Victor N. Uebele

Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel.

We report here a characterization of two families of calcium-activated K+ channel β-subunits, β2 and β3, which are encoded by distinct genes that map to 3q26.2–27. A single β2 family member and four alternatively spliced variants of β3 were investigated. These subunits have predicted molecular masses of 27.1–31.6 kDa, share ∼30–44% amino acid identity with β1, and exhibit distinct but overlapping expression patterns. Coexpression of the β2 or β3a–c subunits with a BK α-subunit altered the functional properties of the current expressed by the α-subunit alone. The β2 subunit rapidly and completely inactivated the current and shifted the voltage dependence for activation to more polarized membrane potentials. In contrast, coexpression of the β3a–c subunits resulted in only partial inactivation of the current, and the β3b subunit conferred an apparent inward rectification. Furthermore, unlike the β1 and β2 subunits, none of the β3 subunits increased channel sensitivity to calcium or voltage. The tissue-specific expression of these β-subunits may allow for the assembly of a large number of distinct BK channels in vivo, contributing to the functional diversity of native BK currents.

Selective T-Type Calcium Channel Block in Thalamic Neurons Reveals Channel Redundancy and Physiological Impact of ITwindow

Although it is well established that low-voltage-activated T-type Ca2+ channels play a key role in many neurophysiological functions and pathological states, the lack of selective and potent antagonists has so far hampered a detailed analysis of the full impact these channels might have on single-cell and neuronal network excitability as well as on Ca2+ homeostasis. Recently, a novel series of piperidine-based molecules has been shown to selectively block recombinant T-type but not high-voltage-activated (HVA) Ca2+ channels and to affect a number of physiological and pathological T-type channel-dependent behaviors. Here we directly show that one of these compounds, 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2), exerts a specific, potent (IC50 = 22 nm), and reversible inhibition of T-type Ca2+ currents of thalamocortical and reticular thalamic neurons, without any action on HVA Ca2+ currents, Na+ currents, action potentials, and glutamatergic and GABAergic synaptic currents. Thus, under current-clamp conditions, the low-threshold Ca2+ potential (LTCP)-dependent high-frequency burst firing of thalamic neurons is abolished by TTA-P2, whereas tonic firing remains unaltered. Using TTA-P2, we provide the first direct demonstration of the presence of a window component of Ca2+ channels in neurons and its contribution to the resting membrane potential of thalamic neurons and to the Up state of their intrinsically generated slow (<1 Hz) oscillation. Moreover, we demonstrate that activation of only a small fraction of the T-type channel population is required to generate robust LTCPs, suggesting that LTCP-driven bursts of action potentials can be evoked at depolarized potentials where the vast majority of T-type channels are inactivated.

Functional Differences in Kv1.5 Currents Expressed in Mammalian Cell Lines Are Due to the Presence of Endogenous Kvβ2.1 Subunits

The voltage-sensitive currents observed following hKv1.5 α subunit expression in HEK 293 and mouse L-cells differ in the kinetics and voltage dependence of activation and slow inactivation. Molecular cloning, immunopurification, and Western blot analysis demonstrated that an endogenous L-cell Kvβ2.1 subunit assembled with transfected hKv1.5 protein. In contrast, both mRNA and protein analysis failed to detect a β subunit in the HEK 293 cells, suggesting that functional differences observed between these two systems are due to endogenous L-cell Kvβ2.1 expression. In the absence of Kvβ2.1, midpoints for activation and inactivation of hKv1.5 in HEK 293 cells were −0.2 ± 2.0 and −9.6 ± 1.8 mV, respectively. In the presence of Kvβ2.1 these values were −14.1 ± 1.8 and −22.1 ± 3.7 mV, respectively. The β subunit also caused a 1.5-fold increase in the extent of slow inactivation at 50 mV, thus completely reconstituting the L-cell current phenotype in the HEK 293 cells. These results indicate that 1) the Kvβ2.1 subunit can alter Kv1.5 α subunit function, 2) β subunits are not required for α subunit expression, and 3) endogenous β subunits are expressed in heterologous expression systems used to study K+ channel function.

High Throughput Ion-Channel Pharmacology: Planar-Array-Based Voltage Clamp

Technological advances often drive major breakthroughs in biology. Examples include PCR, automated DNA sequencing, confocal/single photon microscopy, AFM, and voltage/patch-clamp methods. The patch-clamp method, first described nearly 30 years ago, was a major technical achievement that permitted voltage-clamp analysis (membrane potential control) of ion channels in most cells and revealed a role for channels in unimagined areas. Because of the high information content, voltage clamp is the best way to study ion-channel function; however, throughput is too low for drug screening. Here we describe a novel breakthrough planar-array-based HT patch-clamp technology developed by Essen Instruments capable of voltage-clamping thousands of cells per day. This technology provides greater than two orders of magnitude increase in throughput compared with the traditional voltage-clamp techniques. We have applied this method to study the hERG K(+) channel and to determine the pharmacological profile of QT prolonging drugs.

Presynaptic HCN1 channels regulate CaV3.2 activity and neurotransmission at select cortical synapses

The Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are subthreshold, voltage-gated ion channels that are highly expressed in hippocampal and cortical pyramidal cell dendrites, where they play an important role in regulating synaptic potential integration and plasticity. Here, we demonstrate that HCN1 subunits are also localized to the active zone of mature asymmetric synaptic terminals targeting mouse entorhinal cortical layer III pyramidal neurons. We found that HCN channels inhibit glutamate synaptic release by suppressing the activity of low threshold voltage-gated T- (CaV3.2) type Ca2+ channels. In agreement, electron microscopy showed the co-localisation of pre-synaptic HCN1 and CaV3.2 subunit. This represents a novel mechanism by which HCN channels regulate synaptic strength and thereby neural information processing and network excitability.The hyperpolarization-activated cyclic nucleotide–gated (HCN) channels are subthreshold, voltage-gated ion channels that are highly expressed in hippocampal and cortical pyramidal cell dendrites, where they are important for regulating synaptic potential integration and plasticity. We found that HCN1 subunits are also localized to the active zone of mature asymmetric synaptic terminals targeting mouse entorhinal cortical layer III pyramidal neurons. HCN channels inhibited glutamate synaptic release by suppressing the activity of low-threshold voltage-gated T-type (CaV3.2) Ca2+ channels. Consistent with this, electron microscopy revealed colocalization of presynaptic HCN1 and CaV3.2 subunit. This represents a previously unknown mechanism by which HCN channels regulate synaptic strength and thereby neural information processing and network excitability.

Design, Synthesis, and Evaluation of a Novel 4-Aminomethyl-4-fluoropiperidine as a T-Type Ca2+ Channel Antagonist

The novel T-type antagonist ( S)- 5 has been prepared and evaluated in in vitro and in vivo assays for T-type calcium ion channel activity. Structural modification of the piperidine leads 1 and 2 afforded the fluorinated piperidine ( S)- 5, a potent and selective antagonist that displayed in vivo CNS efficacy without adverse cardiovascular effects.

Essential Thalamic Contribution to Slow Waves of Natural Sleep

Slow waves represent one of the prominent EEG signatures of non-rapid eye movement (non-REM) sleep and are thought to play an important role in the cellular and network plasticity that occurs during this behavioral state. These slow waves of natural sleep are currently considered to be exclusively generated by intrinsic and synaptic mechanisms within neocortical territories, although a role for the thalamus in this key physiological rhythm has been suggested but never demonstrated. Combining neuronal ensemble recordings, microdialysis, and optogenetics, here we show that the block of the thalamic output to the neocortex markedly (up to 50%) decreases the frequency of slow waves recorded during non-REM sleep in freely moving, naturally sleeping-waking rats. A smaller volume of thalamic inactivation than during sleep is required for observing similar effects on EEG slow waves recorded during anesthesia, a condition in which both bursts and single action potentials of thalamocortical neurons are almost exclusively dependent on T-type calcium channels. Thalamic inactivation more strongly reduces spindles than slow waves during both anesthesia and natural sleep. Moreover, selective excitation of thalamocortical neurons strongly entrains EEG slow waves in a narrow frequency band (0.75–1.5 Hz) only when thalamic T-type calcium channels are functionally active. These results demonstrate that the thalamus finely tunes the frequency of slow waves during non-REM sleep and anesthesia, and thus provide the first conclusive evidence that a dynamic interplay of the neocortical and thalamic oscillators of slow waves is required for the full expression of this key physiological EEG rhythm.

Presynaptic CaV3.2 Channels Regulate Excitatory Neurotransmission in Nociceptive Dorsal Horn Neurons

It is generally accepted that presynaptic transmitter release is mainly regulated by subtypes of neuronal high-voltage-activated Ca2+ channels. Here for the first time, we examined the role of T-type Ca2+ channels (T-channels) in synaptic transmission in the dorsal horn (DH) of the spinal cord using patch-clamp recordings from acute spinal cord preparations from both rat and mouse. We found that selective pharmacological antagonism of T-channels inhibited spontaneous synaptic release of glutamate in superficial laminae I-II of the DH, while GABA release was spared. We found similar effect in identified nociceptive projection neurons of lamina I of the DH, but not in inhibitory DH interneurons. In comparison, antagonism of T-channels did not affect excitatory transmission in deeper non-nociceptive DH laminae. Furthermore, we used isoform-specific agents, knock-out mice and immunohistochemistry to specifically implicate presynaptic CaV3.2 channels. We also used an animal model of painful diabetic neuropathy to demonstrate that blocking T-channels in superficial DH neurons suppressed spontaneous excitatory synaptic transmission in diabetic rats in greater degree than in healthy age-matched animals. These studies provide previously unknown information regarding the role of presynaptic T-channels in nociceptive signaling in the spinal cord.

Discovery of 1,4-Substituted Piperidines as Potent and Selective Inhibitors of T-Type Calcium Channels

The discovery of a novel series of potent and selective T-type calcium channel antagonists is reported. Initial optimization of high-throughput screening leads afforded a 1,4-substituted piperidine amide 6 with good potency and limited selectivity over hERG and L-type channels and other off-target activities. Further SAR on reducing the basicity of the piperidine and introducing polarity led to the discovery of 3-axial fluoropiperidine 30 with a significantly improved selectivity profile. Compound 30 showed good oral bioavailability and brain penetration across species. In a rat genetic model of absence epilepsy, compound 30 demonstrated a robust reduction in the number and duration of seizures at 33 nM plasma concentration, with no cardiovascular effects at up to 5.6 microM. Compound 30 also showed good efficacy in rodent models of essential tremor and Parkinsons disease. Compound 30 thus demonstrates a wide margin between CNS and peripheral effects and is a useful tool for probing the effects of T-type calcium channel inhibition.

In Vitro Characterization of T-Type Calcium Channel Antagonist TTA-A2 and In Vivo Effects on Arousal in Mice

T-type calcium channels have been implicated in many behaviorally important neurophysiological processes, and altered channel activity has been linked to the pathophysiology of neurological disorders such as insomnia, epilepsy, Parkinsons disease, depression, schizophrenia, and pain. We have previously identified a number of potent and selective T-type channel antagonists (Barrow et al., 2007; Shipe et al., 2008; Yang et al., 2008). Here we describe the properties of the antagonist TTA-A2 [2-(4-cyclopropylphenyl)-N-((1R)-1-{5-[(2,2,2-trifluoroethyl)oxo]-pyridin-2-yl}ethyl)acetamide], assessed in patch-clamp experiments. TTA-A2 blocks T-type channels (Cav3.1, 3.2, 3.3) voltage dependently and with high potency (IC50 ∼100 nM). Stimulation at 3 Hz revealed additional use dependence of inhibition. A hyperpolarized shift of the channel availability curve and delayed channel recovery from inactivation suggest that the compound preferentially interacts with and stabilizes inactivated channels. The compound showed a ∼300-fold selectivity for Cav3 channels over high-voltage activated calcium channels. Inhibitory effects on native T-type currents were confirmed in brain slice recordings from the dorsal lateral geniculate nucleus and the subthalamic nucleus. Furthermore, we demonstrate that in vivo T-type channel inhibition by TTA-A2 suppresses active wake and promotes slow-wave sleep in wild-type mice but not in mice lacking both Cav3.1 and Cav3.3, suggesting the selective effect of TTA-A2 on recurrent thalamocortical network activity. The discovery of the potent and selective T-type channel antagonist TTA-A2 has enabled us to study the in vivo effects of pharmacological T-channel inhibition on arousal in mice, and it will help to explore the validity of these channels as potential drug targets for sleep-related and other neurological diseases.