Paul Brust

Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris.

The oxidation of methanol follows a well-defined pathway and is similar for several methylotrophic yeasts. The use of methanol as the sole carbon source for the growth of Pichia pastoris stimulates the expression of a family of genes. Three methanol-responsive genes have been isolated; cDNA copies have been made from mRNAs of these genes, and the protein products from in vitro translations have been examined. The identification of alcohol oxidase as one of the cloned, methanol-regulated genes has been made by enzymatic, immunological, and sequence analyses. Methanol-regulated expression of each of these three isolated genes can be demonstrated to occur at the level of transcription. Finally, DNA subfragments of two of the methanol-responsive genomic clones from P. pastoris have been isolated and tentatively identified as containing the control regions involved in methanol regulation.

Functional Consequences of Mutations in the Human α1A Calcium Channel Subunit Linked to Familial Hemiplegic Migraine

Mutations in α1A, the pore-forming subunit of P/Q-type calcium channels, are linked to several human diseases, including familial hemiplegic migraine (FHM). We introduced the four missense mutations linked to FHM into human α1A-2subunits and investigated their functional consequences after expression in human embryonic kidney 293 cells. By combining single-channel and whole-cell patch-clamp recordings, we show that all four mutations affect both the biophysical properties and the density of functional channels. Mutation R192Q in the S4 segment of domain I increased the density of functional P/Q-type channels and their open probability. Mutation T666M in the pore loop of domain II decreased both the density of functional channels and their unitary conductance (from 20 to 11 pS). Mutations V714A and I1815L in the S6 segments of domains II and IV shifted the voltage range of activation toward more negative voltages, increased both the open probability and the rate of recovery from inactivation, and decreased the density of functional channels. Mutation V714A decreased the single-channel conductance to 16 pS. Strikingly, the reduction in single-channel conductance induced by mutations T666M and V714A was not observed in some patches or periods of activity, suggesting that the abnormal channel may switch on and off, perhaps depending on some unknown factor. Our data show that the FHM mutations can lead to both gain- and loss-of-function of human P/Q-type calcium channels.

Structure and Functional Characterization of a Novel Human Low‐Voltage Activated Calcium Channel

Abstract : We have isolated and characterized overlapping cDNAs encoding a novel, voltage‐gated Ca2+ channel α1 subunit, α1H, from a human medullary thyroid carcinoma cell line. The α1H subunit is structurally similar to previously described α1 subunits. Northern blot analysis indicates that α1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba2+ currents recorded from human embryonic kidney 293 cells transiently expressing α1H activated at relatively hyperpolarized potentials (‐50 mV), rapidly inactivated (τ = 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing α1H. Singlechannel measurements in human embryonic kidney 293 cells revealed a single‐channel conductance of ~9 pS. These channels are blocked by Ni2+ (IC50 = 6.6 μM) and the T‐type channel antagonists mibefradil (~50% block at 1 μM) and amiloride (IC50 = 167 μM). Thus, α1H‐containing channels exhibit biophysical and pharmacological properties characteristic of low voltage‐activated, or T‐type, Ca2+ channels.

Human neuronal voltage-dependent calcium channels: studies on subunit structure and role in channel assembly.

Voltage-dependent calcium (Ca2+) channels, expressed in the CNS, appear to be multimeric complexes comprised of at least alpha 1, alpha 2 and beta subunits. Previously, we cloned and expressed human neuronal alpha 1, alpha 2 and beta subunits to study recombinant channel complexes that display properties of those expressed in vivo. The alpha 1B-mediated channel subtype binds omega-conotoxin (CgTx) GVIA with high affinity and exhibits properties of N-type voltage-dependent Ca2+ channels. Here we describe several alpha 2 and beta splice variants and report results on the expression of omega-CgTx GVIA binding sites, assembly of the subunit complex and biophysical function of alpha 1B-mediated channel complexes containing some of these splice variants. We optimized recombinant expression in human embryonic kidney (HEK) 293 cells of alpha 1B alpha 2b beta 1 subunit complexes by controlling the expression levels of subunit mRNAs and monitored cell surface expression by binding of omega-CgTx GVIA to the alpha 1B subunit. Co-expression of either alpha 2b or beta 1 subunits with an alpha 1B subunit increased expression of binding sites while the most efficient expression was achieved when both alpha 2b and beta 1 subunits were co-expressed with an alpha 1B subunit. The presence of alpha 2b affects the affinity of omega-CgTx GVIA binding and barium (Ba2+) current magnitudes, although it does not appear to alter kinetic properties of the Ba2+ current. This is the first evidence of an alpha 2 subunit modulating the binding affinity of a cell-surface Ca2+ channel ligand. Our results demonstrate that alpha 1, alpha 2 and beta subunits together contribute to the efficient assembly and functional expression of voltage-dependent Ca2+ channel complexes.

Positive allosteric modulators of the human sweet taste receptor enhance sweet taste

To identify molecules that could enhance sweetness perception, we undertook the screening of a compound library using a cell-based assay for the human sweet taste receptor and a panel of selected sweeteners. In one of these screens we found a hit, SE-1, which significantly enhanced the activity of sucralose in the assay. At 50 μM, SE-1 increased the sucralose potency by >20-fold. On the other hand, SE-1 exhibited little or no agonist activity on its own. SE-1 effects were strikingly selective for sucralose. Other popular sweeteners such as aspartame, cyclamate, and saccharin were not enhanced by SE-1 whereas sucrose and neotame potency were increased only by 1.3- to 2.5-fold at 50 μM. Further assay-guided chemical optimization of the initial hit SE-1 led to the discovery of SE-2 and SE-3, selective enhancers of sucralose and sucrose, respectively. SE-2 (50 μM) and SE-3 (200 μM) increased sucralose and sucrose potencies in the assay by 24- and 4.7-fold, respectively. In human taste tests, 100 μM of SE-1 and SE-2 allowed for a reduction of 50% to >80% in the concentration of sucralose, respectively, while maintaining the sweetness intensity, and 100 μM SE-3 allowed for a reduction of 33% in the concentration of sucrose while maintaining the sweetness intensity. These enhancers did not exhibit any sweetness when tasted on their own. Positive allosteric modulators of the human sweet taste receptor could help reduce the caloric content in food and beverages while maintaining the desired taste.

Characteristics of a human N-type calcium channel expressed in HEK293 cells

The human alpha 1B-1 alpha 2b beta 1-2 Ca2+ channel was stably expressed in HEK293 cells producing a human brain N-type voltage-dependent calcium channel (VDCC). Whole cell voltage-clamp electrophysiology and fura-2 based microfluorimetry have been used to study its characteristics. Calcium currents (ICa) recorded in transfected HEK293 cells were activated at potentials more depolarized than -20 mV with peak currents occurring at approx + 10 mV in 5 mM extracellular CaCl2. ICa and associated rises in intracellular free calcium concentrations ([Ca2+]i) were sensitive to changes in both the [Ca2+]o and holding potential. Steady-state inactivation was half maximal at a holding potential of -60 mV. Ba2+ was a more effective charge carrier than Ca2+ through the alpha 1B-1 alpha 2b beta 1-2 Ca2+ channel and combinations of both Ba2+ and Ca2+ as charge carriers resulted in the anomalous mole fraction effect. Ca2+ influx into transfected HEK293 cells was irreversibly inhibited by omega-conotoxin-GVIA (omega-CgTx-GVIA; 10 nM-1 microM) and omega-conotoxin-MVIIA; 100 nM-1 microM) whereas 1 microM) whereas no reductions were seen with agents which block P or L-type Ca2+ channels. The inorganic ions, gadolinium (Gd3+), cadmium (Cd2+) and nickel (Ni2+) reduced the ICa under voltage-clamp conditions in a concentration-dependent manner. The order of potency of the three ions was Gd3+ > Cd2+ > Ni2+. These experiments suggest that the cloned and expressed alpha 1B-1 alpha 2b beta 1-2 Ca2+ channel subunits form channels in HEK293 cells that exhibit properties consistent with the activity of the native-N-type VDCC previously described in neurons.

Modal Gating of Human CaV2.1 (P/Q-type) Calcium Channels: I. The Slow and the Fast Gating Modes and their Modulation by β Subunits

The single channel gating properties of human CaV2.1 (P/Q-type) calcium channels and their modulation by the auxiliary β1b, β2e, β3a, and β4a subunits were investigated with cell-attached patch-clamp recordings on HEK293 cells stably expressing human CaV2.1 channels. These calcium channels showed a complex modal gating, which is described in this and the following paper (Fellin, T., S. Luvisetto, M. Spagnolo, and D. Pietrobon. 2004. J. Gen. Physiol. 124:463–474). Here, we report the characterization of two modes of gating of human CaV2.1 channels, the slow mode and the fast mode. A channel in the two gating modes differs in mean closed times and latency to first opening (both longer in the slow mode), in voltage dependence of the open probability (larger depolarizations are necessary to open the channel in the slow mode), in kinetics of inactivation (slower in the slow mode), and voltage dependence of steady-state inactivation (occurring at less negative voltages in the slow mode). CaV2.1 channels containing any of the four β subtypes can gate in either the slow or the fast mode, with only minor differences in the rate constants of the transitions between closed and open states within each mode. In both modes, CaV2.1 channels display different rates of inactivation and different steady-state inactivation depending on the β subtype. The type of β subunit also modulates the relative occurrence of the slow and the fast gating mode of CaV2.1 channels; β3a promotes the fast mode, whereas β4a promotes the slow mode. The prevailing mode of gating of CaV2.1 channels lacking a β subunit is a gating mode in which the channel shows shorter mean open times, longer mean closed times, longer first latency, a much larger fraction of nulls, and activates at more positive voltages than in either the fast or slow mode.

The Role of Autoantibodies in Lambert-Eaton Myasthenic Syndromeaa

The Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder of neuromuscular transmission that causes fatigable muscle weakness, loss of tendon reflexes, and autonomic dysfunction.1–3 LEMS antibodies bind to and induce a downregulation of voltage-gated channels (VGCCs),4,5 resulting in a reduction in the nerve-evoked, Ca2+-dependent release of acetylcholine from motor nerve terminals. Approximately 60% of patients have an associated small cell lung carcinoma (SCLC),6 a tumor that is thought to be neuroendocrine in origin. This strong association with cancer makes LEMS a member of the group of paraneoplastic disorders. SCLC cells have been shown to express functional VGCCs,7,8 and preincubation in LEMS sera or IgG reduces the K+-stimulated 45Ca2+ flux into these cells.8–10

Differential Effect of Lambert‐Eaton Myasthenic Syndrome Immunoglobulin on Cloned Neuronal Voltage‐gated Calcium Channelsa

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder in which antibodies to presynaptic calcium channels at the neuromuscular junction lead to muscle weakness. Around 60% of patients with LEMS have an associated small cell lung carcinoma (SCLC), which is known to express voltage-gated calcium channels (VGCCs). Immunoglobulin (IgG) from patients with LEMS causes inhibition of calcium flux in an SCLC cell line.1 This inhibition has been shown to correlate with disease severity.1 Neuronal VGCCs can be classified into P-, Q-, N-, L-, and R-type according to their electrophysiological and pharmacological properties. Around 92% of LEMS patients have antibodies that immunoprecipitate P/Q-type (125I-CTx-MVIIC labeled) VGCCs and around 30% have antibodies to N-type (125I-CTx-GVIA labeled) VGCCs.2 In addition, antibodies from LEMS patients have been shown to abolish the component of neurotransmitter release subserved by both P-type and Q-type VGCCs in sympathetic and parasympathetic neurons of mice injected with LEMS IgG.3 Neuronal VGCCs consist of an 1 subunit with and 2 subunits. Several of the human genes encoding the different types of VGCC have now been cloned and sequenced, including the 1A, 1B, 1D, 1E, 1–4, and 2 subunits. Human embryonic

Relative contributions of G protein, channel, and receptor to voltage-dependent inhibition of neuronal N-type and P/Q-type calcium channels in HEK 293 cell lines

The voltage-dependent modulation of neuronal voltage-gated calcium channels by heterotrimeric G protein-coupled receptors potentially provides a means for activity-dependent modulation of synaptic efficacy. Recent attention has focused upon the molecular mechanisms by which such G proteins influence the biophysical properties of calcium channels. We have used an HEK 293-based heterologous system which stably expresses human neuronal calcium channels to address the relative contributions of receptor, G protein, and channel to voltage-dependent inhibition. We find that the receptor and channel subtype only insignificantly influence the time it takes to re-establish modulation following voltage-dependent relief of inhibition. In contrast, the G protein subtype mediating inhibition appears to play a significant part in this process. These results emphasize the importance of G protein subtype in the modulation of neuronal calcium channels.