Shaker Mutants Research Articles

Eukaryotic Kv channel Shaker inactivates through selectivity filter dilation rather than collapse

Eukaryotic voltage-gated K + channels have been extensively studied, but the structural bases for some of their most salient functional features remain to be established. C-type inactivation, for example, is an auto-inhibitory mechanism that confers temporal resolution to their signal-firing activity. In a recent breakthrough, studies of a mutant of Shaker that is prone to inactivate indicated that this process entails a dilation of the selectivity filter, the narrowest part of the ion conduction pathway. Here, we report an atomic-resolution cryo–electron microscopy structure that demonstrates that the wild-type channel can also adopt this dilated state. All-atom simulations corroborate this conformation is congruent with the electrophysiological characteristics of the C-type inactivated state, namely, residual K + conductance and altered ion specificity, and help rationalize why inactivation is accelerated or impeded by certain mutations. In summary, this study establishes the molecular basis for an important self-regulatory mechanism in eukaryotic K + channels, laying a solid foundation for further studies.

An activator of voltage-gated K+ channels Kv1.1 as a therapeutic candidate for episodic ataxia type 1

Loss-of-function mutations in the KCNA1(Kv1.1) gene cause episodic ataxia type 1 (EA1), a neurological disease characterized by cerebellar dysfunction, ataxic attacks, persistent myokymia with painful cramps in skeletal muscles, and epilepsy. Precision medicine for EA1 treatment is currently unfeasible, as no drug that can enhance the activity of Kv1.1-containing channels and offset the functional defects caused by KCNA1 mutations has been clinically approved. Here, we uncovered that niflumic acid (NFA), a currently prescribed analgesic and anti-inflammatory drug with an excellent safety profile in the clinic, potentiates the activity of Kv1.1 channels. NFA increased Kv1.1 current amplitudes by enhancing the channel open probability, causing a hyperpolarizing shift in the voltage dependence of both channel opening and gating charge movement, slowing the OFF-gating current decay. NFA exerted similar actions on both homomeric Kv1.2 and heteromeric Kv1.1/Kv1.2 channels, which are formed in most brain structures. We show that through its potentiating action, NFA mitigated the EA1 mutation-induced functional defects in Kv1.1 and restored cerebellar synaptic transmission, Purkinje cell availability, and precision of firing. In addition, NFA ameliorated the motor performance of a knock-in mouse model of EA1 and restored the neuromuscular transmission and climbing ability in Shaker (Kv1.1) mutant Drosophila melanogaster flies (Sh5). By virtue of its multiple actions, NFA has strong potential as an efficacious single-molecule-based therapeutic agent for EA1 and serves as a valuable model for drug discovery.

Effects of memantine on mania-like phenotypes exhibited by Drosophila Shaker mutants.

Increased glutamate levels and electrolytic fluctuations have been observed in acutely manic patients. Despite some efficacy of the non-competitive NMDA receptor antagonist memantine (Mem), such as antidepressant-like and mood-stabilizer drugs in clinical studies, its specific mechanisms of action are still uncertain. The present study aims to better characterize the Drosophila melanogaster fly Shaker mutants (SH), as a translational model of manic episodes within bipolar disorder in humans, and to investigate the potential anti-manic properties of Mem. Our findings showed typical behavioral abnormalities in SH, which mirrored with the overexpression of NMDAR-NR1 protein subunit, matched well to glutamate up-regulation. Such molecular features were associated to a significant reduction of SH brain volume in comparison to Wild Type strain flies (WT). Here we report on the ability of Mem treatment to ameliorate behavioral aberrations of SH (similar to that of Lithium), and its ability to reduce NMDAR-NR1 over-expression. Our results show the involvement of the glutamatergic system in the SH, given the interaction between the Shaker channel and the NMDA receptor, suggesting this model as a promising tool for studying the neurobiology of bipolar disorders. Moreover, our results show Mem as a potential disease-modifying therapy, providing insight on new mechanisms of action.

Programmed cell death in cerebellar Purkinje neurons.

Apoptosis, autophagy and necrosis are the three main types of programmed cell death. One or more of these types of programmed cell death may take place in neurons leading to their death in various neurodegenerative disorders in humans. Purkinje neurons (PNs) are among the most highly vulnerable population of neurons to cell death in response to intrinsic hereditary diseases or extrinsic toxic, hypoxic, ischemic, and traumatic injury. In this review, we will describe the three main types of programmed cell death, including the molecular mechanisms and the sequence of events in each of them, and thus illustrating the intracellular proteins that mediate and regulate each of these types. Then, we will discuss the role of Ca2+ in PN function and increased vulnerability to cell death. Additionally, PN death will be described in animal models, namely lurcher mutant mouse and shaker mutant rat, in order to illustrate the potential therapeutic implications of programmed cell death in PNs by reviewing the previous studies that were carried out to interfere with the programmed cell death in an attempt to rescue PNs from death.

Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel.

C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K+ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K+ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-μs MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D.

Potassium channel Shaker play a protective role against cardiac aging in Drosophila.

Potassium channels, which are the most diverse group of the ion channel family, play an important role in the repolarization of cardiomyocytes. Recent studies showed that potassium channels, such as KCNQ and HERG/eag, play an important role in regulating adult heart function through shaping the action potential and maintaining the rhythm of cardiac contraction. The potassium channel protein Shaker is the first voltage-gated potassium channel found in Drosophila to maintain the electrical excitability of neurons and muscle cells, but its role in adult cardiac function is still unclear. In this study, Drosophila was used as a model to study the role of Shaker channel in the maintenance of cardiac function under stress and aging. The incidence of heart failure was observed in shaker mutant after external electrical pacing, which simulates cardiac stress. Additionally, The cardiac-specific driver hand4.2 Gal4 was used to specifically knock down the expression of the potassium channel shaker in Drosophila. The cardiac parameter was analyzed at 1, 3, 5 weeks of age on cardiac specific knockdown of shaker using Drosophila adult cardiac physiological assay. The results showed that the mutation of shaker gene seriously affect the cardiac function under stress, demonstrated by significant increase in heart failure rate under electrical stimulation. In addition, cardiac specific knockdown of shaker increased the incidence of arrhythmias in Drosophila at the age of 5 weeks. Cardiac-specific knockdown of shaker reduces life span. Therefore, the results of this study suggest a vital role of the potassium channel shaker in maintaining normal cardiac function during aging.

Active caspase-3 upregulation is augmented in at-risk cerebellar Purkinje cells following inferior olive chemoablation in the shaker mutant rat: an immunofluorescence study

ABSTRACTObjectives: Mechanisms underlying Purkinje cell (PC) death, which leads to many diseases in humans, are still poorly elucidated. Progressive PC degeneration occurs in shaker mutant rat due to an X-linked recessive mutation leading to gait ataxia and total-body tremors. Chemoablation of the inferior olive (IO) and olivocerebellar deafferentation temporally accelerated PC death from the natural 6–8 week time-course to 1–2 weeks in the shaker mutant rat. The present study posits that IO chemoablation leads to the accelerated and augmented upregulation of the executioner active caspase-3 that triggers apoptosis of at-risk PCs throughout the ordinary phenotypic manifestation of the shaker mutation.Methods: Immunofluorescence and double labeling for calbindin and active caspase-3 were used in vermal cerebellar sections from IO-chemoablated rats to demonstrate the effect of IO chemoablation on active caspase-3 expression in at-risk PCs.Results: Active caspase-3 expression was enhanced in the anterior degeneration (ADC) and posterior degeneration (PDC) compartments to reach a peak in both degeneration compartments at 24 h following the injections for IO chemoablation.Discussion: Consequently, it can be deduced that active caspase-3 expression in shaker mutant rats is modifiable suggesting the possibility of targeting it therapeutically in an attempt to rescue PCs from death.Abbreviation PC: Purkinje cell; IO: inferior olive; ADC: Anterior degeneration compartment; PDC: Posterior degeneration compartment; ISC: Intermediate survival compartment; FNSC: Flocculonodular survival compartment.

Autophagy precedes apoptosis among at risk cerebellar Purkinje cells in the shaker mutant rat: an ultrastructural study

ABSTRACTCerebellar Purkinje cell (PC) death has been shown to occur in essential tremor, ataxia, and many other neurodegenerative diseases in humans. Shaker mutant rats have an X-linked recessive mutation that causes hereditary degeneration of “at risk” cerebellar PCs. This defect can occur in the restricted anterior (ADC) and posterior (PDC) vermal degeneration compartments postnatally within 7 to 14 weeks of age as a natural phenotype in the shaker mutant rat. “Secure” PCs persist in a flocculonodular survival compartment (FNSC). Because we have previously shown that “at risk” PCs die due to apoptosis in the shaker mutant rat, we hypothesized that the PC death observed in the hereditary shaker mutant rat may be due to the activation of more than one type of death pathway. This ultrastructural investigation suggests that “at risk” PCs die due to apoptosis as a result of autophagic activation. Moreover, our data suggest that both apoptosis and autophagy must be simultaneously inhibited to rescue “at risk” PCs from death.

Cerebellar Purkinje cells die by apoptosis in the shaker mutant rat

Cerebellar Purkinje cells (PCs) die in humans leading to a variety of diseases including ataxia and essential tremor. The etiology underlying PC death is poorly understood. Shaker mutant rat is a unique animal model of progressive PC degeneration that is compartmentalized and of adult-onset. In shaker mutant rats, hereditary degeneration of at risk PCs occurs between 7 and 14 postnatal weeks of age as a natural phenotypic expression of the shaker mutation and at earlier or later ages depending upon experimental conditions in restricted anterior (ADC) and posterior (PDC) vermal degeneration compartments. Secure PCs in a flocculonodular survival compartment (FNSC) always survive. In this study, we investigated DNA fragmentation and active caspase-3 expression characteristic of apoptosis using immunofluorescence and fluorescence microscopy. We also sought to confirm the occurrence of apoptosis in at risk PCs using transmission electron microscope. Purkinje neurons were the only cerebellar cells labeled for TUNEL and immunoreactive to active caspase-3 in the shaker mutant. Active caspase-3 expression observed was spatially and temporally congruous with the pattern of calbindin immunolabeling of degenerating PCs that is characterized by dendritic atrophy, shrinkage of PC cell bodies and the appearance of PC axonal torpedoes. DNA fragmentation, detected by TUNEL, as well as ultrastructural morphological changes characteristic for apoptosis, provided additional evidence for the occurrence of apoptosis in at risk PCs.

Gating Currents of Monomeric Hv Channel Reveals a Permeation Pathway Coupled to the Voltage Activation

Recently, a family of voltage-gated proton channels, called (Hv) was cloned from different organisms. Hv channel shares a common structure with the Voltage Sensor Domain (VSD) of voltage-gated K channels. Despite off the high homology between Hv channel and the VSD, they have distant quaternary structures. Hv channel lacks a pore domain, and is fully functional either as a monomer or dimer. The S4 was proposed as the possible permeation pathway, resembling the omega current present in some mutants of Shaker K+ channel. However the permeation pathway is still elusive. Fluorescence experiments suggests that the voltage activation in Hv is in two steps, where the second step is a cooperative conformational change, involving interactions between subunits that opens both permeation pathways in a concerted way in the dimer. However, the relationship between the H+ pore, and the voltage sensor activation is not fully understood for the monomer. Here we presented the first Hv gating currents recordings from the monomeric Hv form (Hv-ΔNΔC). Interestingly, the voltage sensor activation precedes the H+ conduction, suggesting that voltage sensor and H+ pore, are two different structures allosterically coupled. The Q-V present a zδ of 1.6 e0, in agreement with previous experiments of voltage-clamp fluorometry. The data presented support the hypothesis that the channel opening is associated to a second conformational state of the voltage sensor, associated to an extremely slow component in the gating current. Supported by Fondecyt grants ACT 1104 and Fondecyt 1120802 to CG and 1110430 to R.L.

Mutations in the Cavity Affect the Rate of Slow Inactivation in Shaker K+ Channels

It has been reported earlier that the activation and inactivation gates of Shaker channels are coupled, and this coupling might be mediated by a rotational motion of the S6 helix. We hypothese that either the electrostatics or the volume/hydrophobicity of the side chains pointing into the cavity can affect this coupling. Accordingly, we have introduced side-chains at position 470 in a T449A Shaker-IR background with different hydrophobicities according to the Wimley-White hydrophobicity scale (Ala, Cys, or Trp) and charges (Glu, Asp, Arg, Lys) and measured the inactivation kinetics. Mutant channels were transiently expressed in tsA_201 cells. All ionic current experiments were performed with excised inside-out patches. The introduction of an ionizable cysteine and the small but neutral alanine dramatically slowed entry into the inactivated state (the inactivation time constants for Shaker mutant T449A/I470C, T449A/I470A and T449A were 1.4 s, 250 ms and 50 ms, respectively). We could not detect any current on mutant T449A/I470X (X = Asp, Glu, Trp) but the transfection with a mixture of mutant and wild-type expression plasmids expressed currents. These heterotetramers had slower inactivation kinetics with respect to the T449A channels. Almost all of the channels had a midpoint voltage for activation in the range −50 mV to −20 mV. The only exception was the I470C mutant which had a large shift in voltage dependence (midpoint ∼5 mV). Mutant channels containing Arg or Lys (I470R, I470K) did not give functional expression either as homotetramers or as heterotetramers. Our results show that not the size but the physicochemical properties of the side chains (hydrophobicity and charge) at position 470 determine the inactivation kinetics of voltage-gated K+ channels, which may reflect interaction of the side chains with permeant ions.

The Shaker Potassium Channel Is No Target for Xenon Anesthesia in Short-SleepingDrosophila melanogasterMutants

Background. Xenon seems to be an ideal anesthetic drug. To explore if next to the antagonism at the NMDA-receptor other molecular targets are involved, we tested the xenon requirement in short sleeping Drosophila shaker mutants and in na[har 38]. Methods. The Drosophila melanogaster strains wildtype Canton-S, na[har 38], sh 102 and sh mns, were raised and sleep was measured. Based on the response of the flies at different xenon concentrations, logEC50 values were calculated. Results. The logEC50-values for WT Canton-S were 1.671 (1.601–1.742 95%-confidence intervall; n = 238; P versus sh 102 > 0,05), for sh mns 1.711 (1.650–1.773; n = 242; P versus WT Canton-S > 0,05). The logEC50-value for sh 102 was 1.594 (1.493–1.694; n = 261; P versus sh mns > 0.05). The logEC-value of na[har 38] was 2.076 (1.619–2.532; n = 207; P versus sh mns < 0.05, versus sh 102 < 0.05, versus WT Canton-S < 0.05). P values for all shaker mutants were P > 0.05, while na[har 38] was found to be hyposensitive compared to wildtype (P < 0.05). Conclusions. The xenon requirement in Drosophila melanogaster is not influenced by a single gene mutation at the shaker locus, whereas a reduced expression of a nonselective cation channel leads to an increased xenon requirement. This supports the thesis that xenon mediates its effects not only via an antagonism at the NMDA-receptor.

A Novel Current Pathway Parallel to the Central Pore in a Mutant Voltage-gated Potassium Channel

Voltage-gated potassium channels are proteins composed of four subunits consisting of six membrane-spanning segments S1-S6, with S4 as the voltage sensor. The region between S5 and S6 forms the potassium-selective ion-conducting central α-pore. Recent studies showed that mutations in the voltage sensor of the Shaker channel could disclose another ion permeation pathway through the voltage-sensing domain (S1-S4) of the channel, the ω-pore. In our studies we used the voltage-gated hKv1.3 channel, and the insertion of a cysteine at position V388C (Shaker position 438) generated a current through the α-pore in high potassium outside and an inward current at hyperpolarizing potentials carried by different cations like Na(+), Li(+), Cs(+), and NH(4)(+). The observed inward current looked similar to the ω-current described for the R1C/S Shaker mutant channel and was not affected by some pore blockers like charybdotoxin and tetraethylammonium but was inhibited by a phenylalkylamine blocker (verapamil) that acts from the intracellular side. Therefore, we hypothesize that the hKv1.3_V388C mutation in the P-region generated a channel with two ion-conducting pathways. One, the α-pore allowing K(+) flux in the presence of K(+), and the second pathway, the σ-pore, functionally similar but physically distinct from the ω-pathway. The entry of this new pathway (σ-pore) is presumably located at the backside of Y395 (Shaker position 445), proceeds parallel to the α-pore in the S6-S6 interface gap, ending between S5 and S6 at the intracellular side of one α-subunit, and is blocked by verapamil.

Gate Closure Strictly Follows Voltage-Sensor Movements in K V Channels

Kv channels are voltage-dependent potassium pores that shape the action potential duration and are critical for cell excitability. Detection of membrane potential (V) is done by a charged (Q) voltage sensor domain (VSD) whose reorientations generate a transient gating current (IQ). Prolonged depolarization of Shaker Kv channels pushes the VSD into the relaxed state, characterized by a slowing in IQOff. Kv channels also have two gates (in series) that seal off K+ permeation: the S6 bundle crossing (BC), directly tied to the VSD, and the selectivity filter (SF). Direct comparison of K+-conduction in Shaker, reflecting the status of the BC gate, with IQ shows a strong correlation between both. As IQOff slowed down with prolonged depolarizations, BC gate closure displayed a similar 2-fold slowing when the duration of a +20mV pre-pulse was increased from 0.2 to 10 seconds. Simultaneous monitoring of the VSD movement (fluorescence recordings) and channel gate closure (ionic recordings) in the TMRM-labeled Shaker mutant M356C showed that the slowing in IQOff and gate closure occurs simultaneously. This indicates that the gate is strictly controlled by the movements of the VSD and most importantly that the BC gate remains open even when the VSD relaxes. Consequently, K+ conduction continues as long as the SF gate does not close (inactivation). Interestingly, in Kv3.1 - a channel that regulates high frequency firing in-vivo - the opposite behavior was observed: prolonged depolarization speeded up both IQOff and gate closure. Thus, the effect of VSD relaxation differs between different subtypes of Kv channels suggesting that relaxation affects the excitability of cells differently depending on their depolarization history, either reducing excitability in cells expressing Shaker or increasing it in case of Kv3.1. (Support: NIH-GM030376, FWO-G025608)

Coordinating Different Homeostatic Processes

In this issue of Neuron, Bergquist and colleagues study a rapid form of presynaptic homeostatic regulation at the Drosophila neuromuscular junction. They show that the K(+) channel genes shal and shaker are reciprocally regulated in the central nervous system and suggest a hierarchical organization of intrinsic and synaptic homeostatic regulatory processes.

An Intersubunit Interaction between S4-S5 Linker and S6 Is Responsible for the Slow Off-gating Component in Shaker K+ Channels

Voltage-gated ion channels are controlled by the membrane potential, which is sensed by peripheral, positively charged voltage sensors. The movement of the charged residues in the voltage sensor may be detected as gating currents. In Shaker K(+) channels, the gating currents are asymmetric; although the on-gating currents are fast, the off-gating currents contain a slow component. This slow component is caused by a stabilization of the activated state of the voltage sensor and has been suggested to be linked to ion permeation or C-type inactivation. The molecular determinants responsible for the stabilization, however, remain unknown. Here, we identified an interaction between Arg-394, Glu-395, and Leu-398 on the C termini of the S4-S5 linker and Tyr-485 on the S6 of the neighboring subunit, which is responsible for the development of the slow off-gating component. Mutation of residues involved in this intersubunit interaction modulated the strength of the associated interaction. Impairment of the interaction still led to pore opening but did not exhibit slow gating kinetics. Development of this interaction occurs under physiological ion conduction and is correlated with pore opening. We, thus, suggest that the above residues stabilize the channel in the open state.

A single charged voltage sensor is capable of gating the Shaker K+ channel

We sought to determine the contribution of an individual voltage sensor to Shaker's function. Concatenated heterotetramers of Shaker zH4 Δ(6–46) wild type (wt) in combination with a neutral S4 segment Shaker mutant (mut) with stoichiometries 2wt/2mut and 1wt/3mut were studied and compared with the 4wt concatenated homotetramer. A single charged voltage sensor is sufficient to open Shaker conductance with reduced delay (<1 ms) and at more hyperpolarized voltages compared with 4wt. In addition, the wt-like slow inactivation of 1wt/3mut was almost completely eliminated by mutations T449V-I470C in its single wt subunit, indicating that the subunits bearing a neutral S4 were unable to trigger slow inactivation. Our results strongly suggest that a neutral S4 segment of Shaker's subunit is voltage insensitive and its voltage sensor is in the activated position (i.e., ready for pore opening), and provide experimental support to the proposed model of independent voltage sensors with a final, almost voltage-independent concerted step.

Thalamic Microinfusion of Antibody to a Voltage-gated Potassium Channel Restores Consciousness during Anesthesia

The Drosophila Shaker mutant fruit-fly, with its malfunctioning voltage-gated potassium channel, exhibits anesthetic requirements that are more than twice normal. Shaker mutants with an abnormal Kv1.2 channel also demonstrate significantly reduced sleep. Given the important role the thalamus plays in both sleep and arousal, the authors investigated whether localized central medial thalamic (CMT) microinfusion of an antibody designed to block the pore of the Kv1.2 channel might awaken anesthetized rats. Male Sprague-Dawley rats were implanted with a cannula aimed at the CMT or lateral thalamus. One week later, unconsciousness was induced with either desflurane (3.6 +/- 0.2%; n = 55) or sevoflurane (1.2 +/- 0.1%; n = 51). Arousal effects of a single 0.5-microl infusion of Kv1.2 potassium channel blocking antibody (0.1- 0.2 mg/ml) or a control infusion of Arc-protein antibody (0.2 mg/ml) were then determined. The Kv1.2 antibody, but not the control antibody, temporarily restored consciousness in 17% of all animals and in 75% of those animals where infusions occurred within the CMT (P < 0.01 for each anesthetic). Lateral thalamic infusions showed no effects. Consciousness returned on average (+/- SD) 170 +/- 99 s after infusion and lasted a median time of 398 s (interquartile range: 279-510 s). Temporary seizures, without apparent consciousness, predominated in 33% of all animals. These findings support the idea that the CMT plays a role in modulating levels of arousal during anesthesia and further suggest that voltage-gated potassium channels in the CMT may contribute to regulating arousal or may even be relevant targets of anesthetic action.

Light-Dependent Modulation of Shab Channels via Phosphoinositide Depletion in Drosophila Photoreceptors

The Drosophila phototransduction cascade transforms light into depolarizations that are further shaped by activation of voltage-dependent K+ (Kv) channels. In whole-cell recordings of isolated photoreceptors, we show that light selectively modulated the delayed rectifier (Shab) current. Shab currents were increased by light with similar kinetics to the light-induced current itself (latency approximately 20 ms), recovering to control values with a t(1/2) of approximately 60 s in darkness. Genetic disruption of PLCbeta4, responsible for light-induced PIP(2) hydrolysis, abolished this light-dependent modulation. In mutants of CDP-diaclyglycerol synthase (cds(1)), required for PIP(2) resynthesis, the modulation became irreversible, but exogenously applied PIP(2) restored reversibility. The modulation was accurately and reversibly mimicked by application of PIP(2) to heterologously expressed Shab channels in excised inside-out patches. The results indicate a functionally implemented mechanism of Kv channel modulation by PIP(2) in photoreceptors, which enables light-dependent regulation of signal processing by direct coupling to the phototransduction cascade.

Pre- and post-synaptic mechanisms of synaptic strength homeostasis revealed by slowpoke and shaker K+ channel mutations in Drosophila

We report naturally occurring, systematic variations in synaptic strength at neuromuscular junctions along the dorsal–ventral (D-V) axis of the Drosophila larval body wall. These gradual changes were correlated with differences in presynaptic neurotransmitter release regulated by nerve terminal excitability and in postsynaptic receptor composition influencing miniature excitatory junctional potential (mEJP) amplitude. Surprisingly, synaptic strength and D-V differentials at physiological Ca2+ levels were not significantly altered in slowpoke (slo) and Shaker (Sh) mutants, despite their defects in two major repolarizing forces, Ca2+-activated Slo (BK) and voltage-activated Sh currents, respectively. However, lowering [Ca2+]o levels revealed greatly altered synaptic mechanisms in these mutants, indicated by drastically enhanced excitatory junctional potentials (EJPs) in Sh but paradoxically reduced EJPs in slo. Removal of Sh current in slo mutants by 4-aminopyridine blockade or by combining slo with Sh mutations led to strikingly increased synaptic transmission, suggesting upregulation of presynaptic Sh current to limit excessive neurotransmitter release in the absence of Slo current. In addition, slo mutants displayed altered immunoreactivity intensity ratio between DGluRIIA and DGluRIIB receptor subunits. This modified receptor composition caused smaller mEJP amplitudes, further preventing excessive transmission in the absence of Slo current. Such compensatory regulations were prevented by rutabaga (rut) adenylyl cyclase mutations in rut slo double mutants, demonstrating a novel role of rut in homeostatic plasticity, in addition to its well-established function in learning behavior.