Kenneth A. Stauderman

STIM1, an essential and conserved component of store-operated Ca2+ channel function

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.

Modulation of γ-Secretase Reduces β-Amyloid Deposition in a Transgenic Mouse Model of Alzheimer's Disease

Alzheimers disease (AD) is characterized pathologically by the abundance of senile plaques and neurofibrillary tangles in the brain. We synthesized over 1200 novel gamma-secretase modulator (GSM) compounds that reduced Abeta(42) levels without inhibiting epsilon-site cleavage of APP and Notch, the generation of the APP and Notch intracellular domains, respectively. These compounds also reduced Abeta(40) levels while concomitantly elevating levels of Abeta(38) and Abeta(37). Immobilization of a potent GSM onto an agarose matrix quantitatively recovered Pen-2 and to a lesser degree PS-1 NTFs from cellular extracts. Moreover, oral administration (once daily) of another potent GSM to Tg 2576 transgenic AD mice displayed dose-responsive lowering of plasma and brain Abeta(42); chronic daily administration led to significant reductions in both diffuse and neuritic plaques. These effects were observed in the absence of Notch-related changes (e.g., intestinal proliferation of goblet cells), which are commonly associated with repeated exposure to functional gamma-secretase inhibitors (GSIs).

Complete loss of P/Q calcium channel activity caused by a CACNA1A missense mutation carried by patients with episodic ataxia type 2

Familial hemiplegic migraine, episodic ataxia type 2 (EA2), and spinocerebellar ataxia type 6 are allelic disorders of the CACNA1A gene (coding for the alpha(1A) subunit of P/Q calcium channels), usually associated with different types of mutations (missense, protein truncating, and expansion, respectively). However, the finding of expansion and missense mutations in patients with EA2 has blurred this genotype-phenotype correlation. We report the first functional analysis of a new missense mutation, associated with an EA2 phenotype-that is, T-->C transition of nt 4747 in exon 28, predicted to change a highly conserved phenylalanine residue to a serine at codon 1491, located in the putative transmembrane segment S6 of domain III. Patch-clamp recording in HEK 293 cells, coexpressing the mutagenized human alpha(1A-2) subunit, together with human beta(4) and alpha(2)delta subunits, showed that channel activity was completely abolished, although the mutated protein is expressed in the cell. These results indicate that a complete loss of P/Q channel function is the mechanism underlying EA2, whether due to truncating or to missense mutations.

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.

Characterization of the recombinant human neuronal nicotinic acetylcholine receptors α3β2 and α4β2 stably expressed in HEK293 cells

Abstract HEK293 cells were stably transfected with the cDNAs encoding full-length human neuronal nicotinic acetylcholine receptor (nAChR) subunit combinations α3β2 or α4β2. [ 3 H]-(±)Epibatidine ([ 3 H]-(±)EPI) bound to membranes from A3B2 (α3β2) and A4B2.2 (α4β2) cells with K d values of 7.5 and 33.4 pM and B max values of 497 and 1564 fmol/mg protein, respectively. Concentration-dependent increases in intracellular free Ca 2+ concentration were elicited by nAChR agonists with a rank order of potency of EPI>1,1-dimethyl-4-phenylpiperazinium (DMPP)>nicotine (NIC)=suberyldicholine (SUB)>cytisine (CYT)=acetylcholine (ACh) for A3B2 cells and EPI>CYT=SUB=NIC=DMPP>ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-induced responses with a rank order of potency of d-tubocurarine (d-Tubo)=mecamylamine (MEC)>dihydro-β-erythroidine (DHβE) in A3B2 cells and MEC=DHβE>d-Tubo in A4B2.2 cells. Whole-cell patch clamp recordings indicate that the decay rate of macroscopic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B2 cells are less sensitive to ACh than A4B2.2 cells. ACh currents elicited in α3β2 and α4β2 human nAChRs are maximally potentiated at 20 and 2 mM external Ca 2+ , respectively. Our results indicate that stably expressed α3β2 and α4β2 human nAChRs are pharmacologically and functionally distinct.

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.

Fluoxetine-induced inhibition of synaptosomal [3H] 5-HT release: Possible CA2+-channel inhibition

Fluoxetine, a selective 5-HT uptake inhibitor, inhibited 15 mM K(+)-induced [3H]5-HT release from rat spinal cord and cortical synaptosomes at concentrations greater than 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K+ used to depolarize the synaptosomes and the concentration of external Ca2+. Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of [3H]5-HT release induced by the Ca(2+)-ionophore A 23187 or Ca(2+)-independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K(+)-induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca2+ channels and Ca2+ entry. Whereas fluoxetine and paroxetine inhibited binding of [3H]nitrendipine to the dihydropyridine-sensitive L-type Ca2+ channel, the less selective uptake inhibitors did not alter binding. The dihydropyridine antagonist nimodipine partially blocked fluoxetine-induced inhibition of release. Moreover enhanced K(+)-stimulated release due to the dihydropyridine agonist Bay K 8644 was reversed by fluoxetine. Fluoxetine also inhibited the K(+)-induced increase in intracellular free Ca2+ in fura-2 loaded synaptosomes. These data are consistent with the suggestion that fluoxetine inhibits K(+)-induced [3H]5-HT release by antagonizing voltage-dependent Ca2+ entry into nerve terminals.

Characterization of sodium-dependent, high-affinity serotonin uptake in rat spinal cord synaptosomes

Synaptosomal accumulation of [3H]serotonin was used to determine if the rat spinal cord possesses a high-affinity neuronal uptake system for serotonin. Two temperature-dependent accumulation processes were found, one sodium-dependent, the second sodium-independent. Sodium-dependent [3H]serotonin accumulation was linear with sodium concentrations up to 143 mM, was associated with the purified synaptosomal fraction (P2B), and decreased 76% by osmotic lysis, 88% by sonication, and 96% by 0.1% Triton X-100. Drug inhibition studies demonstrated fluoxetine to be the most potent inhibitor of this system (IC50 0.075 microM) while desipramine (IC50 0.43 microM) and nomifensine (IC50 0.95 microM) were less potent. Kinetic analysis revealed that sodium-dependent accumulation in purified synaptosomes was saturable at low [3H]serotonin concentrations (Ku = 50 nM, Vmax = 4 pmol/mg protein/min). Sodium-independent [3H]5-HT accumulation was substantially less sensitive to fluoxetine, desipramine and nomifensine. While sodium-independent accumulation was not significantly affected by osmotic lysis, it was markedly increased by prior sonication of tissue. Also, in contrast to sodium-dependent accumulation, sodium-independent accumulation was evenly distributed in all tissue fractions, and was not saturable at low [3H]serotonin concentrations. It is concluded that sodium-dependent [3H]serotonin accumulation reflects uptake into spinal serotonergic nerve terminals while sodium-independent accumulation probably reflects a temperature-sensitive binding to membrane fragments. Comparison to brain uptake of serotonin and the necessity for using 37 degrees C sodium-free blanks rather than 0 degree C blanks in spinal cord homogenates is discussed.

Dibutyryl‐Cyclic GMP Stimulation of Ca2+‐ATPase Activity in Rat Brain Synaptic Membranes

Abstract: The effects of dibutyryl cyclic AMP (dbcAMP) and dibutyryl cyclic GMP (db‐cGMP) were tested on Ca2+‐ATPase, Mg2+‐ATPase, and (Ca2++ Mg2+)‐ATPase activities in lysed synaptosomes prepared from whole rat brains (minus cerebellum). At concentrations from 0.1 to 2.0 mM, db‐cGMP produced a selective, concentration‐dependent increase in Ca2+‐ATPase activity. Both db‐cGMP and db‐cAMP slightly reduced Mg2+‐ATPase activity, whereas neither compound had concentration‐dependent effects on (Ca2++ Mg2+)‐ATPase activity. These findings suggest that the Mg2+‐independent, Ca2+‐ATPase activity in rat brain is regulated by a cyclic GMP‐dependent process. Further, the data provide evidence that the Ca2+‐ATPase activity in lysed synaptosomal membranes represents an enzyme that is distinguishable from both the Mg2+‐ and (Ca2++ Mg2+‐ATPase.