide-gated (CNG) channels show sequence homology to corresponding region(s) of the E. coli catabolite gene-activator protein (CAP) and to the regulatory subunit of cAMP-dependent or cGMP-dependent protein kinases (PKAs and PKGs). The structures of CAP and a PKA regulatory subunit have been solved, prompting efforts to generate structural models for the binding domains in CNG channel. These models explicitly predicted that an aromatic residue in the CNG channel aligning with phenylalanine 61 of CAP forms an interaction with the bound cyclic nucleotide. We tested this hypothesis by site-directed mutagenesis in a rat olfactory channel (rOCNC1) and a bovine rod photoreceptor channel (Brcng). We found that mutations at this site had only weak effects which were not specific to the aromatic or the hydrophobic nature of the substituted residue. This result weakens the hypothesis of a strong or specific interaction at this site. We also mutated most of the other aromatic residues in the binding domain to alanine; and most of these individual mutations resulted in channels that either did not function or had only minor changes in sensitivity. However, replacing tyrosine 565 with alanine (Y565A) in rOCNC1 increased agonist sensitivity by ~10-fold. Y565 presumably lies between two a helices in the binding domain; one of these, the C helix, probably rotates during channel activation. The position of Y565 at the "hinge" between the C helix and another portion of the binding domain, and the effects of Y565 mutations, strongly suggest that this portion of the binding domain is involved in channel gating processes.

Single-channel kinetics of the rat olfactory cyclic nucleotide-gated channel expressed in Xenopus oocytes

Jun Li

Cyclic nucleotide-gated channels are nonselective cation channels activated by intracellular cyclic AMP and/or cyclic GMP. It is not known how the binding of agonists opens the channel, or how the presumed four binding sites, one on each subunit, interact to generate cooperativity. We expressed the rat olfactory cyclic nucleotide-gated channel a subunit in Xenopus oocytes and recorded the single-channel currents. We found that the channel had a single conductance state; flickers at -60 mV showed the same power spectrum for cAMP and for cGMP. At steady state, the distribution patterns of open and closed times were relatively simple, usually containing one or two exponential components. The conductance properties and the dwell time distributions were adequately described by models that invoke only one or two binding events. Given the probable tetrameric architecture of cyclic nucleotide-gated channels, possible additional binding and gating states therefore have little effect on channel function. In a comparison between cAMP and cGMP, we find that cGMP has only a slightly greater efficacy than cAMP, and a comparatively much higher affinity. Our data contrast with previous macroscopic studies suggesting that cAMP and cGMP bind with comparable affinities, but that cGMP has a much greater gating efficacy.

Enhancement 0f Neurotransmitter Release Induced by BDNF in Cultured Hippocampal Neurons

Yong-Xin Li, Yinong Zhang, Erin M. Schuman, and Norman Davidson

BDNF, like other neurotrophins, has long-term effects on neuronal survival and differentiation; furthermore, recent work has shown that BDNF can also induce changes in synaptic efficacy on a time scale of min. We have investigated the mechanism(s) of these synaptic effects in studies of embryonic hippocampal neurons in culture. In solutions containing the GABA_A receptor antagonist, picrotoxin, application of BDNF (100 ng/ml) for 1 - 5 min increased the amplitude of evoked synaptic currents by 48 ± 9% in ten out of fifteen pairs of neurons and increased the frequency of excitatory postsynaptic current (EPSC) bursts to 205 ± 20% of the control levels. There was no detectable effect of BDNF on the parameters determining the electrical excitability of the cell, including the resting membrane potential, input resistance, action potential threshold and action potential amplitude. BDNF did not change the postsynaptic currents induced by exogenous application of glutamate. However, BDNF did increase the frequency of miniature EPSCs (mEPSCs) (268.0 ± 46.8 % of control frequency), without affecting the mEPSC amplitude. The effect of BDNF on mEPSC frequency was blocked by the tyrosine kinase inhibitor K252a and also by removal of extracellular calcium ([Ca²⁺]_o). Fura-2 recordings showed that BDNF elicited an increase in intracellular calcium concentration ([Ca²⁺]_c); this effect was dependent on [Ca²⁺]_o; it was blocked by K252a and by thapsigargin but not by caffeine. The results demonstrate that BDNF enhances glutamatergic synaptic transmission at a presynaptic locus and that this effect is accompanied by a rise in [Ca²⁺]_c that requires release of Ca²⁺ from IP₃-gated stores.

Expression of a dominant negative Trk B receptor, T1, reveals a requirement for presynaptic signaling in BDNF-induced synaptic potentiation in cultured hippocampal neurons

Yong-Xin Li, Youfeng Xu, Donghong Ju, Norman Davidson, Erin Schuman

The neurotrophins, including nerve-growth factor (NGF), brain-derived neurotrophin (BDNF) and neurotrophin-3 (NT-3), have fast actions (within minutes) on neurotransmission at central and peripheral synapses. The neurotrophins exert their effects on synaptic strength by interacting with the Trk family of receptor tyrosine kinases. While pharmacological approaches have been useful in elucidating BDNF signaling mechanisms, including Trk receptor kinase activity, they do not provide an unambiguous assessment of the contributions of the pre- or postsynaptic cell to signaling. To address the synaptic locus of BDNF signaling, we constructed an adenovirus vector containing a truncated TrkB receptor gene and a GFP reporter gene (Ad-GFP-TrkB.T1) and a control adenovirus vector containing a GFP reporter gene only (Ad-GFP). Cultured hippocampal neurons (E18) were infected with these adenoviral constructs and dual whole-cell recordings were obtained from synaptically connected pairs. In experiments in which both pre- and postsynaptic neurons were infected by Ad-GFP (n = 11), BDNF induced significant potentiation of evoked EPSCs. In experiments in which both pre- and postsynaptic neurons (n = 8) were infected with Ad-GFP, however, none of the pairs of cells examined exhibited significant synaptic potentiation. Infection of the postsynaptic neuron alone (n = 19) with Ad-GFP-TrkB.T1 did not affect the ability of BDNF to enhance evoked synaptic responses. In contract, infection of the presynaptic neuron alone (n = 15) prevented the BDNF-induced enhancement. Similar results were observed for BDNF-induced increases in mEPSC frequency. These experiments reveal that in cultured hippocampal neurons, BDNF-TrkB signal transduction occurs in the presynaptic neuron, leading to a change in the efficacy of neurotransmitter release.

Extracellular Release and Chemical Conversion of Nitric Oxide†

Brian M. Sullivan, Yong-Xin Li, Erin M. Schuman, and Norman Davidson

Nitric oxide plays an essential role as a signaling molecule in the nervous system, the circulatory system, the immune system and other tissues. Many of the functions of NO involve intercellular signaling: NO is released from the cell of origin and diffuses through the extracellular medium to act on target cells. It is therefore important to understand how the subcellular localization of the NO generating enzymes (NOSs) affect release into the extracellular medium. The nitric oxide synthases of interest here are the calcium/calmodulin dependent neuronal and endothelial isoforms (both of which are expressed in the nervous system). By using recombinant adenovirus mediated gene transfer of e- and nNOS into CHO cells and using a sensitive NO specific electrode for detection, we report data relevant to the following: a) the effect of subcellular location of an NOS on extracellular NO release; b) the lifetime of NO in the extracellular medium.

Analysis of gene expression induced by BDNF in the hippocampus

Markus Lanzrein, W. Bryan Smith, Erin M. Schuman, Norman Davidson

Long lasting synaptic potentiation (LTP) in the hippocampus can be divided into an initial phase (1-3 hr) that relies upon modification of existing proteins and a late phase (>3 hr) that is dependent on transcription and translation. Thus, new genes are induced during the late phase of LTP, and these presumably function in the maintenance of the synaptic potentiation. The neurotrophins BDNF and NT3 can also induce a long-lasting enhancement in synaptic transmission in the Schaffer collateral - CA1 synapses in the rat hippocampus. This enhancement is also sensitive to inhibitors of protein synthesis and therefore requires de novo synthesis of proteins. For a better understanding of neurotrophin action and synaptic plasticity, it would be of value to identify genes which are induced in the hippocampus upon neurotrophin treatment. We have undertaken to identify such genes by performing subtractive hybridization. Hippocampal slices were perfused with BDNF or Ringer solution and the field EPSP was recorded at CA3-CA1 synapses. RNA was isolated from the slices 3.5 h after BDNF application and cDNA libraries were constructed. Suppression subtractive hybridization was used to construct a subtracted library which was enriched in differentially expressed clones. A differential screen led to the isolation of a number of partial sequences of candidate genes. Elevation of the mRNA levels by BDNF was confirmed for most of those genes by RT-PCR. Some of the isolated sequences matched known cDNA sequences, among them synaptic vesicle proteins. The other sequences were either matching sequences from the EST database or with no match. These two groups of cDNA clones correspond to novel genes with a potential function in the maintenance of synaptic potentiation. We are now isolating full-length cDNA clones from our hippocampal library and will characterize the functions of the encoded proteins.

Amino Acid Residues that Control pH Modulation of Transport-Associated Current in Mammalian Serotonin Transporters

Yongwei Cao, Ming Li, Sela Mager

The rat (rSERT) and human (hSERT) serotonin transporters were expressed in Xenopus oocytes and studied using site-directed mutagenesis, electrophysiological recordings, and [³H]5-HT uptake measurements. rSERT, but not hSERT, displayed increased transport-associated current at low pH. Chimeras and point mutations showed that, of the 52 non-identical residues, a single residue at position 490 (threonine in rSERT, lysine in hSERT) governs this difference. Furthermore, potentiation required the glutamate residue at position 493. Cysteine substitution and alkylation experiments showed that residue 493 is extracellular. Cysteine at 493 increased, while aspartate decreased, the net charge movement per transported 5-HT molecule. The mutations at this region did not significantly affect other aspects of SERT function, including agonist-independent leakage current, voltage-dependent transient current, and H⁺ current. This region may therefore be part of an external gate required for rSERT function. The data and analyses show that, in the absence of detailed structural information, a gate-lumen-gate scheme is useful for interpreting results from mutations that alter functional properties of neurotransmitter transporters.

Topological Localization of Cysteine 74 in the GABA transporter, GAT1, and its Importance in Ion Binding and Permeation

Nam Yu, Yongwei Cao, Sela Mager

Xenopus oocytes expressing the GABA transporter GAT1 were exposed to membrane-impermeant sulfhydryl reagents, resulting in decreased the GABA transport current, decreased capacitive charge movements, and increased Na⁺ and Li⁺ leakage currents. Mutation of cysteine 74 to alanine (C74A) eliminated these effects. The W68S and W68L mutations significantly increased and decreased the transporter’s sensitivity, respectively, to sulfhydryl reagents. At each of the positions 73 through 76, cysteine residues were accessible to external MTSET. These findings, together with recent evidence placing the HD2-HD3 loop on the extracellular side, suggest that the HD2 region does not traverse the membrane.

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