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Expression of Kv1.2 within Kv1.x potassium channel complexes is critical in maintaining appropriate neuronal excitability and determining the threshold for action potential firing. This is attributed to the interaction of Kv1.2 with a hitherto unidentified protein that confers bimodal channel activation gating, allowing neurons to adapt to repetitive trains of stimulation and protecting against hyperexcitability. One potential protein candidate is the sigma‐1 receptor (Sig‐1R), which regulates other members of the Kv1.x channel family; however, the biophysical nature of the interaction between Sig‐1R and Kv1.2 has not been elucidated. We hypothesized that Sig‐1R may regulate Kv1.2 and may further act as the unidentified modulator of Kv1.2 activation. In transiently transfected HEK293 cells, we found that ligand activation of the Sig‐1R modulates Kv1.2 current amplitude. More importantly, Sig‐1R interacts with Kv1.2 in baseline conditions to influence bimodal activation gating. These effects are abolished in the presence of the auxiliary subunit Kvβ2 and when the Sig‐1R mutation underlying ALS16 (Sig‐1R‐E102Q), is expressed. These data suggest that Kvβ2 occludes the interaction of Sig‐1R with Kv1.2, and that E102 may be a residue critical for Sig‐1R modulation of Kv1.2. The results of this investigation describe an important new role for Sig‐1R in the regulation of neuronal excitability and introduce a novel mechanism of pathophysiology in Sig‐1R dysfunction. Bimodal activation gating is a unique feature of Kv1.2 containing delayed‐rectifier potassium channels that regulates neuronal excitability. Abraham et al. show, for the first time, that the Sigma‐1 receptor is able to modulate both bimodal activation gating and current amplitude in response to a depolarization. The mutant Sigma‐1 receptor responsible for juvenile ALS uncouples this relationship, suggesting that aberrant modulation of Kv1.2 may underlie neuronal hyperexcitability in ALS.

Inhibitory interneurons are among the most diverse cell types in the brain; the hippocampus itself contains more than 28 different inhibitory interneurons. Interneurons are typically classified using a combination of physiological, morphological, and biochemical observations. One broad separator is action potential firing: low threshold, regular spiking versus higher threshold, fast spiking. We found that spike frequency adaptation (SFA) was highly heterogeneous in low threshold interneurons in the mouse stratum oriens region of area CA1. Analysis with a k‐means clustering algorithm parsed the data set into two distinct clusters based on a constellation of physiological parameters and reliably sorted strong and weak SFA cells into different groups. Interneurons with strong SFA fired fewer action potentials across a range of current inputs and had lower input resistance compared to cells with weak SFA. Strong SFA cells also had higher sag and rebound in response to hyperpolarizing current injections. Morphological analysis shows no difference between the two cell types and the cell types did not segregate along the dorsal–ventral axis of the hippocampus. Strong and weak SFA cells were labeled in hippocampal slices from SST:cre Ai14 mice suggesting both cells express somatostatin. Voltage‐clamp recordings showed hyperpolarization activated current Ih was significantly larger in strong SFA cells compared to weak SFA cells. We suggest that the strong SFA cell represents a previously uncharacterized type of CA1 stratum oriens interneuron. Due to the combination of physiological parameters of these cells, we will refer to them as Low Threshold High Ih (LTH) cells. Spike frequency adaptation (SFA) was highly variable among CA1 stratum oriens interneurons in the mouse. Adapting stratum oriens interneurons were separated into two cell groups using multiple subthreshold and action potential parameters by a k‐means clustering analysis which revealed that cells with strong SFA fired fewer action potentials for a given current injection, had lower input resistance and more sag and rebound compared to weak SFA cells.The physiological differences were not correlated with neuron morphology, location in stratum oriens, or anatomical location along the dorsal‐ventral axis of the hippocampus. Voltage‐clamp recordings revealed that strong SFA cells had higher density of the hyperpolarization activated current Ih compared to weak SFA cells. ​

Excised outside‐out patches from HEK293 cells stably transfected with the human (h) 5‐HT3A receptor cDNA were used to determine the effects of cannabinoid receptor ligands on the 5‐HT‐induced current using the patch clamp technique. In addition, binding studies with radioligands for 5‐HT3 as well as for cannabinoid CB1 and CB2 receptors were carried out. The 5‐HT‐induced current was inhibited by the following cannabinoid receptor agonists (at decreasing order of potency): Δ9‐THC, WIN55,212‐2, anandamide, JWH‐015 and CP55940. The WIN55,212‐2‐induced inhibition was not altered by SR141716A, a CB1 receptor antagonist. WIN55,212‐3, an enantiomer of WIN55,212‐2, did not affect the 5‐HT‐induced current. WIN55,212‐2 did not change the EC50 value of 5‐HT in stimulating current, but reduced the maximum effect. The CB1 receptor ligand [3H]‐SR141716A and the CB1/CB2 receptor ligand [3H]‐CP55940 did not specifically bind to parental HEK293 cells. In competition experiments on membranes of HEK293 cells transfected with the h5‐HT3A receptor cDNA, WIN55,212‐2, CP55940, anandamide and SR141716A did not affect [3H]‐GR65630 binding, but 5‐HT caused a concentration dependent‐inhibition. In conclusion, cannabinoids stereoselectively inhibit currents through recombinant h5‐HT3A receptors independently of cannabinoid receptors. Probably the cannabinoids act allosterically at a modulatory site of the h5‐HT3A receptor. Thus the functional state of the receptor can be controlled by the endogenous ligand anandamide. This site is a potential target for new analgesic and antiemetic drugs. British Journal of Pharmacology (2002) 137, 589–596. doi:10.1038/sj.bjp.0704829

Ranolazine is a novel anti‐ischemic drug that prolongs the QT interval. To evaluate the potential mechanisms and consequences, we studied: (i) Ranolazine's effects on HERG and IsK currents in Xenopus oocytes with two‐electrode voltage clamp; (ii) effects of ranolazine, compared to D‐sotalol, on effective refractory period (ERP), QT interval and ventricular rhythm in a dog model of acquired long QT syndrome; and (iii) effects on selected native currents in canine atrial myocytes with whole‐cell patch‐clamp technique. Ranolazine inhibited HERG and IsK currents with different potencies. HERG was inhibited with an IC50 of 106 μmol l−1, whereas the IC50 for IsK was 1.7 mmol l−1. D‐Sotalol caused reverse use‐dependent ERP and QT interval prolongation, whereas ranolazine produced modest, nonsignificant increases that plateaued at submaximal doses. Neither drug affected QRS duration. D‐Sotalol had clear proarrhythmic effects, with all D‐sotalol‐treated dogs developing torsades de pointes (TdP) ventricular tachyarrhythmias, of which they ultimately died. In contrast, ranolazine did not generate TdP. Effects on IKr and IKs were similar to those on HERG and IsK. Ranolazine blocked ICa with an IC50 of ∼300 μmol l−1. INa was unaffected. We conclude that ranolazine inhibits IKr by blocking HERG currents, inhibits ICa at slightly larger concentrations, and has modest and self‐limited effects on the QT interval. Unlike D‐sotalol, ranolazine does not cause TdP in a dog model. The greater safety of ranolazine may be due to its ability to inhibit ICa at concentrations only slightly larger than those that inhibit IKr, thus producing offsetting effects on repolarization. British Journal of Pharmacology (2004) 142, 1300–1308. doi:10.1038/sj.bjp.0705879

General anesthesia induced by etomidate, barbiturates and propofol is associated with positive modulation of synaptic [alpha][beta][gamma] GABA.sub.A receptors, inhibitory hetero-pentameric ligand-gated ion channels formed from homologous subunits arranged [beta]-[alpha]-[beta]-[alpha]-[gamma] around a central gated chloride channel. Approaches based on mutations, amino-acid level analysis of photolabel incorporation, and cryo-electron micrography (cryo-EM) all indicate that etomidate binds selectively in two outer transmembrane [beta]+/[alpha]- inter-subunit sites per receptor. These approaches also reveal that the potent barbiturate photolabel R-mTFD-MPAB binds selectively in homologous sites formed at [alpha]+/[beta]- and [gamma]+/[beta]- interfaces. The anesthetic photolabel, pTFD-di-iPr-BnOH, was proposed to bind selectively in [alpha]+/[beta]- and [alpha]+/[gamma]- homologs of the etomidate sites, based largely on functional analysis of only 5 point mutations in [alpha]1[beta]3[gamma]2L receptors. To further test the interactions of receptor-bound pTFD-di-iPr-BnOH with outer transmembrane inter-subunit sites, we used voltage-clamp electrophysiology in substituted cysteine modification and protection (SCAMP) experiments at 8 residues located in the five homologous sites, focusing on [alpha]+ and [gamma]- loci. Control SCAMP studies were performed using etomidate and R-mTFD-MPAB. Incorporation of single cysteine mutations ([alpha]1M236C, [alpha]1S280C, [alpha]1A291C, [beta]3L231C, [beta]3M286C, [gamma]2I242C, [gamma]2L246C, and [gamma]2S301C) produced functional GABA-responsive receptors that retained sensitivity to pTFD-di-iPr-BnOH modulation and displayed increased GABA sensitivity following exposure to the covalent sulfhydryl modifier p-chloromercuribenzenesulfonate (pCMBS). In the presence of pTFD-di-iPr-BnOH, pCMBS modification effects were reduced (evidence of steric protection) in receptors with cysteine mutations in [alpha]+, [beta]-, and [gamma]-, but not in [alpha]-, [beta]+, or [gamma]+ interfacial loci. Protection patterns with etomidate and R-mTFD-MPAB mirrored prior results. SCAMP results further support the hypothesis that pTFD-di-iPr-BnOH binds selectively in [alpha]+/[beta]- and [alpha]+/[gamma]- interfacial sites that are homologs of the [beta]+/[alpha]- etomidate sites.

The patch clamp technique has become the gold standard for neuron electrophysiology research in brain science. Brain slices have been widely utilized as the targets of the patch clamp technique due to their higher optical transparency compared to a live brain and their intercellular connectivity in comparison to cultured single neurons. However, the narrow working space, small scope, and depth of the field of view make the positioning of the operation’s micropipette to the target neuron a time-consuming task reliant on a high level of experience, significantly slowing down operation of the patch clamp technique in brain slices. Further, the current poor controllability in gigaseal formation, which is the key to electrophysiology signal recording, significantly lowers the patch clamp success rate. In this paper, a stepwise navigation of the micropipette is conducted to accelerate the positioning process of the micropipette tip to the target neuron in the brain slice. Then, a fuzzy proportional–integral–derivative controller is designed to control the gigaseal formation process along a designed resistance curve. The experimental results demonstrate an almost doubled patch clamp technique speed, with a 25% improvement in the success rate compared to the conventional manual method. The above advantages may promote the application of our method in brain science research based on brain slice platforms.

Two-pore channels (TPCs) are activated by phosphatidylinositol bisphosphate (PIP₂) binding to domain I and/or by voltage sensing in domain II (DII). Little is known about how these two stimuli are integrated, and how each TPC subtype achieves its unique preference. Here, we show that distinct conformations of DII-S4 in the voltage- sensor domain determine the two gating modes. DII-S4 adopts an intermediate conformation, and forced stabilization in this conformation was found to result in a high PIP₂-dependence in primarily voltage-dependent TPC3. In TPC2, which is PIP₂-gated and nonvoltage-dependent, a stabilized intermediate conformation does not affect the PIP₂-gated currents. These results indicate that the intermediate state represents the PIP₂-gating mode, which is distinct from the voltage-gating mode in TPCs. We also found in TPC2 that the tricyclic antidepressant desipramine induces DII-S4-based voltage dependence and that naringenin, a flavonoid, biases the mode preference from PIP₂-gating to desipramine-induced voltage gating. Taken together, our study on TPCs revealed an unprecedented mode-switching mechanism involving conformational changes in DII-S4, and its active role in integrating voltage and PIP₂ stimuli.

De novo variants in SCN2A developmental and epileptic encephalopathy (DEE) show distinctive genotype–phenotype correlations. The two most recurrent SCN2A variants in DEE, R1882Q and R853Q, are associated with different ages and seizure types at onset. R1882Q presents on day 1 of life with focal seizures, while infantile spasms is the dominant seizure type seen in R853Q cases, presenting at a median age of 8 months. Voltage clamp, which characterizes the functional properties of ion channels, predicted gain-of-function for R1882Q and loss-of-function for R853Q. Dynamic action potential clamp, that we implement here as a method for modeling neurophysiological consequences of a given epilepsy variant, predicted that the R1882Q variant would cause a dramatic increase in firing, whereas the R853Q variant would cause a marked reduction in action potential firing. Dynamic clamp was also able to functionally separate the L1563V variant, seen in benign familial neonatal–infantile seizures from R1882Q, seen in DEE, suggesting a diagnostic potential for this type of analysis. Overall, the study shows a strong correlation between clinical phenotype, SCN2A genotype, and functional modeling. Dynamic clamp is well positioned to impact our understanding of pathomechanisms and for development of disease mechanism-targeted therapies in genetic epilepsy.

Two‐electrode voltage‐clamp electrophysiology has been used to study the actions of two amyloid peptides (Aβ1–42, Aβ1–40) on α7, α4β2 and α3β4 recombinant human neuronal nicotinic acetylcholine receptors (nicotinic AChRs), heterologously expressed in Xenopus laevis oocytes. The application of Aβ1–42 or Aβ1–40 (1 pM–100 nM) for 5 s does not directly activate expressed human α7, α4β2 or α3β4 nicotinic AChRs. Aβ1–42 and Aβ1–40 are antagonists of α7 nicotinic AChRs. For example, 10 nM Aβ1–42 and Aβ1–40 both reduced the peak amplitude of currents recorded (3 mM ACh) to 48±5 and 45±10% (respectively) of control currents recorded in the absence of peptide. In both the cases the effect is sustained throughout a 30 min peptide application and is poorly reversible. Aβ1–42 and Aβ1–40 (10 nM) enhance currents recorded in response to ACh (3 mM) from oocytes expressing α4β2 nicotinic AChRs by 195±40 and 195±41% respectively. This effect is transient, reaching a peak after 3 min and returning to control values after a 24 min application of 10 nM Aβ1–42. We observe an enhancement of 157±22% of control ACh‐evoked current amplitude in response to 100 nM Aβ1–42 recorded from oocytes expressing α4β2 nicotinic AChRs. Aβ1–42 and Aβ1–40 (10 nM) were without antagonist actions on the responses of α3β4 nicotinic AChRs to ACh (1 nM–3 mM). British Journal of Pharmacology (2005) 146, 964–971. doi:10.1038/sj.bjp.0706403

Acid-sensing ion channels (ASICs) are proton-activated Na⁺ channels expressed in the nervous system, where they are involved in learning, fear behaviors, neurodegeneration, and pain sensation. In this work, we study the role in pH sensing of two regions of the ectodomain enriched in acidic residues: the acidic pocket, which faces the outside of the protein and is the binding site of several animal toxins, and the palm, a central channel domain. Using voltage clamp fluorometry, we find that the acidic pocket undergoes conformational changes during both activation and desensitization. Concurrently, we find that, although proton sensing in the acidic pocket is not required for channel function, it does contribute to both activation and desensitization. Furthermore, protonation-mimicking mutations of acidic residues in the palm induce a dramatic acceleration of desensitization followed by the appearance of a sustained current. In summary, this work describes the roles of potential pH sensors in two extracellular domains, and it proposes a model of acidification-induced conformational changes occurring in the acidic pocket of ASIC1a.