Two KCNA2 variants (p.H310Y and p.H310R) were discovered in paediatric patients with epilepsy and developmental delay. KCNA2 encodes KV1.2-channel subunits, which regulate neuronal excitability. Both gain and loss of KV1.2 function cause epilepsy, precluding the prediction of variant effects; and while H310 is conserved throughout the KV-channel superfamily, it is largely understudied. We investigated both variants in heterologously expressed, human KV1.2 channels by immunocytochemistry, electrophysiology and voltage-clamp fluorometry. Despite affecting the same channel, at the same position, and being associated with severe neurological disease, the two variants had diametrically opposite effects on KV1.2 functional expression. The p.H310Y variant produced ‘dual gain of function’, increasing both cell-surface trafficking and activity, delaying channel closure. We found that the latter is due to the formation of a hydrogen bond that stabilizes the active state of the voltage-sensor domain. Additionally, H310Y abolished ‘ball and chain’ inactivation of KV1.2 by KVβ1 subunits, enhancing gain of function. In contrast, p.H310R caused ‘dual loss of function’, diminishing surface levels by multiple impediments to trafficking and inhibiting voltage-dependent channel opening. We discuss the implications for KV-channel biogenesis and function, an emergent hotspot for disease-associated variants, and mechanisms of epileptogenesis. 

Significance: A child with epilepsy has a previously unreported, heterozygous mutation in KCNA2, the gene encoding K_V1.2 proteins. Four K_V1.2 assemble into a potassium-selective channel, a protein complex at the neuronal cell surface regulating electrical signaling. K_V1.2 subunits assemble with other K_V1-family members to form heterotetrameric channels, contributing to neuronal potassium-channel diversity. The most striking consequence of this mutation is preventing K_V1.2-subunit trafficking, i.e., their ability to reach the cell surface. Moreover, the mutation is dominant negative, as mutant subunits can assemble with wild-type K_V1.2 and K_V1.4, trapping them into nontrafficking heterotetramers and decreasing their functional expression. Thus, K_V1-family genes’ ability to form heterotetrameric channels is a double-edged sword, rendering K_V1-family members vulnerable to dominant-negative mutations in a single member gene.

Abstract: We report on a heterozygous KCNA2 variant in a child with epilepsy. KCNA2 encodes K_V1.2 subunits, which form homotetrameric potassium channels and participate in heterotetrameric channel complexes with other K_V1-family subunits, regulating neuronal excitability. The mutation causes substitution F233S at the K_V1.2 charge transfer center of the voltage-sensing domain. Immunocytochemical trafficking assays showed that K_V1.2(F233S) subunits are trafficking deficient and reduce the surface expression of wild-type K_V1.2 and K_V1.4: a dominant-negative phenotype extending beyond KCNA2, likely profoundly perturbing electrical signaling. Yet some K_V1.2(F233S) trafficking was rescued by wild-type K_V1.2 and K_V1.4 subunits, likely in permissible heterotetrameric stoichiometries: electrophysiological studies utilizing applied transcriptomics and concatemer constructs support that up to one or two K_V1.2(F233S) subunits can participate in trafficking-capable heterotetramers with wild-type K_V1.2 or K_V1.4, respectively, and that both early and late events along the biosynthesis and secretion pathway impair trafficking. These studies suggested that F233S causes a depolarizing shift of ∼48 mV on K_V1.2 voltage dependence. Optical tracking of the K_V1.2(F233S) voltage-sensing domain (rescued by wild-type K_V1.2 or K_V1.4) revealed that it operates with modestly perturbed voltage dependence and retains pore coupling, evidenced by off-charge immobilization. The equivalent mutation in the Shaker K⁺ channel (F290S) was reported to modestly affect trafficking and strongly affect function: an ∼80-mV depolarizing shift, disrupted voltage sensor activation and pore coupling. Our work exposes the multigenic, molecular etiology of a variant associated with epilepsy and reveals that charge-transfer-center disruption has different effects in K_V1.2 and Shaker, the archetypes for potassium channel structure and function.

Ventricular arrhythmias, a leading cause of sudden cardiac death, can be triggered by cardiomyocyte early afterdepolarizations (EADs). EADs can result from an abnormal late activation of L-type Ca2+ channels (LTCCs). Current LTCC blockers (class IV antiarrhythmics), while effective at suppressing EADs, block both early and late components of I-Ca,I-L, compromising inotropy. However, computational studies have recently demonstrated that selective reduction of late I-Ca,I-L (Ca2+ influx during late phases of the action potential) is sufficient to potently suppress EADs, suggesting that effective antiarrhythmic action can be achieved without blocking the early peak I-Ca,I-L, which is essential for proper excitation-contraction coupling. We tested this new strategy using a purine analogue, roscovitine, which reduces late I-Ca,I-L with minimal effect on peak current. Scaling our investigation from a human Ca(V)1.2 channel clone to rabbit ventricular myocytes and rat and rabbit perfused hearts, we demonstrate that (1) roscovitine selectively reduces I-Ca,I-L noninactivating component in a human Ca(V)1.2 channel clone and in ventricular myocytes native current, (2) the pharmacological reduction of late I-Ca,I-L suppresses EADs and EATs (early after Ca2+ transients) induced by oxidative stress and hypokalemia in isolated myocytes, largely preserving cell shortening and normal Ca2+ transient, and (3) late I-Ca,I-L reduction prevents/suppresses ventricular tachycardia/fibrillation in ex vivo rabbit and rat hearts subjected to hypokalemia and/or oxidative stress. These results support the value of an antiarrhythmic strategy based on the selective reduction of late I-Ca,I-L to suppress EAD-mediated arrhythmias. Antiarrhythmic therapies based on this idea would modify the gating properties of Ca(V)1.2 channels rather than blocking their pore, largely preserving contractility.

Key points K(V)1.2 channels, encoded by theKCNA2gene, regulate neuronal excitability by conducting K(+)upon depolarization. A newKCNA2missense variant was discovered in a patient with epilepsy, causing amino acid substitution F302L at helix S4, in the K(V)1.2 voltage-sensing domain. Immunocytochemistry and flow cytometry showed that F302L does not impair KCNA2 subunit surface trafficking. Molecular dynamics simulations indicated that F302L alters the exposure of S4 residues to membrane lipids. Voltage clamp fluorometry revealed that the voltage-sensing domain of K(V)1.2-F302L channels is more sensitive to depolarization. Accordingly, K(V)1.2-F302L channels opened faster and at more negative potentials; however, they also exhibited enhanced inactivation: that is, F302L causes both gain- and loss-of-function effects. Coexpression of KCNA2-WT and -F302L did not fully rescue these effects. The probands symptoms are more characteristic of patients with loss ofKCNA2function. Enhanced K(V)1.2 inactivation could lead to increased synaptic release in excitatory neurons, steering neuronal circuits towards epilepsy. An exome-based diagnostic panel in an infant with epilepsy revealed a previously unreportedde novomissense variant inKCNA2, which encodes voltage-gated K(+)channel K(V)1.2. This variant causes substitution F302L, in helix S4 of the K(V)1.2 voltage-sensing domain (VSD). F302L does not affect KCNA2 subunit membrane trafficking. However, it does alter channel functional properties, accelerating channel opening at more hyperpolarized membrane potentials, indicating gain of function. F302L also caused loss of K(V)1.2 function via accelerated inactivation onset, decelerated recovery and shifted inactivation voltage dependence to more negative potentials. These effects, which are not fully rescued by coexpression of wild-type and mutant KCNA2 subunits, probably result from the enhancement of VSD function, as demonstrated by optically tracking VSD depolarization-evoked conformational rearrangements. In turn, molecular dynamics simulations suggest altered VSD exposure to membrane lipids. Compared to other encephalopathy patients withKCNA2mutations, the proband exhibits mild neurological impairment, more characteristic of patients withKCNA2loss of function. Based on this information, we propose a mechanism of epileptogenesis based on enhanced K(V)1.2 inactivation leading to increased synaptic release preferentially in excitatory neurons, and hence the perturbation of the excitatory/inhibitory balance of neuronal circuits.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons degenerate, resulting in muscle atrophy, paralysis, and fatality. Studies using mouse models of ALS indicate a protracted period of disease development with progressive motor neuron pathology, evident as early as embryonic and postnatal stages. Key missing information includes concomitant alterations in the sensorimotor circuit essential for normal development and function of the neuromuscular system. Leveraging unique brainstem circuitry, we show in vitro evidence for reflex circuit-specific postnatal abnormalities in the jaw proprioceptive sensory neurons in the well-studied SOD1(G)(93A) mouse. These include impaired and arrhythmic action potential burst discharge associated with a deficit in Nav 1.6 Na+ channels. However, the mechanoreceptive and nociceptive trigeminal ganglion neurons and the visual sensory retinal ganglion neurons were resistant to excitability changes in age-matched SOD1(G)(93A )mice. Computational modeling of the observed disruption in sensory patterns predicted asynchronous self-sustained motor neuron discharge suggestive of imminent reflexive defects, such as muscle fasciculations in ALS. These results demonstrate a novel reflex circuit-specific proprioceptive sensory abnormality in ALS.

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique, a relatively recent addition to the electrophysiologist’s armamentarium, was specifically developed by Drs. Stefani and Bezanilla (Bezanilla et al. 1991) to achieve low-noise recordings of the membrane of Xenopus laevis oocytes with fast clamp speed and thus optimize the most popular transient expression system to reveal the activity voltage-dependent proteins previously difficult to resolve by alternative methods. The high degree of specialization of this technique is complemented by its flexibility; in addition to oocyte perfusion, COVG can be combined with optical measurements (voltage clamp fluorometry and spectroscopy) and flash photolysis for the instantaneous release of intracellular caged compounds, expanding its use beyond electrophysiology.

Neurons utilize bursts of action potentials as an efficient and reliable way to encode information. It is likely that the intrinsic membrane properties of neurons involved in burst generation may also participate in preserving its temporal features. Here we examined the contribution of the persistent and resurgent components of voltage-gated Na⁺ currents in modulating the burst discharge in sensory neurons. Using mathematical modeling, theory and dynamic-clamp electrophysiology, we show that, distinct from the persistent Na⁺ component which is important for membrane resonance and burst generation, the resurgent Na⁺ can help stabilize burst timing features including the duration and intervals. Moreover, such a physiological role for the resurgent Na⁺ offered noise tolerance and preserved the regularity of burst patterns. Model analysis further predicted a negative feedback loop between the persistent and resurgent gating variables which mediate such gain in burst stability. These results highlight a novel role for the voltage-gated resurgent Na⁺ component in moderating the entropy of burst-encoded neural information.

Proteins possess a complex and dynamic structure, which is influenced by external signals and may change as they perform their biological functions. We present an optical approach, distance-encoding photoinduced electron transfer (DEPET), capable of the simultaneous study of protein structure and function. An alternative to FRET-based methods, DEPET is based on the quenching of small conjugated fluorophores by photoinduced electron transfer: a reaction that requires contact of the excited fluorophore with a suitable electron donor. This property allows DEPET to exhibit exceptional spatial and temporal resolution capabilities in the range pertinent to protein conformational change. We report the first implementation of DEPET on human large-conductance K+ (BK) channels under voltage clamp. We describe conformational rearrangements underpinning BK channel sensitivity to electrical excitation, in conducting channels expressed in living cells. Finally, we validate DEPET in synthetic peptide length standards, to evaluate its accuracy in measuring sub-and near-nanometer intramolecular distances.

BK channels are universal regulators of cell excitability, given their exceptional unitary conductance selective for K(+), joint activation mechanism by membrane depolarization and intracellular [Ca(2+)] elevation, and broad expression pattern. In this chapter, we discuss the structural basis and operational principles of their activation, or gating, by membrane potential and calcium. We also discuss how the two activation mechanisms interact to culminate in channel opening. As members of the voltage-gated potassium channel superfamily, BK channels are discussed in the context of archetypal family members, in terms of similarities that help us understand their function, but also seminal structural and biophysical differences that confer unique functional properties.

Excitation-evoked calcium influx across cellular membranes is strictly controlled by voltage-gated calcium channels (Ca_V), which possess four distinct voltage-sensing domains (VSDs) that direct the opening of a central pore. The energetic interactions between the VSDs and the pore are critical for tuning the channel’s voltage dependence. The accessory α₂δ-1 subunit is known to facilitate Ca_V1.2 voltage-dependent activation, but the underlying mechanism is unknown. In this study, using voltage clamp fluorometry, we track the activation of the four individual VSDs in a human L-type Ca_V1.2 channel consisting of α_1C and β₃ subunits. We find that, without α₂δ-1, the channel complex displays a right-shifted voltage dependence such that currents mainly develop at nonphysiological membrane potentials because of very weak VSD–pore interactions. The presence of α₂δ-1 facilitates channel activation by increasing the voltage sensitivity (i.e., the effective charge) of VSDs I–III. Moreover, the α₂δ-1 subunit also makes VSDs I–III more efficient at opening the channel by increasing the coupling energy between VSDs II and III and the pore, thus allowing Ca influx within the range of physiological membrane potentials.

Early afterdepolarizations (EADs) associated with prolongation of the cardiac action potential (AP) can create heterogeneity of repolarization and premature extrasystoles, triggering focal and reentrant arrhythmias. Because the L-type Ca²⁺ current (I_Ca,L) plays a key role in both AP prolongation and EAD formation, L-type Ca²⁺ channels (LTCCs) represent a promising therapeutic target to normalize AP duration (APD) and suppress EADs and their arrhythmogenic consequences. We used the dynamic-clamp technique to systematically explore how the biophysical properties of LTCCs could be modified to normalize APD and suppress EADs without impairing excitation–contraction coupling. Isolated rabbit ventricular myocytes were first exposed to H₂O₂ or moderate hypokalemia to induce EADs, after which their endogenous I_Ca,L was replaced by a virtual I_Ca,L with tunable parameters, in dynamic-clamp mode. We probed the sensitivity of EADs to changes in the (a) amplitude of the noninactivating pedestal current; (b) slope of voltage-dependent activation; (c) slope of voltage-dependent inactivation; (d) time constant of voltage-dependent activation; and (e) time constant of voltage-dependent inactivation. We found that reducing the amplitude of the noninactivating pedestal component of I_Ca,L effectively suppressed both H₂O₂- and hypokalemia-induced EADs and restored APD. These results, together with our previous work, demonstrate the potential of this hybrid experimental–computational approach to guide drug discovery or gene therapy strategies by identifying and targeting selective properties of LTCC.

Excitation-evoked Ca(2+) influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca(2+) entry are thought to be functions of voltage-gated calcium (CaV) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I-IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of CaV1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage- and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in CaV1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ∼50 meV (∼2 kT), amounting to ∼85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (∼16 meV). VSD IV did not appear to participate in channel opening.

The cut-open oocyte Vaseline gap (COVG) voltage-clamp technique, a relatively recent addition to theelectrophysiologist’s armamentarium, was specificallydeveloped by Drs. Stefani and Bezanilla (Bezanillaet al.1991) to achieve low-noise recordings of themembrane ofXenopus laevisoocytes with fast clampspeed and, thus, optimize the most popular transientexpression system to reveal the activity voltage-dependent proteins previously difficult to resolve byalternative methods. The high degree of specializationof this technique is complemented by its flexibility: inaddition  to  oocyte  perfusion,  COVG  can  beencombined  with  optical  measurements  (voltage-clamp fluorometry and spectroscopy) and flash pho-tolysisfor the instantaneous release of intracellular-caged  compounds,  expanding  its  use  beyondelectrophysiology.

Large-conductance Ca²⁺- and voltage-gated K⁺ channels are activated by an increase in intracellular Ca²⁺ concentration and/or depolarization. The channel activation mechanism is well described by an allosteric model encompassing the gate, voltage sensors, and Ca²⁺ sensors, and the model is an excellent framework to understand the influences of auxiliary β and γ subunits and regulatory factors such as Mg²⁺. Recent advances permit elucidation of structural correlates of the biophysical mechanism.

Voltage-activated proteins can sense, and respond to, changes in the electric field pervading the cell membrane by virtue of a transmembrane helix bundle, the voltage-sensing domain (VSD). Canonical VSDs consist of four transmembrane helices (S1-S4) of which S4 is considered a principal component because it possesses charged residues immersed in the electric field. Membrane depolarization compels the charges, and by extension S4, to rearrange with respect to the field. The VSD of large-conductance voltage- and Ca-activated K(+) (BK) channels exhibits two salient inconsistencies from the canonical VSD model: (1) the BK channel VSD possesses an additional nonconserved transmembrane helix (S0); and (2) it exhibits a "decentralized" distribution of voltage-sensing charges, in helices S2 and S3, in addition to S4. Considering these unique features, the voltage-dependent rearrangements of the BK VSD could differ significantly from the standard model of VSD operation. To understand the mode of operation of this unique VSD, we have optically tracked the relative motions of the BK VSD transmembrane helices during activation, by manipulating the quenching environment of site-directed fluorescent labels with native and introduced Trp residues. Having previously reported that S0 and S4 diverge during activation, in this work we demonstrate that S4 also diverges from S1 and S2, whereas S2, compelled by its voltage-sensing charged residues, moves closer to S1. This information contributes spatial constraints for understanding the BK channel voltage-sensing process, revealing the structural rearrangements in a non-canonical VSD.

Large conductance voltage- and Ca²⁺-activated K⁺ (BK) channels are potent regulators of cellular processes including neuronal firing, synaptic transmission, cochlear hair cell tuning, insulin release, and smooth muscle tone. Their unique activation pathway relies on structurally distinct regulatory domains including one transmembrane voltage-sensing domain (VSD) and two intracellular high affinity Ca²⁺-sensing sites per subunit (located in the RCK1 and RCK2 domains). Four pairs of RCK1 and RCK2 domains form a Ca²⁺-sensing apparatus known as the “gating ring.” The allosteric interplay between voltage- and Ca²⁺-sensing apparati is a fundamental mechanism of BK channel function. Using voltage-clamp fluorometry and UV photolysis of intracellular caged Ca²⁺, we optically resolved VSD activation prompted by Ca²⁺ binding to the gating ring. The sudden increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced a hyperpolarizing shift in the voltage dependence of both channel opening and VSD activation, reported by a fluorophore labeling position 202, located in the upper side of the S4 transmembrane segment. The neutralization of the Ca²⁺ sensor located in the RCK2 domain abolished the effect of [Ca²⁺]_i increase on the VSD rearrangements. On the other hand, the mutation of RCK1 residues involved in Ca²⁺ sensing did not prevent the effect of Ca²⁺ release on the VSD, revealing a functionally distinct interaction between RCK1 and RCK2 and the VSD. A statistical-mechanical model quantifies the complex thermodynamics interplay between Ca²⁺ association in two distinct sites, voltage sensor activation, and BK channel opening.

Large-conductance voltage- and Ca²⁺-dependent K⁺ (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca²⁺ homeostasis. Their activation results from the complex synergy between the transmembrane voltage sensors and a large (>300 kDa) C-terminal, cytoplasmic complex (the “gating ring”), which confers sensitivity to intracellular Ca²⁺ and other ligands. However, the molecular and biophysical operation of the gating ring remains unclear. We have used spectroscopic and particle-scale optical approaches to probe the metal-sensing properties of the human BK gating ring under physiologically relevant conditions. This functional molecular sensor undergoes Ca²⁺- and Mg²⁺-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba²⁺-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca²⁺-binding site (the “calcium bowl”) reduced the Ca²⁺ and abolished the Mg²⁺ dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca²⁺-binding site and that Mg²⁺ can bind to the calcium bowl with less affinity than Ca²⁺. Dynamic light scattering analysis revealed a reversible Ca²⁺-dependent decrease of the hydrodynamic radius of the gating ring, consistent with a more compact overall shape. These structural changes, resolved under physiologically relevant conditions, likely represent the molecular transitions that initiate the ligand-induced activation of the human BK channel.

Non‐technical summary Diseases, genetic defects, or ionic imbalances can alter the normal electrical activity of cardiac myocytes causing an anomalous heart rhythm, which can degenerate to ventricular fibrillation (VF) and sudden cardiac death. Well‐recognized triggers for VF are aberrations of the cardiac action potential, known as early afterdepolarizations (EADs). In this study, combining mathematical modelling and experimental electrophysiology in real‐time (dynamic clamp), we investigated the dependence of EADs on the biophysical properties of the L‐type Ca²⁺ current (I_Ca,L) and identified modifications of I_Ca,L properties which effectively suppress EAD. We found that minimal changes in the voltage dependence of activation or inactivation of I_Ca,L can dramatically reduce the occurrence of EADs in cardiac myocytes exposed to different EAD‐inducing conditions. This work assigns a critical role to the L‐type Ca²⁺ channel biophysical properties for EADs formation and identifies the L‐type Ca²⁺ channel as a promising therapeutic target to suppress EADs and their arrhythmogenic effects.

Voltage sensor domains (VSDs) are structurally and functionally conserved protein modules that consist of four transmembrane segments (S1–S4) and confer voltage sensitivity to many ion channels. Depolarization is sensed by VSD-charged residues residing in the membrane field, inducing VSD activation that facilitates channel gating. S4 is typically thought to be the principal functional component of the VSD because it carries, in most channels, a large portion of the VSD gating charge. The VSDs of large-conductance, voltage- and Ca²⁺-activated K⁺ channels are peculiar in that more gating charge is carried by transmembrane segments other than S4. Considering its “decentralized” distribution of voltage-sensing residues, we probed the BK_Ca VSD for evidence of cooperativity between charge-carrying segments S2 and S4. We achieved this by optically tracking their activation by using voltage clamp fluorometry, in channels with intact voltage sensors and charge-neutralized mutants. The results from these experiments indicate that S2 and S4 possess distinct voltage dependence, but functionally interact, such that the effective valence of one segment is affected by charge neutralization in the other. Statistical-mechanical modeling of the experimental findings using allosteric interactions demonstrates two mechanisms (mechanical coupling and dynamic focusing of the membrane electric field) that are compatible with the observed cross-segment effects of charge neutralization.

Large-conductance voltage- and Ca²⁺-activated K⁺ (BK_Ca) channel α subunits possess a unique transmembrane helix referred to as S0 at their N terminus, which is absent in other members of the voltage-gated channel superfamily. Recently, S0 was found to pack close to transmembrane segments S3 and S4, which are important components of the BK_Ca voltage-sensing apparatus. To assess the role of S0 in voltage sensitivity, we optically tracked protein conformational rearrangements from its extracellular flank by site-specific labeling with an environment-sensitive fluorophore, tetramethylrhodamine maleimide (TMRM). The structural transitions resolved from the S0 region exhibited voltage dependence similar to that of charge-bearing transmembrane domains S2 and S4. The molecular determinant of the fluorescence changes was identified in W203 at the extracellular tip of S4: at hyperpolarized potential, W203 quenches the fluorescence of TMRM labeling positions at the N-terminal flank of S0. We provide evidence that upon depolarization, W203 (in S4) moves away from the extracellular region of S0, lifting its quenching effect on TMRM fluorescence. We suggest that S0 acts as a pivot component against which the voltage-sensitive S4 moves upon depolarization to facilitate channel activation.

Large-conductance voltage- and Ca²⁺-activated K⁺ (BK_Ca) channels play a fundamental role in cellular function by integrating information from their voltage and Ca²⁺ sensors to control membrane potential and Ca²⁺ homeostasis. The molecular mechanism of Ca²⁺-dependent regulation of BK_Ca channels is unknown, but likely relies on the operation of two cytosolic domains, regulator of K⁺ conductance (RCK)1 and RCK2. Using solution-based investigations, we demonstrate that the purified BK_Ca RCK1 domain adopts an α/β fold, binds Ca²⁺, and assembles into an octameric superstructure similar to prokaryotic RCK domains. Results from steady-state and time-resolved spectroscopy reveal Ca²⁺-induced conformational changes in physiologically relevant [Ca²⁺]. The neutralization of residues known to be involved in high-affinity Ca²⁺ sensing (D362 and D367) prevented Ca²⁺-induced structural transitions in RCK1 but did not abolish Ca²⁺ binding. We provide evidence that the RCK1 domain is a high-affinity Ca²⁺ sensor that transduces Ca²⁺ binding into structural rearrangements, likely representing elementary steps in the Ca²⁺-dependent activation of human BK_Ca channels.

Histamine (HA) is the photoreceptor neurotransmitter in arthropods, directly gating chloride channels on large monopolar cells (LMCs), postsynaptic to photoreceptors in the lamina. Two histamine-gated channel genes that could contribute to this channel in Drosophila are hclA (also known as ort) and hclB (also known as hisCl1), both encoding novel members of the Cys-loop receptor superfamily. Drosophila S2 cells transfected with these genes expressed both homomeric and heteromeric histamine-gated chloride channels. The electrophysiological properties of these channels were compared with those from isolated Drosophila LMCs. HCLA homomers had nearly identical HA sensitivity to the native receptors (EC50 = 25 ÎŒm). Single-channel analysis revealed further close similarity in terms of single-channel kinetics and subconductance states (~25, 40, and 60 pS, the latter strongly voltage dependent). In contrast, HCLB homomers and heteromeric receptors were more sensitive to HA (EC50 = 14 and 1.2 ÎŒm, respectively), with much smaller single-channel conductances (~4 pS). Null mutations of hclA (ortUS6096) abolished the synaptic transients in the electroretinograms (ERGs). Surprisingly, the ERG “on” transients in hclB mutants transients were approximately twofold enhanced, whereas intracellular recordings from their LMCs revealed altered responses with slower kinetics. However, HCLB expression within the lamina, assessed by both a GFP (green fluorescent protein) reporter gene strategy and mRNA tagging, was exclusively localized to the glia cells, whereas HCLA expression was confirmed in the LMCs. Our results suggest that the native receptor at the LMC synapse is an HCLA homomer, whereas HCLB signaling via the lamina glia plays a previously unrecognized role in shaping the LMC postsynaptic response.

Neuronal depolarization causes larger intracellular pH (pH_i) shifts in axonal and dendritic regions than in the cell body. In this paper, we present evidence relating the time for collapse of these gradients to neuronal morphology. We have used ratiometric pH_i measurements using 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) in whole-cell patch-clamped snail neurons to study the collapse of longitudinal pH gradients. Using depolarization to open voltage-gated proton channels, we produced alkaline pH_i microdomains. In the absence of added mobile buffers, facilitated H⁺ diffusion down the length of the axon plays a critical role in determining pH_i microdomain lifetime, with axons of ∼100 μm allowing pH differences to be maintained for >60 s. An application of mobile, membrane-permeant pH buffers accelerated the collapse of the alkaline-pH gradients but, even at 30 mM, was unable to abolish them. Modeling of the pH_i dynamics showed that both the relatively weak effect of the weak acid/base on the peak size of the pH gradient and the accelerated collapse of the pH gradient could be due to the time taken for equilibration of the weak acid and base across the cell. We propose that appropriate weak acid/base mixes may provide a simple method for studying the role of local pH_i signals without perturbing steady-state pH_i. Furthermore, an extrapolation of our in vitro data to longer and thinner neuronal structures found in the mammalian nervous system suggests that dendritic and axonal pH_i are likely to be dominated by local pH_i-regulating mechanisms rather than simply following the soma pH_i.