Apocalmodulin itself promotes ion channel opening and Ca(2+) regulation.

The Ca(2+)-free form of calmodulin (apoCaM) often appears inert, modulating target molecules only upon conversion to its Ca(2+)-bound form. This schema has appeared to govern voltage-gated Ca(2+) channels, where apoCaM has been considered a dormant Ca(2+) sensor, associated with channels but awaiting the binding of Ca(2+) ions before inhibiting channel opening to provide vital feedback inhibition. Using single-molecule measurements of channels and chemical dimerization to elevate apoCaM, we find that apoCaM binding on its own markedly upregulates opening, rivaling the strongest forms of modulation. Upon Ca(2+) binding to this CaM, inhibition may simply reverse the initial upregulation. As RNA-edited and -spliced channel variants show different affinities for apoCaM, the apoCaM-dependent control mechanisms may underlie the functional diversity of these variants and explain an elongation of neuronal action potentials by apoCaM. More broadly, voltage-gated Na channels adopt this same modulatory principle. ApoCaM thus imparts potent and pervasive ion-channel regulation. PAPERCLIP:

Large Ca²âº-dependent facilitation of Ca(V)2.1 channels revealed by Ca²âº photo-uncaging.

KEY POINTS: CaV 2.1 channels constitute a dominant Ca(2+) entry pathway into brain neurons, triggering downstream Ca(2+) -dependent processes such as neurotransmitter release. CaV 2.1 is itself modulated by Ca(2+) , resulting in activity-dependent enhancement of channel opening termed Ca(2+) -dependent facilitation (CDF). Real-time Ca(2+) imaging and Ca(2+) uncaging here reveal that CDF turns out to be strikingly faster, more Ca(2+) sensitive, and larger than anticipated on previous grounds. Robust resolution of the quantitative profile of CDF enables deduction of a realistic biophysical model for this process. These results suggest that CaV 2.1 CDF would figure most prominently in short-term synaptic plasticity and cerebellar Purkinje cell rhythmicity. ABSTRACT: CaV 2.1 (P-type) voltage-gated Ca(2+) channels constitute a major source of neuronal Ca(2+) current, strongly influencing rhythmicity and triggering neurotransmitter release throughout the central nervous system. Fitting with such stature among Ca(2+) entry pathways, CaV 2.1 is itself feedback regulated by intracellular Ca(2+) , acting through calmodulin to facilitate channel opening. The precise neurophysiological role of this calcium-dependent facilitation (CDF) remains uncertain, however, in large measure because the very magnitude, Ca(2+) dependence and kinetics of CDF have resisted quantification by conventional means. Here, we utilize the photo-uncaging of Ca(2+) with CaV 2.1 channels fluxing Li(+) currents, so that voltage-dependent activation of channel gating is no longer conflated with Ca(2+) entry, and CDF is then driven solely by light-induced increases in Ca(2+) . By using this strategy, we now find that CDF can be unexpectedly large, enhancing currents by as much as twofold at physiological voltages. CDF is steeply Ca(2+) dependent, with a Hill coefficient of approximately two, a half-maximal effect reached by nearly 500 nm Ca(2+) , and Ca(2+) on/off kinetics in the order of milliseconds to tens of milliseconds. These properties were established for both native P-type currents in cerebellar Purkinje neurons, as well as their recombinant channel counterparts under heterologous expression. Such features suggest that CDF of CaV 2.1 channels may substantially enhance the regularity of rhythmic firing in cerebellar Purkinje neurons, where regularity is believed crucial for motor coordination. In addition, this degree of extensive CDF would be poised to exert large order-of-magnitude effects on short-term synaptic plasticity via rapid modulation of presynaptic Ca(2+) entry.

Dysbiosis not observed in Canadian horse with free fecal liquid (FFL) using 16S rRNA sequencing.

Free Fecal Liquid (FFL), also termed Fecal Water Syndrome (FWS), is an ailment in horses characterized by variable solid and liquid (water) phases at defecation. The liquid phase can be excreted before, during, or after the solid defecation phase. While the underlying causes of FFL are unknown, hindgut dysbiosis is suggested to be associated with FFL. Three European studies investigated dysbiosis in horses with FFL using 16S rRNA sequencing and reported results that conflicted between each other. In the present study, we also used 16S rRNA sequencing to study the fecal microbial composition in 14 Canadian horses with FFL, and 11 healthy stable mate controls. We found no significant difference in fecal microbial composition between FFL and healthy horses, which further supports that dysbiosis is not associated with FFL.

Contribution of calcium-dependent facilitation to synaptic plasticity revealed by migraine mutations in the P/Q-type calcium channel.

The dynamics, computational power, and strength of neural circuits are essential for encoding and processing information in the CNS and rely on short and long forms of synaptic plasticity. In a model system, residual calcium (Ca(2+)) in presynaptic terminals can act through neuronal Ca(2+) sensor proteins to cause Ca(2+)-dependent facilitation (CDF) of P/Q-type channels and induce short-term synaptic facilitation. However, whether this is a general mechanism of plasticity at intact central synapses and whether mutations associated with human disease affect this process have not been described to our knowledge. In this report, we find that, in both exogenous and native preparations, gain-of-function missense mutations underlying Familial Hemiplegic Migraine type 1 (FHM-1) occlude CDF of P/Q-type Ca(2+) channels. In FHM-1 mutant mice, the alteration of P/Q-type channel CDF correlates with reduced short-term synaptic facilitation at cerebellar parallel fiber-to-Purkinje cell synapses. Two-photon imaging suggests that P/Q-type channels at parallel fiber terminals in FHM-1 mice are in a basally facilitated state. Overall, the results provide evidence that FHM-1 mutations directly affect both P/Q-type channel CDF and synaptic plasticity and that together likely contribute toward the pathophysiology underlying FHM-1. The findings also suggest that P/Q-type channel CDF is an important mechanism required for normal synaptic plasticity at a fast synapse in the mammalian CNS.

Faster time to automated elevation of the head and thorax during cardiopulmonary resuscitation increases the probability of return of spontaneous circulation.

OBJECTIVES: Resuscitation in the Head Up position improves outcomes in animals treated with active compression decompression cardiopulmonary resuscitation and an impedance threshold device (ACD + ITD CPR).We assessed impact of time to deployment of an automated Head Up position (AHUP) based bundle of care after out-of-hospital cardiac arrest on return of spontaneous circulation (ROSC). METHODS: Observational data were analyzed from a patient registry. Patients received treatment with 1) ACD + and/or automated CPR 2) an ITD and 3) an AHUP device. Probability of ROSC (ROSCprob) from the 9-1-1 call to AHUP device placement was assessed with a restricted cubic spline model and linear regression. RESULTS: Of 11 sites, 6 recorded the interval from 9-1-1 to AHUP device (n = 227). ROSCprobfor all rhythms was 34%(77/227). Median age (range) was 66 years (19-101) and 68% men. TheROSCprobfor shockable rhythms was 47%(18/38). Minutes from 9-1-1 to AHUP device (median, range) varied between sites: 1) 6.4(4,15), 2) 8.0(5,19), 3) 9.9(4, 12), 4) 14.1(6, 36), 5) 15.9(6, 34), 6) 19.0(8, 38),(p = 0.0001).ROSCprobalso varied; 1) 55.1%(16/29), 2) 60%(3/5), 3) 50%(3/6), 4) 22.7%(17/75), 5) 26.4%(9/34), and 6) 37.1%(29/78), (p = 0.019). For all rhythms between 4 and 12 min (n = 85),ROSCprobdeclined 5.6% for every minute elapsed (p = 0.024). For shockable rhythms, between 6 and 15 min (n = 23),ROSCprobdeclined 9.0% for every minute elapsed (p = 0.006). CONCLUSIONS: Faster time to deployment of an AHUP based bundle of care is associated with higher incidence of ROSC. This must be considered when evaluating and implementing this bundle.

Head and thorax elevation during cardiopulmonary resuscitation using circulatory adjuncts is associated with improved survival.

BACKGROUND: Survival after out-of-hospital cardiac arrest (OHCA) remains poor. A physiologically distinct cardiopulmonary resuscitation (CPR) strategy consisting of (1) active compression-decompression CPR and/or automated CPR, (2) an impedance threshold device, and (3) automated controlled elevation of the head and thorax (ACE) has been shown to improve neurological survival significantly versus conventional (C) CPR in animal models. This resuscitation device combination, termed ACE-CPR, is now used clinically. OBJECTIVES: To assess the probability of OHCA survival to hospital discharge after ACE-CPR versus C-CPR. METHODS: As part of a prospective registry study, 227 ACE-CPR OHCA patients were enrolled 04/2019-07/2020 from 6 pre-hospital systems in the United States. Individual C-CPR patient data (n = 5196) were obtained from three large published OHCA randomized controlled trials from high-performing pre-hospital systems. The primary study outcome was survival to hospital discharge. Secondary endpoints included return of spontaneous circulation (ROSC) and favorable neurological survival. Propensity-score matching with a 1:4 ratio was performed to account for imbalances in baseline characteristics. RESULTS: Irrespective of initial rhythm, ACE-CPR (n = 222) was associated with higher adjusted odds ratios (OR) of survival to hospital discharge relative to C-CPR (n = 860), when initiated in <11 min (3.28, 95 % confidence interval [CI], 1.55-6.92) and < 18 min (1.88, 95 % CI, 1.03-3.44) after the emergency call, respectively. Rapid use of ACE-CPR was also associated with higher probabilities of ROSC and favorable neurological survival. CONCLUSIONS: Compared with C-CPR controls, rapid initiation of ACE-CPR was associated with a higher likelihood of survival to hospital discharge after OHCA.

A rendezvous with the queen of ion channels: Three decades of ion channel research by David T Yue and his Calcium Signals Laboratory.

David T. Yue was a renowned biophysicist who dedicated his life to the study of Ca(2+) signaling in cells. In the wake of his passing, we are left not only with a feeling of great loss, but with a tremendous and impactful body of work contributed by a remarkable man. David's research spanned the spectrum from atomic structure to organ systems, with a quantitative rigor aimed at understanding the fundamental mechanisms underlying biological function. Along the way he developed new tools and approaches, enabling not only his own research but that of his contemporaries and those who will come after him. While we cannot hope to replicate the eloquence and style we are accustomed to in David's writing, we nonetheless undertake a review of David's chosen field of study with a focus on many of his contributions to the calcium channel field.

Towards a Unified Theory of Calmodulin Regulation (Calmodulation) of Voltage-Gated Calcium and Sodium Channels.

Voltage-gated Na and Ca(2+) channels represent two major ion channel families that enable myriad biological functions including the generation of action potentials and the coupling of electrical and chemical signaling in cells. Calmodulin regulation (calmodulation) of these ion channels comprises a vital feedback mechanism with distinct physiological implications. Though long-sought, a shared understanding of the channel families remained elusive for two decades as the functional manifestations and the structural underpinnings of this modulation often appeared to diverge. Here, we review recent advancements in the understanding of calmodulation of Ca(2+) and Na channels that suggest a remarkable similarity in their regulatory scheme. This interrelation between the two channel families now paves the way towards a unified mechanistic framework to understand vital calmodulin-dependent feedback and offers shared principles to approach related channelopathic diseases. An exciting era of synergistic study now looms.

Continuously tunable Ca(2+) regulation of RNA-edited CaV1.3 channels.

CaV1.3 ion channels are dominant Ca(2+) portals into pacemaking neurons, residing at the epicenter of brain rhythmicity and neurodegeneration. Negative Ca(2+) feedback regulation of CaV1.3 channels (CDI) is therefore critical for Ca(2+) homeostasis. Intriguingly, nearly half the CaV1.3 transcripts in the brain are RNA edited to reduce CDI and influence oscillatory activity. It is then mechanistically remarkable that this editing occurs precisely within an IQ domain, whose interaction with Ca(2+)-bound calmodulin (Ca(2+)/CaM) is believed to induce CDI. Here, we sought the mechanism underlying the altered CDI of edited channels. Unexpectedly, editing failed to attenuate Ca(2+)/CaM binding. Instead, editing weakened the prebinding of Ca(2+)-free CaM (apoCaM) to channels, which proves essential for CDI. Thus, editing might render CDI continuously tunable by fluctuations in ambient CaM, a prominent effect we substantiate in substantia nigral neurons. This adjustability of Ca(2+) regulation by CaM now looms as a key element of CNS Ca(2+) homeostasis.

Ca(V)2.1 P/Q-type calcium channel alternative splicing affects the functional impact of familial hemiplegic migraine mutations: implications for calcium channelopathies.

Alternative splicing is known to generate multiple functionally distinct calcium channel variants that exhibit unique spatial and temporal expression patterns. In humans, naturally occurring mutations in genes encoding calcium channel pore forming alpha(1)-subunits are associated with several severe hereditary disorders although it remains to be described whether there exists any relationship between the physiological effects of these mutations and calcium channel splice variation. In the present study, we systematically compare the biophysical effects of three type-1 familial hemiplegic migraine (FHM-1) mutations in two predominant splice variants of the neuronal Ca(V)2.1 P/Q-type channel. All three FHM-1 mutations cause a greater hyperpolarizing shift in voltage-dependent properties when expressed in the short carboxyl terminus variant (Ca(V)2.1 Delta47) compared to the long variant (Ca(V)2.1 +47). Furthermore, the FHM-1 mutations also exhibit differential splice variant-specific effects on recovery from inactivation and accumulation of inactivation during tonic and burst firing. Our findings provide important insight concerning the role of calcium channel alternatively spliced variants and the molecular pathophysiology of FHM-1 and potentially of other calcium channelopathies.