Requirement for vacuolar H+ -ATPase activity and Ca2+ gradient during entry of rotavirus into MA104 cells

The mechanism by which rotavirus and other nonenveloped viruses enter the cell is still not clear. We have proposed an endocytosis model where the critical step for virus uncoating and membrane permeabilization is the decrease in Ca(2+) concentration in the endosome. In this paper, we monitored rotavirus entry by measuring alpha-sarcin-rotavirus coentry and infectivity in MA104 cells. The participation of endocytosis, acidification, and endosomal Ca(2+) concentration on virus entry was studied by inhibiting the endosomal H(+)-ATPase with bafilomycin A1 and/or increasing the extracellular calcium reservoir by addition of 10 mM CaEGTA. Rotavirus-alpha-sarcin coentry was inhibited by bafilomycin A1 and by addition of 10 mM CaEGTA. These effects were additive. These substances induced a significant inhibition of infectivity without affecting virus binding and postentry steps. These results are compatible with the interpretation that bafilomycin A1 and CaEGTA block rotavirus penetration from the endosome into the cytoplasm and support our hypothesis of a Ca(2+)-dependent endocytosis model.     

Inhibitors of poliovirus uncoating efficiently block the early membrane permeabilization induced by virus particles.

The entry of animal viruses into cells is associated with permeabilization of the infected cells to protein toxins such as alpha-sarcin (C. Fernández-Puentes and L. Carrasco, Cell 20:769-775, 1980). This phenomenon has been referred to as "the early permeabilization by animal viruses" (L. Carrasco, Virology 113:623-629, 1981). A number of inhibitors of poliovirus growth such as WIN 51711 6-(3,4-dichlorophenoxy)-3-(ethylthio)-2-pyridincarbonitrile (DEPC) and Ro 09-0410 specifically block the uncoating step of poliovirus but have no effect on attachment or entry of poliovirus particles into cells. These agents are potent inhibitors of the early permeabilization induced by poliovirus to the toxin alpha-sarcin. Thus, the uncoating of poliovirus is required for the permeabilization of cell membranes to proteins. The increased entry of labeled heparin promoted by virus entry is not blocked by these agents, indicating that poliovirus binds to its receptor and is internalized along with heparin in endosomes in the presence of WIN 51711, DEPC, or Ro 09-0410. We conclude that the delivery to the cytoplasm of some molecules that coenter with virion particles does not take place if the uncoating process is hindered.     

Rotavirus interaction with isolated membrane vesicles.

To gain information about the mechanism of epithelial cell infection by rotavirus, we studied the interaction of bovine rotavirus, RF strain, with isolated membrane vesicles from apical membrane of pig enterocytes. Vesicles were charged with high (quenching) concentrations of either carboxyfluorescein or calcein, and the rate of fluorophore release (dequenching) was monitored as a function of time after mixing with purified virus particles. Purified single-shelled particles and untrypsinized double-shelled ones had no effect. Trypsinized double-shelled virions induced carboxyfluorescein release according to sigmoid curves whose lag period and amplitude were a function of virus concentration and depended on both temperature and pH. The presence of 100 mM salts (Tris Cl, NaCl, or KCl) was required, since there was no reaction in isoosmotic salt-free sorbitol media. Other membrane vesicle preparations such as apical membranes of piglet enterocyte and rat placenta syncytiotrophoblasts, basolateral membranes of pig enterocytes, and the undifferentiated plasma membrane of cultured MA104 cells all gave qualitatively similar responses. Inhibition by a specific monoclonal antibody suggests that the active species causing carboxyfluorescein release is VP5*. Ca2+ (1 mM), but not Mg2+, inhibited the reaction. In situ solubilization of the outer capsid of trypsinized double-shelled particles changed release kinetics from sigmoidal to hyperbolic and was not inhibited by Ca2+. Our results indicate that membrane destabilization caused by trypsinized outer capsid proteins of rotavirus leads to fluorophore release. From the data presented here, a hypothetical model of the interaction of the various states of the viral particles with the membrane lipid phase is proposed. Membrane permeabilization induced by rotavirus may be related to the mechanism of entry of the virus into the host cell.     

Cross-linking of rotavirus outer capsid protein VP7 by antibodies or disulfides inhibits viral entry

Antibodies that neutralize rotavirus infection target outer coat proteins VP4 and VP7 and inhibit viral entry. The structure of a VP7-Fab complex (S. T. Aoki, et al., Science 324:1444-1447, 2009) led us to reclassify epitopes into two binding regions at inter- and intrasubunit boundaries of the calcium-dependent trimer. It further led us to show that antibodies binding at the intersubunit boundary inhibit uncoating of the virion outer layer. We have now tested representative antibodies for each of the defined structural epitope regions and find that antibodies recognizing epitopes in either binding region neutralize by cross-linking VP7 trimers. Antibodies that bind at the intersubunit junction neutralize as monovalent Fabs, while those that bind at the intrasubunit region require divalency. The VP7 structure has also allowed us to design a disulfide cross-linked VP7 mutant which recoats double-layered particles (DLPs) as efficiently as does wild-type VP7 but which yields particles defective in cell entry as determined both by lack of infectivity and by loss of α-sarcin toxicity in the presence of recoated particles. We conclude that dissociation of the VP7 trimer is an essential step in viral penetration into cells.     

Assembly of highly infectious rotavirus particles recoated with recombinant outer capsid proteins

Assembly of the rotavirus outer capsid is the final step of a complex pathway. In vivo, the later steps include a maturational membrane penetration that is dependent on the scaffolding activity of a viral nonstructural protein. In vitro, simply adding the recombinant outer capsid proteins VP4 and VP7 to authentic double-layered rotavirus subviral particles (DLPs) in the presence of calcium and acidic pH increases infectivity by a factor of up to 10(7), yielding particles as infectious as authentic purified virions. VP4 must be added before VP7 for high-level infectivity. Steep dependence of infectious recoating on VP4 concentration suggests that VP4-VP4 interactions, probably oligomerization, precede VP4 binding to particles. Trypsin sensitivity analysis identifies two populations of VP4 associated with recoated particles: properly mounted VP4 that can be specifically primed by trypsin, and nonspecifically associated VP4 that is degraded by trypsin. A full complement of properly assembled VP4 is not required for efficient infectivity. Minimal dependence of recoating on VP7 concentration suggests that VP7 binds DLPs with high affinity. The parameters for efficient recoating and the characterization of recoated particles suggest a model in which, after a relatively weak interaction between oligomeric VP4 and DLPs, VP7 binds the particles and locks VP4 in place. Recoating will allow the use of infectious modified rotavirus particles to explore rotavirus assembly and cell entry and could lead to practical applications in novel immunization strategies.     

Rhesus rotavirus entry into a polarized epithelium is endocytosis dependent and involves sequential VP4 conformational changes

Rotavirus (RV) cell entry is an incompletely understood process, involving VP4 and VP7, the viral proteins composing the outermost layer of the nonenveloped RV triple-layered icosahedral particle (TLP), encasing VP6. VP4 can exist in three conformational states: soluble, cleaved spike, and folded back. In order to better understand the events leading to RV entry, we established a detection system to image input virus by monitoring the rhesus RV (RRV) antigens VP4, VP6, and VP7 at very early times postinfection. We provide evidence that decapsidation occurs directly after cell membrane penetration. We also demonstrate that several VP4 and VP7 conformational changes take place during entry. In particular, we detected, for the first time, the generation of folded-back VP5 in the context of the initiation of infection. Folded-back VP5 appears to be limited to the entry step. We furthermore demonstrate that RRV enters the cell cytoplasm through an endocytosis pathway. The endocytosis hypothesis is supported by the colocalization of RRV antigens with the early endosome markers Rab4 and Rab5. Finally, we provide evidence that the entry process is likely dependent on the endocytic Ca(2+) concentration, as bafilomycin A1 treatment as well as an augmentation of the extracellular calcium reservoir using CaEGTA, which both lead to an elevated intraendosomal calcium concentration, resulted in the accumulation of intact virions in the actin network. Together, these findings suggest that internalization, decapsidation, and cell membrane penetration involve endocytosis, calcium-dependent uncoating, and VP4 conformational changes, including a fold-back.     

Effects of methotrexate on calcium flux in rat liver mitochondria, microsomes and plasma membrane vesicles

The metabolic effects of methotrexate in perfused livers are similar to those exerted by hormones acting through Ca(2+)-dependent mechanisms. The aim of the present study was to determine whether the effects of methotrexate are mediated by a direct action on cellular Ca(2+) fluxes. Methotrexate did not affect the ATP-dependent (45)Ca(2+) uptake by mitochondria, microsomes and inside-out plasma membrane vesicles and Ca(2+) efflux from plasma membrane vesicles. However, methotrexate was able to stimulate (45)Ca(2+) release from preloaded microsomes. The amount of Ca(2+) released by methotrexate was similar to that induced by IP(3). Methotrexate could be acting through the capacitative calcium entry mechanism.     

Ion channels in smooth muscle: regulation by the sarcoplasmic reticulum and mitochondria

In smooth muscle, Ca(2+) regulates cell division, growth and cell death as well as providing the main trigger for contraction. Ion channels provide the major access route to elevate the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) in smooth muscle by permitting Ca(2+) entry across the plasma membrane and release of the ion from intracellular Ca(2+) stores. The control of [Ca(2+)](c) relies on feedback modulation of the entry and release channels by Ca(2+) itself. Local rises in [Ca(2+)](c) may promote or inhibit channel activity directly or indirectly. The latter may arise from Ca(2+) regulation of ionic conductances in the plasma membrane to provide control of cell excitability and so [Ca(2+)](c) entry. Organelles such as mitochondria may also contribute significantly to the feedback regulation of ion channel activity by the control of Ca(2+) or redox status of the cell. This brief review describes the feedback regulation of Ca(2+) release from the internal Ca(2+) store and of plasma membrane excitability in smooth muscle.     

Physical coupling supports the local Ca2+ transfer between sarcoplasmic reticulum subdomains and the mitochondria in heart muscle

In many cell types, transfer of Ca(2+) released via ryanodine receptors (RyR) to the mitochondrial matrix is locally supported by high [Ca(2+)] microdomains at close contacts between the sarcoplasmic reticulum (SR) and mitochondria. Here we studied whether the close contacts were secured via direct physical coupling in cardiac muscle using isolated rat heart mitochondria (RHMs). "Immuno-organelle chemistry" revealed RyR2 and calsequestrin-positive SR particles associated with mitochondria in both crude and Percoll-purified "heavy" mitochondrial fractions (cRHM and pRHM), to a smaller extent in the latter one. Mitochondria-associated vesicles were also visualized by electron microscopy in the RHMs. Western blot analysis detected greatly reduced presence of SR markers (calsequestrin, SERCA2a, and phospholamban) in pRHM, suggesting that the mitochondria-associated particles represented a small subfraction of the SR. Fluorescence calcium imaging in rhod2-loaded cRHM revealed mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) responses to caffeine-induced Ca(2+) release that were prevented when thapsigargin was added to predeplete the SR or by mitochondrial Ca(2+) uptake inhibitors. Importantly, caffeine failed to increase [Ca(2+)] in the large volume of the incubation medium, suggesting that local Ca(2+) transfer between the SR particles and mitochondria mediated the [Ca(2+)](m) signal. Despite the substantially reduced SR presence, pRHM still displayed a caffeine-induced [Ca(2+)](m) rise comparable with the one recorded in cRHM. Thus, a relatively small fraction of the total SR is physically coupled and transfers Ca(2+) locally to the mitochondria in cardiac muscle. The transferred Ca(2+) stimulates dehydrogenase activity and affects mitochondrial membrane permeabilization, indicating the broad significance of the physical coupling in mitochondrial function.     

Proteolysis of Monomeric Recombinant Rotavirus VP4 Yields an Oligomeric VP5* Core

Rotavirus particles are activated for cell entry by trypsin cleavage of the outer capsid spike protein, VP4, into a hemagglutinin, VP8*, and a membrane penetration protein, VP5*. We have purified rhesus rotavirus VP4, expressed in baculovirus-infected insect cells. Purified VP4 is a soluble, elongated monomer, as determined by analytical ultracentrifugation. Trypsin cleaves purified VP4 at a number of sites that are protected on the virion and yields a heterogeneous group of protease-resistant cores of VP5*. The most abundant tryptic VP5* core is trimmed past the N terminus associated with activation for virus entry into cells. Sequential digestion of purified VP4 with chymotrypsin and trypsin generates homogeneous VP8* and VP5* cores (VP8CT and VP5CT, respectively), which have the authentic trypsin cleavages in the activation region. VP8CT is a soluble monomer composed primarily of beta-sheets. VP5CT forms sodium dodecyl sulfate-resistant dimers. These results suggest that trypsinization of rotavirus particles triggers a rearrangement in the VP5* region of VP4 to yield the dimeric spikes observed in icosahedral image reconstructions from electron cryomicroscopy of trypsinized rotavirus virions. The solubility of VP5CT and of trypsinized rotavirus particles suggests that the trypsin-triggered conformational change primes VP4 for a subsequent rearrangement that accomplishes membrane penetration. The domains of VP4 defined by protease analysis contain all mapped neutralizing epitopes, sialic acid binding residues, the heptad repeat region, and the membrane permeabilization region. This biochemical analysis of VP4 provides sequence-specific structural information that complements electron cryomicroscopy data and defines targets and strategies for atomic-resolution structural studies.     

Regulation of adenovirus membrane penetration by the cytoplasmic tail of integrin beta5

Adenovirus (Ad) cell entry involves sequential interactions with host cell receptors that mediate attachment (CAR), internalization (alphavbeta3 and alphavbeta5), and penetration (alphavbeta5) of the endosomal membrane. These events allow the virus to deliver its genome to the nucleus. While integrins alphavbeta3 and alphavbeta5 both promote Ad internalization into cells, integrin alphavbeta5 selectively facilitates Ad-mediated membrane permeabilization and endosome rupture. In the experiments reported herein, we demonstrate that the intracellular domain of the integrin beta5 subunit specifically regulates Ad-mediated membrane permeabilization and gene delivery. CS-1 melanoma cells expressing a truncated integrin beta5 or a chimeric (beta5-beta3) cytoplasmic tail (CT) supported normal levels of Ad endocytosis but had reduced Ad-mediated gene delivery and membrane permeabilization relative to cells expressing a wild-type integrin beta5. Thin-section electron microscopy revealed that virion particles were capable of being endocytosed into cells expressing a truncated beta5CT, but they failed to escape cytoplasmic vesicles and translocate to the nucleus. Site-specific mutagenesis studies suggest that a C-terminal TVD motif in the beta5CT plays a major role in Ad membrane penetration.     

Localization and functions of native and eGFP-tagged capsid proteins in HIV-1 particles

In infectious HIV-1 particles, the capsid protein (CA) forms a cone-shaped shell called the capsid, which encases the viral ribonucleoprotein complex (vRNP). Following cellular entry, the capsid is disassembled through a poorly understood process referred to as uncoating, which is required to release the reverse transcribed HIV-1 genome for integration into host chromatin. Whereas single virus imaging using indirect CA labeling techniques suggested uncoating to occur in the cytoplasm or at the nuclear pore, a recent study using eGFP-tagged CA reported uncoating in the nucleus. To delineate the HIV-1 uncoating site, we investigated the mechanism of eGFP-tagged CA incorporation into capsids and the utility of this fluorescent marker for visualizing HIV-1 uncoating. We find that virion incorporated eGFP-tagged CA is effectively excluded from the capsid shell, and that a subset of the tagged CA is vRNP associated. These results thus imply that eGFP-tagged CA is not a direct marker for capsid uncoating. We further show that native CA co-immunoprecipitates with vRNP components, providing a basis for retention of eGFP-tagged and untagged CA by sub-viral complexes in the nucleus. Moreover, we find that functional viral replication complexes become accessible to integrase-interacting host factors at the nuclear pore, leading to inhibition of infection and demonstrating capsid permeabilization prior to nuclear import. Finally, we find that HIV-1 cores containing a mixture of wild-type and mutant CA interact differently with cytoplasmic versus nuclear pools of the CA-binding host cofactor CPSF6. Our results suggest that capsid remodeling (including a loss of capsid integrity) is the predominant pathway for HIV-1 nuclear entry and provide new insights into the mechanism of CA retention in the nucleus via interaction with vRNP components.     

Orientation of synaptic plasma membrane vesicles containing calcium pump and sodium-calcium exchange activities

Sidedness of synaptic plasma membrane vesicles isolated from brain synaptosomes has been assessed by two distinct experimental approaches: first, analysis of (Na+ + K+)-ATPase, Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities before and after permeabilization of vesicles; second, analysis of Ca2+ fluxes via the Na+/Ca2+ exchanger, before and after modification of an imposed Na+ gradient by penetrating or nonpenetrating Na+ channel-modifying drugs. 0.05% saponin, which completely permeabilizes the vesicles, increases digitoxigenin-sensitive (Na+ + K+)-ATPase, basal Mg2+-ATPase, and (Ca2+ + Mg2+)-ATPase activities by 51.0, 47.4, and 83.6%, respectively. Saponin increases only the Vmax of the latter activity, the Km for Ca2+ (0.13 microM; the same as that for Ca2+-pumping) being unaltered by saponin. An increment of 20.5% in the Vmax of (Ca2+ + Mg2+)-ATPase activity with 10 microM A23187, reveals that the enzyme activity in nonpermeabilized vesicles is limited by the formation of a Ca2+ gradient. Thus, the saponin-induced increment in (Ca2+ + Mg2+)-ATPase due only to exposure of occluded sites (as opposed to Ca2+ gradient dissipation) is actually 52%, which is similar to values for both other ATPases, and suggests that 32-35% of plasma membranes exist in an inverted orientation. Vesicle orientation was independently assessed by the differential actions of tetrodotoxin (a membrane impermeant blocker) and veratridine (a membrane permeant agonist) on Na+-channel opening measured indirectly by dissipation of an imposed Na+ gradient utilized to drive a large 45Ca2+ accumulation via the Na+/Ca2+ exchanger. Tetrodotoxin reverses 35-44% of veratridine-mediated Na+ gradient-dissipation, the relative membrane-permeability of the two channel modifiers, suggesting that 56-65% of sealed vesicles are inverted. The concurrence of these two independent measurements of vesicle orientation reinforces their validity.     

Ca(2+) sequestering in the early-branching amitochondriate protozoan Tritrichomonas foetus: an important role of the Golgi complex and its Ca(2+)-ATPase

Total membrane vesicles isolated from Tritrichomonas foetus showed an ATP-dependent Ca(2+) uptake, which was not sensitive to 10 microM protonophore FCCP but was blocked by orthovanadate, the inhibitor of P-type ATPases (I(50)=130 microM), and by the Ca(2+)/H(+) exchanger, A-23187. The Ca(2+) uptake was prevented also by thapsigargin, an inhibitor of the SERCA Ca(2+)-ATPases. The sensitivity of the Ca(2+) uptake by the protozoan membrane vesicles to thapsigargin was similar to that of Ca(2+)-ATPase from rabbit muscle sarcoplasmic reticulum. Fractionation of the total membrane vesicles in sucrose density gradient revealed a considerable peak of Ca(2+) transport activity that co-migrated with the Golgi marker guanosine diphosphatase (GDPase). Electron microscopy confirmed that membrane fractions of the peak were enriched with the Golgi membranes. The Golgi Ca(2+)-ATPase contributed to the Ca(2+) uptake by all membrane vesicles 80-85%. We conclude that: (i) the Golgi and/or Golgi-like vesicles form the main Ca(2+) store compartment in T. foetus; (ii) Ca(2+) ATPase is responsible for the Ca(2+) sequestering in this protozoan, while Ca(2+)/H(+) antiporter is not involved in the process; (iii) the Golgi pump of this ancient eukaryotic microorganism appears to be similar to the enzymes of the SERCA family by its sensitivity to thapsigargin.     

Invited review: mechanisms of calcium handling in smooth muscles.

The concentration of cytoplasmic Ca(2+) regulates the contractile state of smooth muscle cells and tissues. Elevations in global cytoplasmic Ca(2+) resulting in contraction are accomplished by Ca(2+) entry and release from intracellular stores. Pathways for Ca(2+) entry include dihydropyridine-sensitive and -insensitive Ca(2+) channels and receptor and store-operated nonselective channels permeable to Ca(2+). Intracellular release from the sarcoplasmic reticulum (SR) is accomplished by ryanodine and inositol trisphosphate receptors. The impact of Ca(2+) entry and release on cytoplasmic concentration is modulated by Ca(2+) reuptake into the SR, uptake into mitochondria, and extrusion into the extracellular solution. Highly localized Ca(2+) transients (i.e., sparks and puffs) regulate ionic conductances in the plasma membrane, which can provide feedback to cell excitability and affect Ca(2+) entry. This short review describes the major transport mechanisms and compartments that are utilized for Ca(2+) handling in smooth muscles.     

Ca<Superscript>2+</Superscript>-Induced Phase Separation in the Membrane of Palmitate-Containing Liposomes and Its Possible Relation to Membrane Permeabilization

A Ca(2+)-induced phase separation of palmitic acid (PA) in the membrane of azolectin unilamellar liposomes has been demonstrated with the fluorescent membrane probe nonyl acridine orange (NAO). It has been shown that NAO, whose fluorescence in liposomal membranes is quenched in a concentration-dependent way, can be used to monitor changes in the volume of lipid phase. The incorporation of PA into NAO-labeled liposomes increased fluorescence corresponding to the expansion of membrane. After subsequent addition of Ca(2+), fluorescence decreased, which indicated separation of PA/Ca(2+) complexes into distinct membrane domains. The Ca(2+)-induced phase separation of PA was further studied in relation to membrane permeabilization caused by Ca(2+) in the PA-containing liposomes. A supposition was made that the mechanism of PA/Ca(2+)-induced membrane permeabilization relates to the initial stage of Ca(2+)-induced phase separation of PA and can be considered as formation of fast-tightening lipid pores due to chemotropic phase transition in the lipid bilayer.     

Characterization of the ion channels formed by poliovirus in planar lipid membranes.

The steps in poliovirus infection leading to viral entry and uncoating are not well understood. Current evidence suggests that the virus first binds to a plasma membrane-bound receptor present in viable cells, leading to a conformational rearrangement of the viral proteins such that the virus crosses the membrane and releases the genomic RNA. The studies described in this report were undertaken to determine if poliovirus (160S) as well as one of the subviral particles (135S) could interact with membranes lacking poliovirus receptors in an effort to begin to understand the process of uncoating of the virus. We report that both forms of viral particles, 160S and 135S, interact with lipid membranes and induce the formation of ion-permeable channels in a manner that does not require acid pH. The channels induced by the viral particles 160S have a voltage-dependent conductance which depends on the ionic composition of the medium. Our findings raise the possibility that viral entry into cells may be mediated by direct interaction of viral surface proteins with membrane lipids.     

A role for the actin cytoskeleton in the initiation and maintenance of store-mediated calcium entry in human platelets. Evidence for conformational coupling

The nature of the mechanism underlying store-mediated Ca(2+) entry has been investigated in human platelets through a combination of cytoskeletal modifications. Inhibition of actin polymerization by cytochalasin D or latrunculin A had a biphasic time-dependent effect on Ca(2+) entry, showing an initial potentiation followed by inhibition of Ca(2+) entry. Moreover, addition of these agents after induction of store-mediated Ca(2+) entry inhibited the Ca(2+) influx mechanism. Jasplakinolide, which reorganizes actin filaments into a tight cortical layer adjacent to the plasma membrane, prevented activation of store-mediated Ca(2+) entry but did not modify this process after its activation. In addition, jasplakinolide prevented cytochalasin D-induced inhibition of store-mediated Ca(2+) entry. Calyculin A, an inhibitor of protein serine/threonine phosphatases 1 and 2 which activates translocation of existing F-actin to the cell periphery without inducing actin polymerization, also prevented activation of store-mediated Ca(2+) entry. Finally, inhibition of vesicular transport with brefeldin A inhibited activation of store-mediated Ca(2+) entry but did not alter this mechanism once initiated. These data suggest that store-mediated Ca(2+) entry in platelets may be mediated by a reversible trafficking and coupling of the endoplasmic reticulum with the plasma membrane, which shows close parallels to the events mediating secretion.     

Amplitude-dependent spike-broadening and enhanced Ca(2+) signaling in GnRH-secreting neurons

In GnRH-secreting (GT1) neurons, activation of Ca(2+)-mobilizing receptors induces a sustained membrane depolarization that shifts the profile of the action potential (AP) waveform from sharp, high-amplitude to broad, low-amplitude spikes. Here we characterize this shift in the firing pattern and its impact on Ca(2+) influx experimentally by using prerecorded sharp and broad APs as the voltage-clamp command pulse. As a quantitative test of the experimental data, a mathematical model based on the membrane and ionic current properties of GT1 neurons was also used. Both experimental and modeling results indicated that inactivation of the tetrodotoxin-sensitive Na(+) channels by sustained depolarization accounted for a reduction in the amplitude of the spike upstroke. The ensuing decrease in tetraethylammonium-sensitive K(+) current activation slowed membrane repolarization, leading to AP broadening. This change in firing pattern increased the total L-type Ca(2+) current and facilitated AP-driven Ca(2+) entry. The leftward shift in the current-voltage relation of the L-type Ca(2+) channels expressed in GT1 cells allowed the depolarization-induced AP broadening to facilitate Ca(2+) entry despite a decrease in spike amplitude. Thus the gating properties of the L-type Ca(2+) channels expressed in GT1 neurons are suitable for promoting AP-driven Ca(2+) influx in receptor- and non-receptor-depolarized cells.