A Two-test Protocol for the Precise Determination of the Maximal Lactate Steady State.

The purpose of this study was to determine the efficacy of a two-test method for precisely identifying the Maximal Lactate Steady State (MLSS). Eight male competitive cyclists performed two bouts on a cycle ergometer. Following a maximal oxygen consumption (V̇O2max) test (66.91 ± 5.29 mL·kg-1·min-1) we identified the lactate deflection point using the visual deflection (TVis), Log-Log (TLog), Dmax (TDmax), RER = 1.00 (TRER), ventilatory threshold (TVent), and the 1.0 mmol·L-1 increase above baseline (T+1) methods. The second incremental test (SIT) consisted of 6-7 stages (5 min each) starting 20-30 W below to 20-30 W above the predetermined deflection point, in 10 W increments. Comparison of the two tests yielded different threshold estimates (range 11-46W) for all methods (P = 0.001-0.019) except the TLog (P = 0.194) and TRER (P = 0.100). The SIT resulted in significantly (P = 0.007) more narrow range of thresholds (27.5 ± 11.01W) compared to the V̇O2max test (70 ± 42.51W). The TVis from the SIT was identified as the MLSS and was verified using three 45-minute steady-state exercise bouts at 95%, 100%, and 105% of MLSS intensity (average increment 12.8 W). Blood lactate and V̇O2 were recorded every 5 minutes and differed between the three intensities at every time point (P < 0.001). V̇O2 increased from the 5th to the 45th minute by 7.02 mL·kg-1·min-1 (100% MLSS), 3.63 mL·kg-1·min-1 (95% MLSS) and 7.5 mL·kg-1·min-1 (105% MLSS, to the 30th minute). These results indicate that the MLSS was identified correctly by the SIT, the single incremental test overestimated the MLSS intensity, and the TVis provides a very accurate determination of the lactate breakpoint. The use of a second submaximal test is required for a precise identification of MLSS.

Calcium-induced calcium release activates spontaneous miniature outward currents in newt medullary reticular formation neurons.

Neurons in the medullary reticular formation are involved in the control of postural and locomotor behaviors in all vertebrates. Reticulospinal neurons in this brain region provide one of the major descending projections to the spinal cord. Although neurons in the newt medullary reticular formation have been extensively studied using in vivo extracellular recordings, little is known of their intrinsic biophysical properties or of the underlying circuitry of this region. Using whole cell patch-clamp recordings in brain slices containing the rostromedial reticular formation from adult male newts, we observed spontaneous miniature outward currents (SMOCs) in ~2/3 of neurons. Although SMOCs superficially resembled inhibitory postsynaptic currents (IPSCs), they had slower risetimes and decay times than spontaneous IPSCs. SMOCs required intracellular Ca2+ release from ryanodine receptors and were also dependent on the influx of extracellular Ca2+. SMOCs were unaffected by apamin but were partially blocked by iberiotoxin and charybdotoxin, indicating that SMOCs were mediated by big-conductance Ca2+-activated K+ channels. Application of the sarco/endoplasmic Ca2+ ATPase inhibitor cyclopiazonic acid blocked the generation of SMOCs and also increased neural excitability. Neurons with SMOCs had significantly broader action potentials, slower membrane time constants, and higher input resistance than neurons without SMOCs. Thus, SMOCs may serve as a mechanism to regulate action potential threshold in a majority of neurons within the newt medullary reticular formation. NEW & NOTEWORTHY The medullary reticular formation exerts a powerful influence on sensorimotor integration and subsequent motor behavior, yet little is known about the neurons involved. In this study, we identify a transient potassium current that regulates action potential threshold in a majority of medullary reticular neurons.

Heteromeric KV2/KV8.2 Channels Mediate Delayed Rectifier Potassium Currents in Primate Photoreceptors.

Silent voltage-gated potassium channel subunits (KVS) interact selectively with members of the KV2 channel family to modify their functional properties. The localization and functional roles of these silent subunits remain poorly understood. Mutations in the KVS subunit, KV8.2 (KCNV2), lead to severe visual impairment in humans, but the basis of these deficits remains unclear. Here, we examined the localization, native interactions, and functional properties of KV8.2-containing channels in mouse, macaque, and human photoreceptors of either sex. In human retina, KV8.2 colocalized with KV2.1 and KV2.2 in cone inner segments and with KV2.1 in rod inner segments. KV2.1 and KV2.2 could be coimmunoprecipitated with KV8.2 in retinal lysates indicating that these subunits likely interact directly. Retinal KV2.1 was less phosphorylated than cortical KV2.1, a difference expected to alter the biophysical properties of these channels. Using voltage-clamp recordings and pharmacology, we provide functional evidence for Kv2-containing channels in primate rods and cones. We propose that the presence of KV8.2, and low levels of KV2.1 phosphorylation shift the activation range of KV2 channels to align with the operating range of rod and cone photoreceptors. Our data indicate a role for KV2/KV8.2 channels in human photoreceptor function and suggest that the visual deficits in patients with KCNV2 mutations arise from inadequate resting activation of KV channels in rod and cone inner segments.SIGNIFICANCE STATEMENT Mutations in a voltage-gated potassium channel subunit, KV8.2, underlie a blinding inherited photoreceptor dystrophy, indicating an important role for these channels in human vision. Here, we have defined the localization and subunit interactions of KV8.2 channels in primate photoreceptors. We show that the KV8.2 subunit interacts with different Kv2 channels in rods and cones, giving rise to potassium currents with distinct functional properties. Our results provide a molecular basis for retinal dysfunction in patients with mutations in the KCNV2 gene encoding KV8.2.

Electrochemical Na+ and Ca2+ gradients drive coupled-clock regulation of automaticity of isolated rabbit sinoatrial nodal pacemaker cells.

Coupling of an intracellular Ca(2+) clock to surface membrane ion channels, i.e., a "membrane clock, " via coupling of electrochemical Na(+) and Ca(2+) gradients (ENa and ECa, respectively) has been theorized to regulate sinoatrial nodal cell (SANC) normal automaticity. To test this hypothesis, we measured responses of [Na(+)]i, [Ca(2+)]i, membrane potential, action potential cycle length (APCL), and rhythm in rabbit SANCs to Na(+)/K(+) pump inhibition by the digitalis glycoside, digoxigenin (DG, 10-20 µmol/l). Initial small but significant increases in [Na(+)]i and [Ca(2+)]i and reductions in ENa and ECa in response to DG led to a small reduction in maximum diastolic potential (MDP), significantly enhanced local diastolic Ca(2+) releases (LCRs), and reduced the average APCL. As [Na(+)]i and [Ca(2+)]i continued to increase at longer times following DG exposure, further significant reductions in MDP, ENa, and ECa occurred; LCRs became significantly reduced, and APCL became progressively and significantly prolonged. This was accompanied by increased APCL variability. We also employed a coupled-clock numerical model to simulate changes in ENa and ECa simultaneously with ion currents not measured experimentally. Numerical modeling predicted that, as the ENa and ECa monotonically reduced over time in response to DG, ion currents (ICaL, ICaT, If, IKr, and IbNa) monotonically decreased. In parallel with the biphasic APCL, diastolic INCX manifested biphasic changes; initial INCX increase attributable to enhanced LCR ensemble Ca(2+) signal was followed by INCX reduction as ENCX (ENCX = 3ENa - 2ECa) decreased. Thus SANC automaticity is tightly regulated by ENa, ECa, and ENCX via a complex interplay of numerous key clock components that regulate SANC clock coupling.

Auditory Golgi cells are interconnected predominantly by electrical synapses.

The mossy fiber-granule cell-parallel fiber system conveys proprioceptive and corollary discharge information to principal cells in cerebellum-like systems. In the dorsal cochlear nucleus (DCN), Golgi cells inhibit granule cells and thus regulate information transfer along the mossy fiber-granule cell-parallel fiber pathway. Whereas excitatory synaptic inputs to Golgi cells are well understood, inhibitory and electrical synaptic inputs to Golgi cells have not been examined. Using paired recordings in a mouse brain slice preparation, we find that Golgi cells of the cochlear nucleus reliably form electrical synapses onto one another. Golgi cells were only rarely electrically coupled to superficial stellate cells, which form a separate network of electrically coupled interneurons in the DCN. Spikelets had a biphasic effect on the excitability of postjunctional Golgi cells, with a brief excitatory phase and a prolonged inhibitory phase due to the propagation of the prejunctional afterhyperpolarization through gap junctions. Golgi cells and stellate cells made weak inhibitory chemical synapses onto Golgi cells with low probability. Electrical synapses are therefore the predominant form of synaptic communication between auditory Golgi cells. We propose that electrical synapses between Golgi cells may function to regulate the synchrony of Golgi cell firing when electrically coupled Golgi cells receive temporally correlated excitatory synaptic input.

Single granule cells excite Golgi cells and evoke feedback inhibition in the cochlear nucleus.

In cerebellum-like circuits, synapses from thousands of granule cells converge onto principal cells. This fact, combined with theoretical considerations, has led to the concept that granule cells encode afferent input as a population and that spiking in individual granule cells is relatively unimportant. However, granule cells also provide excitatory input to Golgi cells, each of which provide inhibition to hundreds of granule cells. We investigated whether spiking in individual granule cells could recruit Golgi cells and thereby trigger widespread inhibition in slices of mouse cochlear nucleus. Using paired whole-cell patch-clamp recordings, trains of action potentials at 100 Hz in single granule cells was sufficient to evoke spikes in Golgi cells in ∼40% of paired granule-to-Golgi cell recordings. High-frequency spiking in single granule cells evoked IPSCs in ∼5% of neighboring granule cells, indicating that bursts of activity in single granule cells can recruit feedback inhibition from Golgi cells. Moreover, IPSPs mediated by single Golgi cell action potentials paused granule cell firing, suggesting that inhibitory events recruited by activity in single granule cells were able to control granule cell firing. These results suggest a previously unappreciated relationship between population coding and bursting in single granule cells by which spiking in a small number of granule cells may have an impact on the activity of a much larger number of granule cells.

Coactivation of multiple tightly coupled calcium channels triggers spontaneous release of GABA.

Voltage-activated Ca(2+) channels (VACCs) mediate Ca(2+) influx to trigger action potential-evoked neurotransmitter release, but the mechanism by which Ca(2+) regulates spontaneous transmission is unclear. We found that VACCs are the major physiological triggers for spontaneous release at mouse neocortical inhibitory synapses. Moreover, despite the absence of a synchronizing action potential, we found that spontaneous fusion of a GABA-containing vesicle required the activation of multiple tightly coupled VACCs of variable type.

Designing calcium release channel inhibitors with enhanced electron donor properties: stabilizing the closed state of ryanodine receptor type 1.

New drugs with enhanced electron donor properties that target the ryanodine receptor from skeletal muscle sarcoplasmic reticulum (RyR1) are shown to be potent inhibitors of single-channel activity. In this article, we synthesize derivatives of the channel activator 4-chloro-3-methyl phenol (4-CmC) and the 1,4-benzothiazepine channel inhibitor 4-[-3{1-(4-benzyl) piperidinyl}propionyl]-7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine (K201, JTV519) with enhanced electron donor properties. Instead of activating channel activity (~100 µM), the 4-methoxy analog of 4-CmC [4-methoxy-3-methyl phenol (4-MmC)] inhibits channel activity at submicromolar concentrations (IC(50) = 0.34 ± 0.08 µM). Increasing the electron donor characteristics of K201 by synthesizing its dioxole congener results in an approximately 16 times more potent RyR1 inhibitor (IC(50) = 0.24 ± 0.05 µM) compared with K201 (IC(50) = 3.98 ± 0.79 µM). Inhibition is not caused by an increased closed time of the channel but seems to be caused by an open state block of RyR1. These alterations to chemical structure do not influence the ability of these drugs to affect Ca(2+)-dependent ATPase activity of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase type 1. Moreover, the FKBP12 protein, which stabilizes RyR1 in a closed configuration, is shown to be a strong electron donor. It seems as if FKBP12, K201, its dioxole derivative, and 4-MmC inhibit RyR1 channel activity by virtue of their electron donor characteristics. These results embody strong evidence that designing new drugs to target RyR1 with enhanced electron donor characteristics results in more potent channel inhibitors. This is a novel approach to the design of new, more potent drugs with the aim of functionally modifying RyR1 single-channel activity.

The cytosolic protein kinase CK2 phosphorylates cardiac calsequestrin in intact cells.

The luminal SR protein CSQ2 contains phosphate on roughly half of the serines found in its C-terminus. The sequence around phosphorylation sites in CSQ2 suggest that the in vivo kinase is protein kinase CK2, even though this enzyme is thought to be present only in the cytoplasm and nucleus. To test whether CSQ2 kinase is CK2, we combined approaches that reduced CK2 activity and CSQ2 phosphorylation in intact cells. Tetrabromocinnamic acid, a specific inhibitor of CK2, inhibited both the CSQ2 kinase and CK2 in parallel across a range of concentrations. In intact primary adult rat cardiomyocytes and COS cells, 24 h of drug treatment reduced phosphorylation of overexpressed CSQ2 by 75%. Down-regulation of CK2α subunits in COS cells using siRNA, produced a 90% decrease in CK2α protein levels, and CK2-silenced COS cells exhibited a twofold reduction in CSQ2 kinase activity. Phosphorylation of CSQ2 overexpressed in CK2-silenced cells was also reduced by a factor of two. These data suggested that CSQ2 in intact cells is phosphorylated by CK2, a cytosolic kinase. When phosphorylation site mutants were analyzed in COS cells, the characteristic rough endoplasmic reticulum form of the CSQ2 glycan (GlcNAc2Man9,8) underwent phosphorylation site dependent processing such that CSQ2-nonPP (Ser to Ala mutant) and CSQ2-mimPP (Ser to Glu mutant) produced apparent lower and greater levels of ER retention, respectively. Taken together, these data suggest CK2 can phosphorylate CSQ2 co-translationally at biosynthetic sites in rough ER, a process that may result in changes in its subsequent trafficking through the secretory pathway.