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E Stefani

Publications and source records attributed to E Stefani.

At least 55 records · Page 3Linked to original sources
PubMed1997

Voltage-dependent proton transport by the voltage sensor of the Shaker K+ channel.

In voltage-dependent ion channels, pore opening is initiated by electrically driven movements of charged residues, and this movement generates a gating current. To examine structural rearrangements in the Shaker K+ channel, basic residues R365 and R368 in the S4 segment were replaced with histidine, and gating currents were recorded. Changes in gating charge displacement with solvent pH reveal voltage-dependent changes in exposure of the histidine to solvent protons. This technique directly monitors accessibility changes during gating, probes the environment even in confined locations, and introduces minimal interference of gating charge motion. The results indicate that charges 365 and 368 traverse the entire electric field during gating. The remarkable implication of the successive exposure of histidine to each side of the membrane is that in a pH gradient, the voltage sensor transports protons.

Amino Acid Substitution↗
PubMed1997

Correlation between charge movement and ionic current during slow inactivation in Shaker K+ channels.

Prolonged depolarization induces a slow inactivation process in some K+ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Delta(6-46) K+ channel and in the nonconducting mutant (Shaker H4-Delta(6-46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547-556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to -90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non-slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K+ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011-1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.

Animals↗
PubMed1996

On the molecular basis and regulation of cellular capacitative calcium entry: roles for Trp proteins.

During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of capacitative calcium entry (CCE) into cells. Specifically, we tested the hypothesis that CCE channels are formed of subunits encoded in genes related to the Drosophila trp gene. The first step in this pursuit was to search for mammalian trp genes. We found not one but six mammalian genes and cloned several of their cDNAs, some in their full length. As assayed in mammalian cells, overexpression of some mammalian Trps increases CCE, while expression of partial trp cDNAs in antisense orientation can interfere with endogenous CCE. These findings provided a firm connection between CCE and mammalian Trps. This article reviews the known forms of CCE and highlights unanswered questions in our understanding of intracellular Ca2+ homeostasis and the physiological roles of CCE.

Animals↗
PubMed1996

Coupling between charge movement and pore opening in vertebrate neuronal alpha 1E calcium channels.

1. Neuronal alpha 1E Ca2+ channels were expressed alone and in combination with the beta 2a subunit in Xenopus laevis oocytes. 2. The properties of ionic and gating currents of alpha 1E were investigated: ionic currents were measured in 10 mM external Ba2+; gating currents were isolated in 2 mM external Co2+. 3. Charge movement preceded channel opening. The charge movement voltage curve (Q(V)) preceded the ionic conductance voltage dependence (G(V)) by approximately 20 mV. 4. Coexpression of alpha 1E with the beta 2a subunit did not modify the voltage dependence of charge movement but shifted the G(V) curve to more negative potentials. The voltage gap between Q(V) and G(V) curves was reduced by the beta 2a subunit and both curves overlapped at potentials near 0 mV. 5. The coupling efficiency between the charge movement and pore opening was estimated by the ration between limiting conductance and maximum charge movement (Gmax/Qmax). Coexpression of the beta 2a subunit increased the Gmax/Qmax ratio from 9.2 x 10(5) +/- 1.4 x 10(5) to 21.9 x 10(5) +/- 2.8 X 10(5) S C-1 for alpha 1E and alpha 1E + beta 2a, respectively. 6. We conclude that in the neuronal alpha 1E the charge movement is tightly coupled with the pore opening and that the beta 2a subunit coexpression further improves this coupling.

Animals↗
PubMed1996

Molecular determinants of external barium block in Shaker potassium channels.

Mutations in the outer pore region of Shaker K+ channels (T449 and D447) can influence external Ba2+ block. Substitution of T449 by A, V or Y differentially reduced Ba2+ block primarily by decreasing the blocking rate. Substitution of D447 by N resulted in a non-conducting channel with apparently normal gating currents. External Ba2+ can speed the OFF gating current of a different non-conducting mutant, W434F; this effect was markedly attenuated by the D447N substitution. These results suggest that D447 contributes to an external Ba2+ binding site while T449 imposes a barrier to the access of that site.

Animals↗
PubMed1996

trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2+ entry.

SUMMARY: Capacitative calcium entry (CCE) describes CA2+ influx into cells that replenishes CA2+ stores emptied through the action of IP3 and other agents. It is an essential component of cellular responses to many hormones and growth factors. The molecular basis of this form of Ca2+ entry is complex and may involve more than one type of channel. Studies on visual signal transduction in Drosophila led to the hypothesis that a protein encoded in trp may be a component of CCE channels. We reported the existence of six trp-related genes in the mouse genome. Expression in L cells of small portions of these genes in antisense orientation suppressed CCE. Expression in COS cells of two full-length cDNAs encoding human trp homologs, Htrp1 and Htrp3, increased CCE. This identifies mammalian gene products that participate in CCE. We propose that trp homologs are subunits of CCE channels, not unlike those of classical voltage-gated ion channels.

Amino Acid Sequence↗
PubMed1996

The clinical measurement of volumes using helical CT.

The object of this preliminary study is to evaluate the new techniques of measurement by helical CT which allow direct assessment of the volume of a lesion in clinical practice particularly by obtaining direct macroscopic anatomical correlation. Its primary application is anatomical, with measurement of the volumes of organs or anatomical structures, the clinical importance of which relates primarily to oncology. We present our initial results, including their applications and limits, before extending this study to a larger series so that it may be compared with other multicentre evaluations.

Feasibility Studies↗
PubMed1996

Interaction of internal Ba2+ with a cloned Ca(2+)-dependent K+ (hslo) channel from smooth muscle.

We have studied potassium currents through a cloned Ca(2+)-dependent K+ channel (hslo) from human myometrium. Currents were recorded in inside-out macropatches from membranes of Xenopus laevis oocytes. In particular, the inactivation-like process that these channels show at high positive potentials was assessed in order to explore its molecular nature. This current inhibition conferred a bell shape to the current-voltage curves. The kinetic and voltage dependence of this process suggested the possibility of a Ba2+ block. There were the following similarities between the inactivation process observed at zero-added Ba2+ and the internal Ba2+ block of hslo channels: (a) in the steady state, the voltage dependence of the current inhibition observed at zero-added Ba2+ was the same as the voltage dependence of the Ba2+ block; (b) the time constant for recovery from current decay at zero-added Ba2+ was the same as the time constant for current recovery from Ba2+ blockade; and (c) current decay was largely suppressed in both cases by adding a Ba2+ chelator [(+)-18-crown-6-tetracarboxylic acid] to the internal solution. In our experimental conditions, we determined that the Kd for the complex chelator-Ba2+ is 1.6 x 10(-10) M. We conclude that the current decay observed at zero-added Ba2+ to the internal solution is due to contaminant Ba2+ present in our solutions (approximately 70 nM) and not to an intrinsic gating process. The Ba2+ blocking reaction in hslo channels is bimolecular. Ba2+ binds to a site (Kd = 0.36 +/- 0.05 mM at zero applied voltage) that senses 92 +/- 25% of the potential drop from the internal membrane surface.

Animals↗
PubMed1996

Effective gating charges per channel in voltage-dependent K+ and Ca2+ channels.

In voltage-dependent ion channels, the gating of the channels is determined by the movement of the voltage sensor. This movement reflects the rearrangement of the protein in response to a voltage stimulus, and it can be thought of as a net displacement of elementary charges (e0) through the membrane (z: effective number of elementary charges). In this paper, we measured z in Shaker IR (inactivation removed) K+ channels, neuronal alpha 1E and alpha 1A, and cardiac alpha 1C Ca2+ channels using two methods: (a) limiting slope analysis of the conductance-voltage relationship and (b) variance analysis, to evaluate the number of active channels in a patch, combined with the measurement of charge movement in the same patch. We found that in Shaker IR K+ channels the two methods agreed with a z congruent to 13. This suggests that all the channels that gate can open and that all the measured charge is coupled to pore opening in a strictly sequential kinetic model. For all Ca2+ channels the limiting slope method gave consistent results regardless of the presence or type of beta subunit tested (z = 8.6). However, as seen with alpha 1E, the variance analysis gave different results depending on the beta subunit used. alpha 1E and alpha 1E beta 1a gave higher z values (z = 14.77 and z = 15.13 respectively) than alpha 1E beta 2a (z = 9.50, which is similar to the limiting slope results). Both the beta 1a and beta 2a subunits, coexpressed with alpha 1E Ca2+ channels facilitated channel opening by shifting the activation curve to more negative potentials, but only the beta 2a subunit increased the maximum open probability. The higher z using variance analysis in alpha 1E and alpha 1E beta 1a can be explained by a set of charges not coupled to pore opening. This set of charges moves in transitions leading to nulls thus not contributing to the ionic current fluctuations but eliciting gating currents. Coexpression of the beta 2a subunit would minimize the fraction of nulls leading to the correct estimation of the number of channels and z.

Animals↗
PubMed1996

Identification of a second region of the beta-subunit involved in regulation of calcium channel inactivation.

Previous studies have shown that NH2 termini of the type 1 and 2 beta-subunits modulate the rate at which the neuronal alpha 1E calcium channel inactivates in response to voltage and that they do so independently of their common effect to stimulate activation by voltage (R. Olcese, N. Qin, T. Schneider, A. Neely, X. Wei, E. Stefani, and L. Birnbaumer, Neuron 13: 1433-1438, 1994). By constructing NH2-terminal deletions of several splice variants of beta-subunits, we have now found differences in the way they affect the rate of alpha 1E inactivation that lead us to identify a second domain that also regulates the rate of voltage-induced inactivation of the Ca2+ channel. This second domain, named segment 3, lies between two regions of high-sequence identity between all known beta-subunits and exists in two lengths (long and short), each encoded in a separate exon. Beta-Subunits with the longer 45- to 53-amino acid version cause the channel to inactivate more slowly than subunits with the shorter 7-amino acid version. As is the case for the NH2 terminus, the segment 3 does not affect the regulation of channel activation by the beta-subunit. In addition, the effect of the NH2-terminal segment prevails over that of the internal segment. This raises the possibility that phosphorylation, other types of posttranslational modification, or interaction with other auxiliary calcium channel subunits may be necessary to unmask the regulatory effect of the internal segment.

Alternative Splicing↗
PubMed1996

Increase in Ca2+ channel expression by deletions at the amino terminus of the cardiac alpha 1C subunit.

The alpha 1 subunit of the cardiac L-type Ca2+ channel (alpha 1C) is one of the many alternatively spliced products of a single gene that is expressed in a number of excitable tissues. Sequence comparison indicates that the amino terminus is a site of significant structural diversity. To explore the role of the amino terminus of alpha 1C in expression and function of Ca2+ channels, we constructed a series of deletion mutants of the rabbit cardiac alpha 1C subunit and expressed them in Xenopus oocytes. Deletions of up to 120 amino acids from the amino terminus increased both ionic and gating currents by 5- to 8-fold. Ca2+ currents induced by these mutants had voltage-dependent activation, inactivation, modulation by beta subunits, and single channel conductance similar to the wild type cardiac alpha 1C (wt alpha 1C). Thus, deletion of a major portion of the amino terminus of alpha 1C did not alter the three dimensional conformation essential for channel function, but enhanced the expression of Ca2+ channels in Xenopus oocytes. A deletion mutant lacking the first 171 amino acids did not yield any measurable current.

Amino Acid Sequence↗
PubMed1995

Amyotrophic lateral sclerosis immunoglobulins increase Ca2+ currents in a motoneuron cell line.

The sporadic form of amyotrophic lateral sclerosis (ALS) is an idiopathic and eventually lethal disorder causing progressive degeneration of cortical and spinal motoneurons. Recent studies have shown that the majority of patients with sporadic ALS have serum antibodies that bind to purified L-type voltage-gated calcium channels and that antibody titer correlates with the rate of disease progression. Furthermore, antibodies purified from ALS patient sera have been found to alter the physiologic function of voltage-gated calcium channels in nonmotoneuron cell types. Using whole-cell patch-clamp techniques, immunoglobulins purified from sera of 5 of 6 patients with sporadic ALS are now shown to increase calcium currents in a hybrid motoneuron cell line, VSC4.1. These calcium currents are blocked by the polyamine funnel-web spider toxin FTX, which has previously been shown to block Ca2+ currents and evoked transmitter release at mammalian motoneuron terminals. These data provide additional evidence linking ALS to an autoimmune process and suggest that antibody-induced increases in calcium entry through voltage-gated calcium channels may occur in motoneurons in this disease, with possible deleterious effects in susceptible neurons.

Adult↗
PubMed1995

Altered muscle calcium channel binding kinetics in autoimmune motoneuron disease.

While skeletal muscle is not apparently affected directly in amyotrophic lateral sclerosis (ALS), immunoglobulin G fractions purified from patients with ALS (ALS IgG) bind dihydropyridine (DHP)-sensitive L-type voltage-gated calcium channel (VGCC) antigen isolated from skeletal muscle in ELISA and Western immunoblot, and alter VGCC function in vitro. To determine whether muscle VGCC properties are altered in ALS, VGCC-enriched subsarcolemmal membrane fractions were prepared from biopsied quadriceps muscle of patients with ALS, with other neurologic diseases, or without apparent muscle disease, and tested for DHP binding with [3H]PN200-110. ALS muscle VGCCs possessed eightfold higher binding affinities for [3H]PN200-110 than did VGCCs from muscle fractions of most other patients, independent of denervation-induced increases in DHP binding site number. Similarly elevated DHP binding affinities were observed in specimens from patients with autoimmune motor neuropathies, suggesting that ALS and immune mediated motoneuron disease share skeletal muscle L-type VGCC alterations.

Amyotrophic Lateral Sclerosis↗
PubMed1995

External barium block of Shaker potassium channels: evidence for two binding sites.

External barium ions inhibit K+ currents of Xenopus oocytes expressing ShH4 delta 6-46, the non-inactivating deletion of the Shaker K+ channel. At the macroscopic level, Ba2+ block comprises both a fast and a slow component. The fast component is less sensitive to Ba2+ (apparent dissociation constant at 0 mV, K(0), approximately 19.1 mM) than the slow component and is also less voltage dependent (apparent electrical distance, delta, approximately 0.14). The slow component (K(0), approximately 9.4 mM, delta approximately 0.25) is relieved by outward K+ current, which suggests that the corresponding binding site resides within the channel conduction pathway. At the single channel level, the fast component of block is evidenced as an apparent reduction in amplitude, suggesting an extremely rapid blocking and unblocking reaction. In contrast, the slow component appears to be associated with long blocked times that are present from the beginning of a depolarizing command. Installation of the slow component is much slower than a diffusion limited process; for example, the blocking time constant (tau) produced by 2 mM Ba2+ is approximately 159 s (holding potential, HP = -90 mV). However, the blocking rate of this slow component is not a linear function of external Ba2+ and tends to saturate at higher concentrations. This is inconsistent with a simple bi-molecular blocking reaction. These features of external Ba2+ block can be accounted for by a simple model of two sequential Ba2+ binding sites, where the deeper of the two sites produces the slow component of block.

Animals↗
PubMed1995

Dual activation of the cardiac Ca2+ channel alpha 1C-subunit and its modulation by the beta-subunit.

Ca2+ channels are heteromultimeric proteins in which the alpha 1-subunit forms the voltage-dependent Ca(2+)-selective ionic channel. We reported recently that coexpression of the beta-subunit with the cardiac alpha 1-subunit (alpha 1C) facilitates channel opening without affecting either the amplitude or the time course of the gating currents (13). Here we present evidence for the existence of two modes of channel opening. Xenopus oocytes expressing the alpha 1C-subunit alone display two modes of activation as indicated by the double-exponential time course of macroscopic ionic currents and the two open-time distributions of single channels. Coexpression of the beta-subunit potentiates Ca2+ currents by a relative increase of the fast-activating component, an acceleration of the slow component, and a larger proportion of long openings. We propose that multiple modes of gating are encoded in the alpha 1-subunit and that the beta-subunit increases Ca2+ channel opening by favoring a willing mode of gating in which the final transitions leading to channel opening are facilitated. In addition, we show that the carboxy terminus of alpha 1C also modulates the channel-gating behavior.

Animals↗
PubMed1995

Relaxin stimulates myometrial calcium-activated potassium channel activity via protein kinase A.

Relaxin, a hormone that is elevated during pregnancy, can suppress myometrial contractile activity. Ca(2+)-activated K+ channels (KCa) play a role in the modulation of uterine contractions and myometrial Ca2+ homeostasis and have been implicated in the control of smooth muscle excitability. We now show that relaxin stimulates KCa channels in cell-attached patches in a cell line derived from term pregnant human myometrium. This effect was prevented by the protein kinase A (PKA) antagonist, the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). After patch excision, the channel was activated by PKA and inhibited by alkaline phosphatase. These data suggest that relaxin may promote myometrial quiescence in part by stimulation of KCa channels via a PKA-mediated mechanism.

Calcium↗
PubMed1995

Characterization of and modulation by a beta-subunit of a human maxi KCa channel cloned from myometrium.

cDNAs encoding functional maxi KCa channel alpha-subunits (hslo) were cloned from human myometrium. Northern blot analysis revealed a high abundance of mRNA in human uterine smooth muscle. Calcium- and voltage-activated K+ currents were recorded from Xenopus laevis oocytes injected with hslo cRNA and compared with currents after reconstitution of oocyte membranes expressing cloned maxi KCa channels. The expressed channels displayed characteristics of native maxi KCa channels, including large conductance (280 pS in symmetrical 110 mM K+), calcium sensitivity, kinetics and pharmacology. Currents were activated by niflumic acid; blocked by tetraethylammonium, charybdotoxin and iberiotoxin; and were insensitive to lemakalim, pinacidil, apamin and 4-aminopyridine. Coexpression with the beta-subunit, cloned from bovine trachea smooth muscle, dramatically increased the apparent calcium sensitivity as evident from a leftward shift of the voltage-activation curves. Half maximal activation (V1/2), measured in 10 microM Ca2+, was 12 +/- 18 mV (+/- SD, n = 62) for the alpha-subunit alone and -87 +/- 10 mV (+/- SD, n = 39) in presence of the beta-subunit.

Animals↗