Channel Desensitization Research Articles

Involvement of Ectodomain Leu 214 in ATP Binding and Channel Desensitization of the P2X4 Receptor

P2X receptors are trimeric ATP-gated cation permeable ion channels. When ATP binds, the extracellular head and dorsal fin domains are predicted to move closer to each other. However, there are scant functional data corroborating the role of the dorsal fin in ligand binding. Here using site-directed mutagenesis and electrophysiology, we show that a dorsal fin leucine, L214, contributes to ATP binding. Mutant receptors containing a single substitution of alanine, serine, glutamic acid, or phenylalanine at L214 of the rat P2X4 receptor exhibited markedly reduced sensitivities to ATP. Mutation of other dorsal fin side chains, S216, T223, and D224, did not significantly alter ATP sensitivity. Exposure of L214C to sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) or (2-aminoethyl) methanethiosulfonate hydrobromide in the absence of ATP blocked responses evoked by subsequent ATP application. In contrast, when MTSES(-) was applied in the presence of ATP, no current inhibition was observed. Furthermore, L214A also slightly reduced the inhibitory effect of the antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP, and the blockade was more rapidly reversible after washout. Certain L214 mutants also showed effects on current desensitization in the continued presence of ATP. L214I exhibited an accelerated current decline, whereas L214M exhibited a slower rate. Taken together, these data reveal that position L214 participates in both ATP binding and conformational changes accompanying channel opening and desensitization, providing compelling evidence that the dorsal fin domain indeed has functional properties that are similar to those previously reported for the body domains.

A Charge-inverting Mutation in the “Linker” Region of α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Alters Agonist Binding and Gating Kinetics Independently of Allosteric Modulators

AMPA receptors are gated through binding of glutamate to a solvent-accessible ligand-binding domain. Upon glutamate binding, these receptors undergo a series of conformational rearrangements regulating channel function. Allosteric modulators can bind within a pocket adjacent to the ligand-binding domain to stabilize specific conformations and prevent desensitization. Yelshansky et al. (Yelshansky, M. V., Sobolevsky, A. I., Jatzke, C., and Wollmuth, L. P. (2004) J. Neurosci. 24, 4728-4736) described a model of an electrostatic interaction between the ligand-binding domain and linker region to the pore that regulated channel desensitization. To test this hypothesis, we have conducted a series of experiments focusing on the R628E mutation. Using ultrafast perfusion with voltage clamp, we applied glutamate to outside-out patches pulled from transiently transfected HEK 293 cells expressing wild type or R628E mutant GluA2. In response to a brief pulse of glutamate (1 ms), mutant receptors deactivated with significantly slower kinetics than wild type receptors. In addition, R628E receptors showed significantly more steady-state current in response to a prolonged (500-ms) glutamate application. These changes in receptor kinetics occur through a pathway that is independent of that of allosteric modulators, which show an additive effect on R628E receptors. In addition, ligand binding assays revealed the R628E mutation to have increased affinity for agonist. Finally, we reconciled experimental data with computer simulations that explicitly model mutant and modulator interactions. Our data suggest that R628E stabilizes the receptor closed cleft conformation by reducing agonist dissociation and the transition to the desensitized state. These results suggest that the AMPA receptor external vestibule is a viable target for new positive allosteric modulators.

Sensory nerves and transient receptor potential vanilloid 1 channels in CO2 regulation of cerebrovascular tone

This study investigated the involvement of sensory nerves and, in particular, neuronal transient receptor potential vanilloid (TRPV) 1 channels, in the CO2-mediated regulation of cerebrovascular tone. Basilar artery diameter and blood flow velocity in the ventral midbrain were determined in a rat cranial window preparation by digital imaging and laser-Doppler flowmetry, respectively. Superfusion of the basilar artery with capsaicin, a selective TRPV1 receptor agonist, caused a transient relaxation, consistent with acute desensitization of neuronal TRPV1 channels. Constriction to respiratory hypocapnia remained unaffected following capsaicin superfusion. Denervation of sensory nerves by repeated capsaicin injection of neonates also did not reduce the respiratory hypocapnia constriction of the basilar artery as well as the decreased flow velocity in the ventral midbrain in adults. These findings suggest that sensory nerves and, in particular, neuronal TRPV1 channels, do not play a role in respiratory hypocapnia constriction and decreased flow, at least in rat basilar artery and ventral midbrain.

Intranasal capsaicin in management of nonallergic (vasomotor) rhinitis.

Capsaicin is a selective transient receptor potential vanilloid 1 (TRPV1) ion channel agonist and has been demonstrated to reduce nerve conduction of nociceptive C fibers in the trigeminal nerve without affecting conduction in Adelta fibers. This chapter reviews the classification of chronic rhinitis subtypes, the prevalence and epidemiology of nonallergic rhinitis (NAR), postulated pathophysiology and mechanisms of NAR including the role of transient receptor potential (TRP) ion channels and discusses the potential therapeutic benefits of capsaicin in the treatment of chronic rhinitis subtypes, specifically NAR. Evidence supports that hypersensitivity of TRP ion channels on sensory afferent neurons innervating nasal mucosa is responsible for inducing NAR symptoms. These symptoms, characterized as excessive nasal glandular secretion, nasal congestion, and headache, are mediated through neuropeptide release during axonal and parasympathetic reflexes which are initiated by a spectrum of nonspecific irritants that activate TRP channels. Rational approaches to treat the pathophysiology of NAR would be to develop therapies with selective TRPV1 agonist activity like capsaicin that target desensitization of TRP ion channels on sensory afferent nerves.

Functional Chimeras of the Human α7 Acetylcholine Receptor Provide Insights into Allosteric Modulation

The human α7 neuronal acetylcholine receptor (α7nAChR) is a promising drug target for treatment of psychiatric and neurological disorders. A group of positive allosteric modulators (PAMs) specific to α7nAChR are thought to act through the transmembrane domain (TMD), but the structural basis of functional modulation remains unclear. In this study, we constructed multiple chimeras between the TMD of human α7nAChR and the extracellular domain (ECD) of a bacterial homolog, ELIC, for which a high-resolution structure has been determined. Functions of the chimeras were evaluated in Xenopus oocytes by two-electrode voltage clamp electrophysiology. Functional ELIC-α7nAChR chimeras were obtained when their ECD-TMD interfaces were modified to resemble either the ELIC or α7nAChR interface, but only the latter chimera with the entirely native α7nAChR TMD retained the unique pharmacology of α7nAChR evoked by the modulators, including insensitivity to the anesthetic propofol, potentiation by the PAMs ivermectin, PNU-120596, and TQS, as well as activation by 4BP-TQS. None of the ELIC-α7nAChR chimeras exhibited the fast desensitization characteristic of α7nAChR. Functional analysis of a series of chimeras indicated that efficient coupling at loop 7, in addition to efficient coupling at loop 2 or loop 9, was essential for a channel to be functional. The most efficient coupling at the ECD-TMD interface required optimization of all three loops. Altogether, the study suggests the interdependent of the TMD on the ECD-TMD interface for channel gating, desensitization, and allosteric modulation. (Funded in part by grants from the NIH: R01GM069766, R01GM057257, and R37GM049202)

Coordinated Movements During ASIC1A Activity

Acid-sensing ion channels (ASICs) are H+-gated and Na+-conducting channels predominantly expressed in the nervous system. They are involved in H+ sensing in many physiological and pathological states such as ischemia and pain perception. The crystal structure of the chicken ASIC1 isoform has been revealed in the desensitized and the open state. Functional studies indicate that protonation of key residues in the extracellular loop triggers conformational changes leading to channel opening. However the molecular mechanisms linking protonation to the opening and closing of the gate have not been clarified yet. In this study we used voltage-clamp fluorometry (VCF) to reveal activity-associated movements occurring on the different ASIC1a domains. 20 different fluorophore positions located in the thumb, palm, finger and knuckle domains and in the extracellular pore entry showed VCF signals related to conformational changes. The timing of fluorescence changes suggests a complex sequence of movements upon pH change. When the pH of the extracellular solution was lowered to activate ASICs, rapid conformational changes were observed in the thumb, finger, knuckle and extracellular pore entry, followed by slower movements in the palm. The kinetics of fluorescence and current signals were compared to each other in order to assess whether the timing of the fluorescence signal corresponded to an effective channel transition. Some of the residues tested were found to be closely related to channel opening, desensitization or recovery from desensitization. Moreover we found that an endogenous tryptophan of the β-ball quenched the fluorescence signal of probes positioned in the finger and knuckle domains. The observed increase of this signal during channel opening indicates a movement of these domains away from the β-ball. This is the first extensive analysis of activity-dependent conformational changes in ASICs which could be applied to other ENaC-Deg related channels.

Dynamics of Cleft Closure of the GluA2 Ligand-binding Domain in the Presence of Full and Partial Agonists Revealed by Hydrogen-Deuterium Exchange

The majority of excitatory neurotransmission in the CNS is mediated by tetrameric AMPA receptors. Channel activation begins with a series of interactions with an agonist that binds to the cleft between the two lobes of the ligand-binding domain of each subunit. Binding leads to a series of conformational transitions, including the closure of the two lobes of the binding domain around the ligand, culminating in ion channel opening. Although a great deal has been learned from crystal structures, determining the molecular details of channel activation, deactivation, and desensitization requires measures of dynamics and stabilities of hydrogen bonds that stabilize cleft closure. The use of hydrogen-deuterium exchange at low pH provides a measure of the variation of stability of specific hydrogen bonds among agonists of different efficacy. Here, we used NMR measurements of hydrogen-deuterium exchange to determine the stability of hydrogen bonds in the GluA2 (AMPA receptor) ligand-binding domain in the presence of several full and partial agonists. The results suggest that the stabilization of hydrogen bonds between the two lobes of the binding domain is weaker for partial than for full agonists, and efficacy is correlated with the stability of these hydrogen bonds. The closure of the lobes around the agonists leads to a destabilization of the hydrogen bonding in another portion of the lobe interface, and removing an electrostatic interaction in Lobe 2 can relieve the strain. These results provide new details of transitions in the binding domain that are associated with channel activation and desensitization.

Conformational Changes in the Lower Palm Domain of ASIC1a Contribute to Desensitization and RFamide Modulation

Acid-sensing ion channel 1a (ASIC1a) is a proton-gated cation channel that contributes to fear and pain as well as neuronal damage following persistent cerebral acidosis. Neuropeptides can affect acid-induced neuronal injury by altering ASIC1a inactivation and/or steady-state desensitization. Yet, exactly how ASIC1a inactivation and desensitization occur or are modulated by peptides is not completely understood. We found that regions of the extracellular palm domain and the β11-12 linker are important for inactivation and steady-state desensitization of ASIC1a. The single amino acid substitutions L280C and L415C dramatically enhanced the rate of inactivation and altered the pH-dependence of steady-state desensitization. Further, the use of methanethiosulfonate (MTS) reagents suggests that the lower palm region (L280C) undergoes a conformational change when ASIC1a transitions from closed to desensitized. We determined that L280C also displays an altered response to the RFamide peptide, FRRFamide. Further, the presence of FRRFamide limited MTS modification of L280C. Together, these results indicate a potential role of the lower palm domain in peptide modulation and suggest RFamide-related peptides promote conformational changes within this region. These data provide empirical support for the idea that L280, and likely this region of the central vestibule, is intimately involved in channel inactivation and desensitization.

Validation of Alexa-647-ATP as a powerful tool to study P2X receptor ligand binding and desensitization

Ion channel opening and desensitization is a fundamental process in neurotransmission. The ATP-gated P2X1 receptor (P2X1R) shows rapid and long-lasting desensitization upon agonist binding. This makes the electrophysiological investigation of its desensitization process, agonist unbinding, and recovery from desensitization a challenging task. Here, we show that the fluorescent agonist Alexa-647-ATP is a potent agonist at the P2X1R and a versatile tool to directly visualize agonist binding and unbinding. We demonstrate that the long-lasting desensitization of the P2X1R is due to both slow unbinding of agonist from the desensitized receptor and agonist mediated receptor internalization. Furthermore, the unbinding of the agonist Alexa-647-ATP from the desensitized receptor is accelerated in the continuous presence of competitive ligand. Modeling of our data indicates that three agonist molecules are required to drive the receptor into desensitization. Direct visualization of ligand unbinding from the desensitized receptor demonstrates the cooperativity of this process.

P2X Receptor Chimeras Highlight Roles of the Amino Terminus to Partial Agonist Efficacy, the Carboxyl Terminus to Recovery from Desensitization, and Independent Regulation of Channel Transitions

P2X receptor subtypes can be distinguished by their sensitivity to ATP analogues and selective antagonists. We have used chimeras between human P2X1 and P2X2 receptors to address the contribution of the extracellular ligand binding loop, transmembrane segments (TM1 and TM2), and intracellular amino and carboxyl termini to the action of partial agonists (higher potency and efficacy of BzATP and Ap5A at P2X1 receptors) and antagonists. Sensitivity to the antagonists NF449, suramin, and PPADS was conferred by the nature of the extracellular loop (e.g. nanomolar for NF449 at P2X1 and P2X2-1EXT and micromolar at P2X2 and P2X1-2EXT). In contrast, the effectiveness of partial agonists was similar to P2X1 levels for both of the loop transfers, suggesting that interactions with the rest of the receptor played an important role. Swapping TM2 had reciprocal effects on partial agonist efficacy. However, TM1 swaps increased partial agonist efficacy at both chimeras, and this was similar for swaps of both TM1 and 2. Changing the amino terminus had no effect on agonist potency but increased partial agonist efficacy at P2X2-1N and decreased it at P2X1-2N chimeras, demonstrating that potency and efficacy can be independently regulated. Chimeras and point mutations also identified residues in the carboxyl terminus that regulated recovery from channel desensitization. These results show that interactions among the intracellular, transmembrane, and extracellular portions of the receptor regulate channel properties and suggest that transitions to channel opening, the behavior of the open channel, and recovery from the desensitized state can be controlled independently.

Point-to-point ligand–receptor interactions across the subunit interface modulate the induction and stabilization of conformational states of alpha7 nAChR by benzylidene anabaseines

The homomeric α7 nicotinic acetylcholine receptor is a well-studied therapeutic target, though its characteristically rapid desensitization complicates the development of drugs with specific agonist effects. Moreover, some experimental compounds such as GTS-21 (2,4diMeOBA), a derivative of the α7-selective partial agonist benzylidene anabaseine (BA), produce a prolonged residual desensitization (RD) in which the receptor remains non-activatable long after the drug has been removed from extracellular solution. In contrast, the desensitization caused by GTS-21's dihydroxy metabolite (2,4diOHBA) is relatively short-lived. RD is hypothetically due to stable binding of the ligand to the receptor in its desensitized state. We can attribute the reduction in RD to a single BA hydroxyl group on the 4′ benzylidene position. Computational prediction derived from homology modeling showed the serine36 (S36) residue of α7 as a reasonable candidate for point-to-point interaction between BA compounds and the receptor. Through evaluating the activity of BA and simple derivatives on wild-type and mutant α7 receptors, it was observed that the drug–receptor pairs which were capable of hydrogen bonding at residue 36 exhibited significantly less stable desensitization. Further experiments involving the type II positive allosteric modulator (PAM) PNU-120596 showed that the various BA compounds’ preference to induce either a PAM-sensitive (Ds) or PAM-insensitive (Di) desensitized state is concentration dependent and suggested that both states are destabilized by S36 H-bonding. These results indicate that the fine-tuning of agonists for specific interaction with S36 can facilitate the development of therapeutics with targeted effects on ion channel desensitization properties and conformational state stability.

Structural Insights into the Dynamics of the TRPA1 Activation Mechanism

TRPA1 is a Ca2+-permeable, non-selective cation channel and one of the key pain sensors in mammals. Pain sensation mediated by TRPA1 involves modification of N-terminal cysteine residues on the channel by thiol-reactive compounds and inflammatory mediators. Recent mutagenesis and chimeric studies have suggested that N-terminal cysteine residues (C622, C642, C666) located on the flexible linker region of the channel are involved in channel activation and desensitization, but how conformational changes in the flexible linker region lead to activation and desensitization is unresolved.Capitalizing on our ability to isolate pure and functional TRPA1 channel protein, we recently provided the first insight into the channel architecture by reconstructing a TRPA1 structure to 16 A resolution in resting state using electron microscopy (EM). Fitting TRPA1 homology model into the EM density lead us to hypothesize that the critical cysteines on the flexible linker region (C622, C642, C666) are in close proximity to one another and that covalent modification of cysteines within this pocket could promote conformational changes leading to channel gating and desensitization. Furthermore, we performed a mass spectrometry analysis of the in vivo TRPA1 thiol status and discovered that, when treated with a thiol-reactive TRPA1 agonist, C622 and C666 could form a disulfide bond with each other or with two other cysteine residues.Our current structural and biophysical analyses using EM, mass spectrometry and homology modeling are elucidating ligand-induced conformational changes in the channel's flexible linker region that occur during activation and desensitization. These insights bring us a greater understanding of the structural rearrangements involved in the gating and desensitization mechanisms of the TRPA1 ion channel.

Fast Photoswitching of Tethered Ligands to Study Ionotropic Glutamate Receptor Activation and Desensitization

Binding of glutamate triggers both the activation and subsequent desensitization of ionotropic glutamate receptors (iGluRs). Matching their role in excitatory neurotransmission, iGluR activation and desensitization are fast processes occurring on the submillisecond and millisecond timescale, respectively. However, little is known about how ligand binding to the four subunits of the tetrameric channel assemblies mediates these processes. Here we address this question using photo-switchable ligands (MAGs) that can be tethered to individual subunits via a cysteine-reactive maleimide group. An azobenzene group serves as photo-switch that allows binding of the glutamate head group in its cis, but not its trans state [1]. This allows us to control ligand binding and unbinding with short (<100 μs) pulses of light and to follow channel activation and desensitization in real-time. The experiments give insight into the gating mechanism of GluK2 homotetramers and will be extended to study subunit specific activation in heteromeric GluK2/GluK5 complexes, which assemble with defined 2:2 subunit stoichiometry [2].1. Gorostiza P. et al., Proc. Natl. Acad. Sci. USA (2007) 104: 10865.2. Reiner A. et al., Cell. Rep. (2012) 1: 234.

Avoiding TRP Channel Desensitization using Brief Agonist Applications or Temperature Steps

Transient receptor potential (TRP) channels are an important class of receptors found widely distributed throughout the mammalian central and peripheral nervous systems. They have been shown to be activated by many stimuli including temperature, mechano-stimulation, divalent cations and pH. TRP channels are receiving much attention as potential targets for the treatment of, for example, chronic pain, asthma and diabetes isipidus.View Large Image | View Hi-Res Image | Download PowerPoint SlideRepetitive activation by heat of TRP channels often tends to desensitize the channels. But as shown in this figure, it is possible to avoid it.Exemplar traces different TRP channels including TRPV1, TRPA1,TRPM3, TRPM7 and TRPM8 will be presented.

Slo1 Channel Desensitization to Ethanol is Coupled to Calcium-Sensing Structures in the Channel Cytosolic Domain

Data from native BK (Dopico et al., 1996; Pietrzykowski et al., 2004) and recombinant slo1 channels (Dopico et al., 1998; Yuan et al., 2008) consistently show that ethanol-induced increase in channel steady-state activity (NPo) disappears within several minutes of continuous alcohol exposure. Ionotropic receptor desensitization is usually a monotonic function of activating ligand (Dopico & Lovinger, 2009), and slo1 channels enter a low activity mode in presence of high levels of activating ligand (i.e., Ca2+i) (Rothberg et al., 1996). Moreover, Ca2+i is required for ethanol to activate slo1 channels (Liu et al., 2008). In this work, we began to examine the Ca2+i-dependence and structural bases of slo1 channel desensitization to protracted ethanol exposure. Following BK channel-forming protein expression in membrane patches from Xenopus laevis oocytes, exposure to 50 mM ethanol increased NPo×1.5-2 fold within 1-2 min of drug application, a response that totally disappeared within 5 min of alcohol exposure (n=7). Experimental conditions rule out a major role of genetic adaptation, change in channel protein composition, channel internalization, cytosolic signaling and ethanol metabolism in slo1 channel desensitization to alcohol. Rather, the phenomenon must be attributed to ligand interaction(s) with the slo1 subunit and/or its immediate lipid environment. Desensitization to ethanol was observed whether the slo1 channel was studied at 0.3, 1 or 10 μM Ca2+i, indicating that slo1 channel desensitization to ethanol is not significantly modified within Ca2+i levels that correspond to the physiological range. However, two constructs containing amino acid substitutions in the channel cytosolic domain that alter Ca2+i-sensing by slo1 (5D5N in calcium bowl; D362A/D367A in RCK1) displayed a significant delay in channel desensitization to ethanol. Thus, Ca2+i-sensing structures in the slo1 cytosolic domain are functionally coupled to ethanol-desensitization processes.Support: R37-AA011560 (AMD).

Acid-sensing ion channels (ASICs) are differentially modulated by anions dependent on their subunit composition

Acid-sensing ion channels (ASICs) are sodium channels gated by extracellular protons. ASIC1a channels possess intersubunit Cl(-)-binding sites in the extracellular domain, which are highly conserved between ASIC subunits. We previously found that anions modulate ASIC1a gating via these sites. Here we investigated the effect of anion substitution on native ASICs in rat sensory neurons and heterologously expressed ASIC2a and ASIC3 channels by whole cell patch clamp. Similar to ASIC1a, anions modulated the kinetics of desensitization of other ASIC channels. However, unlike ASIC1a, anions also modulated the pH dependence of activation. Moreover, the order of efficacy of different anions to modulate ASIC2a and -3 was very different from that of ASIC1a. More surprising, mutations of conserved residues that form an intersubunit Cl(-)-binding site in ASIC1a only partially abrogated the effects of anion modulation of ASIC2a and had no effect on anion modulation of ASIC3. The effects of anions on native ASICs in rat dorsal root ganglion neurons mimicked those in heterologously expressed ASIC1a/3 heteromeric channels. Our data show that anions modulate a variety of ASIC properties and are dependent on the subunit composition, and the mechanism of modulation for ASIC2a and -3 is distinct from that of ASIC1a. We speculate that modulation of ASIC gating by Cl(-) is a novel mechanism to sense shifts in extracellular fluid composition.

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

Involvement of the cysteine-rich head domain in activation and desensitization of the P2X1 receptor

P2X receptors (P2XRs) are ligand-gated ion channels activated by extracellular ATP. Although the crystal structure of the zebrafish P2X4R has been solved, the exact mode of ATP binding and the conformational changes governing channel opening and desensitization remain unknown. Here, we used voltage clamp fluorometry to investigate movements in the cysteine-rich head domain of the rat P2X1R (A118-I125) that projects over the proposed ATP binding site. On substitution with cysteine residues, six of these residues (N120-I125) were specifically labeled by tetramethyl-rhodamine-maleimide and showed significant changes in the emission of the fluorescence probe on application of the agonists ATP and benzoyl-benzoyl-ATP. Mutants N120C and G123C showed fast fluorescence decreases with similar kinetics as the current increases. In contrast, mutants P121C and I125C showed slow fluorescence increases that seemed to correlate with the current decline during desensitization. Mutant E122C showed a slow fluorescence increase and fast decrease with ATP and benzoyl-benzoyl-ATP, respectively. Application of the competitive antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP) resulted in large fluorescence changes with the N120C, E122C, and G123C mutants and minor or no changes with the other mutants. Likewise, TNP-ATP-induced changes in control mutants distant from the proposed ATP binding site were comparably small or absent. Combined with molecular modeling studies, our data confirm the proposed ATP binding site and provide evidence that ATP orients in its binding site with the ribose moiety facing the solution. We also conclude that P2XR activation and desensitization involve movements of the cysteine-rich head domain.

Interaction of Voltage-gated Sodium Channel Nav1.6 (SCN8A) with Microtubule-associated Protein Map1b

The mechanism by which voltage-gated sodium channels are trafficked to the surface of neurons is not well understood. Our previous work implicated the cytoplasmic N terminus of the sodium channel Na(v)1.6 in this process. We report that the N terminus plus the first transmembrane segment (residues 1-153) is sufficient to direct a reporter to the cell surface. To identify proteins that interact with the 117-residue N-terminal domain, we carried out a yeast two-hybrid screen of a mouse brain cDNA library. Three clones containing overlapping portions of the light chain of microtubule-associated protein Map1b (Mtap1b) were recovered from the screen. Interaction between endogenous Na(v)1.6 channels and Map1b in mouse brain was confirmed by co-immunoprecipitation. Map1b did not interact with the N terminus of the related channel Na(v)1.1. Alanine-scanning mutagenesis of the Na(v)1.6 N terminus demonstrated that residues 77-80 (VAVP) contribute to interaction with Map1b. Co-expression of Na(v)1.6 with Map1b in neuronal cell line ND7/23 resulted in a 50% increase in current density, demonstrating a functional role for this interaction. Mutation of the Map1b binding site of Na(v)1.6 prevented generation of sodium current in transfected cells. The data indicate that Map1b facilitates trafficking of Na(v)1.6 to the neuronal cell surface.

Length and Amino Acid Sequence of Peptides Substituted for the 5-HT3A Receptor M3M4 Loop May Affect Channel Expression and Desensitization

5-HT3A receptors are pentameric neurotransmitter-gated ion channels in the Cys-loop receptor family. Each subunit contains an extracellular domain, four transmembrane segments (M1, M2, M3, M4) and a 115 residue intracellular loop between M3 and M4. In contrast, the M3M4 loop in prokaryotic homologues is <15 residues. To investigate the limits of M3M4 loop length and composition on channel function we replaced the 5-HT3A M3M4 loop with two to seven alanine residues (5-HT3A-An = 2–7). Mutants were expressed in Xenopus laevis oocytes and characterized using two electrode voltage clamp recording. All mutants were functional. The 5-HT EC50's were at most 5-fold greater than wild-type (WT). The desensitization rate differed significantly among the mutants. Desensitization rates for 5-HT3A-A2, 5-HT3A-A4, 5-HT3A-A6, and 5-HT3A-A7 were similar to WT. In contrast, 5-HT3A-A3 and 5-HT3A-A5 had desensitization rates at least an order of magnitude faster than WT. The one Ala loop construct, 5-HT3A-A1, entered a non-functional state from which it did not recover after the first 5-HT application. These results suggest that the large M3M4 loop of eukaryotic Cys-loop channels is not required for receptor assembly or function. However, loop length and amino acid composition can effect channel expression and desensitization. We infer that the cytoplasmic ends of the M3 and M4 segments may undergo conformational changes during channel gating and desensitization and/or the loop may influence the position and mobility of these segments as they undergo gating-induced conformational changes. Altering structure or conformational mobility of the cytoplasmic ends of M3 and M4 may be the basis by which phosphorylation or protein binding to the cytoplasmic loop alters channel function.