Synaptic activity is followed within seconds by a local surge in lactate concentration, a phenomenon that underlies functional magnetic resonance imaging and whose causal mechanisms are unclear, partly because of the limited spatiotemporal resolution of standard measurement techniques. Using a novel Förster resonance energy transfer-based method that allows real-time measurement of the glycolytic rate in single cells, we have studied mouse astrocytes in search for the mechanisms responsible for the lactate surge. Consistent with previous measurements with isotopic 2-deoxyglucose, glutamate was observed to stimulate glycolysis in cultured astrocytes, but the response appeared only after a lag period of several minutes. Na ϩ overloads elicited by engagement of the Na ϩ -glutamate cotransporter with D-aspartate or application of the Na ϩ ionophore gramicidin also failed to stimulate glycolysis in the short term. In marked contrast, K ϩ stimulated astrocytic glycolysis by fourfold within seconds, an effect that was observed at low millimolar concentrations and was also present in organotypic hippocampal slices. After removal of the agonists, the stimulation by K ϩ ended immediately but the stimulation by glutamate persisted unabated for Ͼ20 min. Both stimulations required an active Na ϩ /K ϩ ATPase pump. By showing that small rises in extracellular K ϩ mediate short-term, reversible modulation of astrocytic glycolysis and that glutamate plays a long-term effect and leaves a metabolic trace, these results support the view that astrocytes contribute to the lactate surge that accompanies synaptic activity and underscore the role of these cells in neurometabolic and neurovascular coupling.

KCNMA1 encodes the pore-forming α subunit of the “Big K+” (BK) large conductance calcium and voltage-activated K+ channel. BK channels are widely distributed across tissues, including both excitable and nonexcitable cells. Expression levels are highest in brain and muscle, where BK channels are critical regulators of neuronal excitability and muscle contractility. A global deletion in mouse (KCNMA1−/−) is viable but exhibits pathophysiology in many organ systems. Yet despite the important roles in animal models, the consequences of dysfunctional BK channels in humans are not well characterized. Here, we summarize 16 rare KCNMA1 mutations identified in 37 patients dating back to 2005, with an array of clinically defined pathological phenotypes collectively referred to as “KCNMA1-linked channelopathy.” These mutations encompass gain-of-function (GOF) and loss-of-function (LOF) alterations in BK channel activity, as well as several variants of unknown significance (VUS). Human KCNMA1 mutations are primarily associated with neurological conditions, including seizures, movement disorders, developmental delay, and intellectual disability. Due to the recent identification of additional patients, the spectrum of symptoms associated with KCNMA1 mutations has expanded but remains primarily defined by brain and muscle dysfunction. Emerging evidence suggests the functional BK channel alterations produced by different KCNMA1 alleles may associate with semi-distinct patient symptoms, such as paroxysmal nonkinesigenic dyskinesia (PNKD) with GOF and ataxia with LOF. However, due to the de novo origins for the majority of KCNMA1 mutations identified to date and the phenotypic variability exhibited by patients, additional evidence is required to establish causality in most cases. The symptomatic picture developing from patients with KCNMA1-linked channelopathy highlights the importance of better understanding the roles BK channels play in regulating cell excitability. Establishing causality between KCNMA1-linked BK channel dysfunction and specific patient symptoms may reveal new treatment approaches with the potential to increase therapeutic efficacy over current standard regimens.

Summary Several transient receptor potential (TRP) ion channels are activated with high sensitivity by either cold or hot temperatures. However, structures and mechanism that determine temperature-directionality (cold vs. heat) are not established. Here we screened 12,000 random mutant clones of the cold-activated mouse TRPA1 ion channel with a heat stimulus. We identified three single-point mutations that are individually sufficient to make mouse TRPA1 warm-activated, while leaving sensitivity to chemicals unaffected. Mutant channels have high temperature-sensitivity of voltage-activation, specifically of channel opening, but not channel closing, which is reminiscent of other heat-activated TRP channels. All mutations are located in ankyrin repeat six, which identifies this domain as a sensitive modulator of thermal activation. We propose that a change in the coupling of temperature-sensing to channel-gating generates this sensitivity to warm temperatures. Our results demonstrate that minimal changes in protein sequence are sufficient to generate a wide diversity of thermal sensitivities in TRPA1.

In recent years, the use of fluorescent glucose analogs has allowed the study of rapid transport modulation in heterogeneous cell cultures and complex tissues. However, the kinetic behavior of these tracers is not conventional. For instance, the fluorescent glucose analog 6-NBDG permeates the cell 50-100 times slower than glucose but the uptake of 6-NBDG is almost insensitive to glucose, an observation that casts doubts as to the specificity of the uptake pathway. To investigate this apparent anomaly in cultured astrocytes, which are rich in the glucose transporter GLUT1, we first estimated the kinetic parameters of 6-NBDG uptake, which were then incorporated into the kinetic model of GLUT1. The main outcome of the analysis was that 6-NBDG binds to GLUT1 with 300 times higher affinity than glucose, which explains why its uptake is not efficiently displaced by glucose. The high binding affinity of 6-NBDG also explains why cytochalasin B is less effective at inhibiting 6-NBDG uptake than at inhibiting glucose uptake. We conclude that 6-NBDG, used at low concentrations, permeates into astrocytes chiefly through GLUT1, and advise that the exofacial GLUT1 inhibitor 4,6-ethylidine-D-glucose be used, instead of glucose, as the tool of choice to confirm the specificity of 6-NBDG uptake.

KCNMA1-linked channelopathy is an emerging neurological disorder characterized by heterogeneous and overlapping combinations of movement disorder, seizure, developmental delay, and intellectual disability. KCNMA1 encodes the BK K + channel, which contributes to both excitatory and inhibitory neuronal and muscle activity. Understanding the basis of the disorder is an important area of active investigation; however, the rare prevalence has hampered the development of large patient cohorts necessary to establish genotype-phenotype correlations. In this review, we summarize 37 KCNMA1 alleles from 69 patients currently defining the channelopathy and assess key diagnostic and clinical hallmarks. At present, 3 variants are classified as gain-offunction with respect to BK channel activity, 14 loss-of-function, 15 variants of uncertain significance, and putative benign/VUS. Symptoms associated with these variants were curated from patient-provided information and prior publications to define the spectrum of clinical phenotypes. In this newly expanded cohort, seizures showed no differential distribution between patients harboring GOF and LOF variants, while movement disorders segregated by mutation type. Paroxysmal non-kinesigenic dyskinesia was predominantly observed among patients with GOF alleles of the BK channel, although not exclusively so, while additional movement disorders were observed in patients with LOF variants. Neurodevelopmental and structural brain abnormalities were prevalent in patients with LOF mutations. In contrast to mutations, disease-associated KCNMA1 single nucleotide polymorphisms were not predominantly related to neurological phenotypes but covered a wider set of peripheral physiological functions. Together, this review provides additional evidence exploring the genetic and biochemical basis for KCNMA1-linked channelopathy and summarizes the clinical repository of patient symptoms across multiple types of KCNMA1 gene variants.

Ion channels are membrane proteins that mediate efficient ion transport across the hydrophobic core of cell membranes, an unlikely process in their absence. K+ channels discriminate K+ over cations with similar radii with extraordinary selectivity and display a wide diversity of ion transport rates, covering differences of two orders of magnitude in unitary conductance. The pore domains of large- and small-conductance K+ channels share a general architectural design comprising a conserved narrow selectivity filter, which forms intimate interactions with permeant ions, flanked by two wider vestibules toward the internal and external openings. In large-conductance K+ channels, the inner vestibule is wide, whereas in small-conductance channels it is narrow. Here we raise the idea that the physical dimensions of the hydrophobic internal vestibule limit ion transport in K+ channels, accounting for their diversity in unitary conductance.

KCNMA1, encoding the voltage- and calcium-activated potassium channel, has a pivotal role in brain physiology. Mutations in KCNMA1 are associated with epilepsy and/or dyskinesia (PNKD3). Two KCNMA1 mutations correlated with these phenotypes, D434G and N999S, were previously identified as producing gain-of-function (GOF) effects on BK channel activity. Three new patients have been reported harboring N999S, one carrying a second mutation, R1128W, but the effects of these mutations have not yet been reported under physiological K+ conditions or compared to D434G. In this study, we characterize N999S, the novel N999S/R1128W double mutation, and D434G in a brain BK channel splice variant, comparing the effects on BK current properties under a physiological K+ gradient with action potential voltage commands. N999S, N999S/R1128W, and D434G cDNAs were expressed in HEK293T cells and characterized by patch-clamp electrophysiology. N999S BK currents were shifted to negative potentials, with faster activation and slower deactivation compared with wild type (WT) and D434G. The double mutation N999S/R1128W did not show any additional changes in current properties compared with N999S alone. The antiepileptic drug acetazolamide was assessed for its ability to directly modulate WT and N999S channels. Neither the WT nor N999S channels were sensitive to the antiepileptic drug acetazolamide, but both were sensitive to the inhibitor paxilline. We conclude that N999S is a strong GOF mutation that surpasses the D434G phenotype, without mitigation by R1128W. Acetazolamide has no direct modulatory action on either WT or N999S channels, indicating that its use may not be contraindicated in patients harboring GOF KCNMA1 mutations. NEW & NOTEWORTHY KCNMA1-linked channelopathy is a new neurological disorder characterized by mutations in the BK voltage- and calcium-activated potassium channel. The epilepsy- and dyskinesia-associated gain-of-function mutations N999S and D434G comprise the largest number of patients in the cohort. This study provides the first direct comparison between D434G and N999S BK channel properties as well as a novel double mutation, N999S/R1128W, from another patient, defining the functional effects during an action potential stimulus.

KCNMA1 encodes the pore-forming α subunit of the ‘Big K+’ (BK) large conductance calcium and voltage-activated K+ channel. BK channels are widely distributed across tissues, including both excitable and non-excitable cells. Expression levels are highest in brain and muscle, where BK channels are critical regulators of neuronal excitability and muscle contractility. A global deletion in mouse (KCNMA1–/–) is viable but exhibits pathophysiology in many organ systems. Yet despite the important roles in animal models, the consequences of dysfunctional BK channels in humans are not well-characterized. Here, we summarize 16 rare KCNMA1 mutations identified in 37 patients dating back to 2005, with an array of clinically defined pathological phenotypes collectively referred to as ‘KCNMA1-linked channelopathy.’ These mutations encompass gain-of-function (GOF) and loss-of-function (LOF) alterations in BK channel activity, as well as several variants of unknown significance (VUS). Human KCNMA1 mutations are primarily associated with neurological conditions, including seizures, movement disorders, developmental delay, and intellectual disability. Due to the recent identification of additional patients, the spectrum of symptoms associated with KCNMA1 mutations has expanded but remains primarily defined by brain and muscle dysfunction. Emerging evidence suggests the functional BK channel alterations produced by different KCNMA1 alleles may associate with semi-distinct patient symptoms, such as paroxysmal non-kinesigenic dyskinesia (PNKD) with GOF and ataxia with LOF. However, due to the de novo origins for the majority of KCNMA1 mutations identified to date, and the phenotypic variability exhibited by patients, additional evidence is required to establish causality in most cases. The symptomatic picture developing from patients with KCNMA1­­-linked channelopathy highlights the importance of better understanding the roles BK channels play in regulating cell excitability. Establishing causality between KCNMA1-linked BK channel dysfunction and specific patient symptoms may reveal new treatment approaches with the potential to increase therapeutic efficacy over current standard regimens.