Effects of rapid antigen degradation and VEE glycoprotein specificity on immune responses induced by a VEE replicon vaccine.

Genetic vaccines are engineered to produce immunogens de novo in the cells of the host for stimulation of a protective immune response. In some of these systems, antigens engineered for rapid degradation have produced an enhanced cellular immune response by more efficient entry into pathways for processing and presentation of MHC class I peptides. VEE replicon particles (VRP), single cycle vaccine vectors derived from Venezuelan equine encephalitis virus (VEE), are examined here for the effect of an increased rate of immunogen degradation on VRP vaccine efficacy. VRP expressing the matrix capsid (MA/CA) portion of SIV Gag were altered to promote rapid degradation of MA/CA by various linkages to co-translated ubiquitin or by destabilizing mutations and were used to immunize BALB/c mice for quantitation of anti-MA/CA cellular and humoral immune responses. Rapid degradation by the N-end rule correlated with a dampened immune response relative to unmodified MA/CA when the VRP carried a glycoprotein spike from an attenuated strain of VEE. In contrast, statistically equivalent numbers of IFNgamma(+)T-cells resulted when VRP expressing unstable MA/CA were packaged with the wild-type VEE glycoproteins. These results suggest that the cell types targeted in vivo by VRP carrying mutant or wild type glycoprotein spikes are functionally different, and are consistent with previous findings suggesting that wild-type VEE glycoproteins preferentially target professional antigen presenting cells that use peptides generated from the degraded antigen for direct presentation on MHC.

Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release.

Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.

Anion transport in heart.

Anion transport proteins in mammalian cells participate in a wide variety of cell and intracellular organelle functions, including regulation of electrical activity, pH, volume, and the transport of osmolites and metabolites, and may even play a role in the control of immunological responses, cell migration, cell proliferation, and differentiation. Although significant progress over the past decade has been achieved in understanding electrogenic and electroneutral anion transport proteins in sarcolemmal and intracellular membranes, information on the molecular nature and physiological significance of many of these proteins, especially in the heart, is incomplete. Functional and molecular studies presently suggest that four primary types of sarcolemmal anion channels are expressed in cardiac cells: channels regulated by protein kinase A (PKA), protein kinase C, and purinergic receptors (I(Cl.PKA)); channels regulated by changes in cell volume (I(Cl.vol)); channels activated by intracellular Ca(2+) (I(Cl.Ca)); and inwardly rectifying anion channels (I(Cl.ir)). In most animal species, I(Cl.PKA) is due to expression of a cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator Cl(-) channel. New molecular candidates responsible for I(Cl.vol), I(Cl.Ca), and I(Cl.ir) (ClC-3, CLCA1, and ClC-2, respectively) have recently been identified and are presently being evaluated. Two isoforms of the band 3 anion exchange protein, originally characterized in erythrocytes, are responsible for Cl(-)/HCO(3)(-) exchange, and at least two members of a large vertebrate family of electroneutral cotransporters (ENCC1 and ENCC3) are responsible for Na(+)-dependent Cl(-) cotransport in heart. A 223-amino acid protein in the outer mitochondrial membrane of most eukaryotic cells comprises a voltage-dependent anion channel. The molecular entities responsible for other types of electroneutral anion exchange or Cl(-) conductances in intracellular membranes of the sarcoplasmic reticulum or nucleus are unknown. Evidence of cardiac expression of up to five additional members of the ClC gene family suggest a rich new variety of molecular candidates that may underlie existing or novel Cl(-) channel subtypes in sarcolemmal and intracellular membranes. The application of modern molecular biological and genetic approaches to the study of anion transport proteins during the next decade holds exciting promise for eventually revealing the actual physiological, pathophysiological, and clinical significance of these unique transport processes in cardiac and other mammalian cells.

Patterned library analysis: a method for the quantitative assessment of hypotheses concerning the determinants of protein structure.

Site-directed mutagenesis and combinatorial libraries are powerful tools for providing information about the relationship between protein sequence and structure. Here we report two extensions that expand the utility of combinatorial mutagenesis for the quantitative assessment of hypotheses about the determinants of protein structure. First, we show that resin-splitting technology, which allows the construction of arbitrarily complex libraries of degenerate oligonucleotides, can be used to construct more complex protein libraries for hypothesis testing than can be constructed from oligonucleotides limited to degenerate codons. Second, using eglin c as a model protein, we show that regression analysis of activity scores from library data can be used to assess the relative contributions to the specific activity of the amino acids that were varied in the library. The regression parameters derived from the analysis of a 455-member sample from a library wherein four solvent-exposed sites in an alpha-helix can contain any of nine different amino acids are highly correlated (P < 0.0001, R(2) = 0. 97) to the relative helix propensities for those amino acids, as estimated by a variety of biophysical and computational techniques.

Relationship between L-type Ca2+ current and unitary sarcoplasmic reticulum Ca2+ release events in rat ventricular myocytes.

1. The time courses of Ca2+ current and Ca2+ spark occurrence were determined in single rat ventricular myocytes voltage clamped with patch pipettes containing 0.1 microM fluo-3. Acquisition of line-scan images on a laser scanning confocal microscope was synchronized with measurement of Cd2+-sensitive Ca2+ currents. In most cells, individual Ca2+ sparks were observed by reducing Ca2+ current density with nifedipine (0.1-8 microM). 2. Ca2+ sparks elicited by depolarizing voltage-clamp pulses had a peak [Ca2+] amplitude of 289 +/- 3 nM with a decay half-time of 20.8 +/- 0.2 ms and a full width at half-maximum of 1.40 +/- 0.03 microm (mean +/- s. e.m., n = 345), independent of the membrane potential. 3. The time between the beginning of a depolarization and the initiation of each Ca2+ spark was calculated and data were pooled to construct waiting time histograms. Exponential functions were fitted to these histograms and to the decaying phase of the Ca2+ current. This analysis showed that the time constants describing Ca2+ current and Ca2+ spark occurrence at membrane potentials between -30 mV and +30 mV were not significantly different. At +50 mV, in the absence of nifedipine, the time constant describing Ca2+ spark occurrence was significantly larger than the time constant of the Ca2+ current. 4. A simple model is developed using Poisson statistics to relate macroscopic Ca2+ current to the opening of single L-type Ca2+ channels at the dyad junction and to the time course of Ca2+ spark occurrence. The model suggests that the time courses of macroscopic Ca2+ current and Ca2+ spark occurrence should be closely related when opening of a single L-type Ca2+ channel initiates a Ca2+ spark. By comparison with the data, the model suggests that Ca2+ sparks are initiated by the opening of a single L-type Ca2+ channel at all membrane potentials encountered during an action potential.

Regulation of recombinant cardiac cystic fibrosis transmembrane conductance regulator chloride channels by protein kinase C.

We investigated the regulation of cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by protein kinase C (PKC) in Xenopus oocytes injected with cRNA encoding the cardiac (exon 5-) CFTR Cl- channel isoform. Membrane currents were recorded using a two-electrode voltage clamp technique. Activators of PKC or a cAMP cocktail elicited robust time-independent Cl- currents in cardiac CFTR-injected oocytes, but not in control water-injected oocytes. The effects of costimulation of both pathways were additive; however, maximum protein kinase A (PKA) activation occluded further activation by PKC. In oocytes expressing either the cardiac (exon 5-) or epithelial (exon 5+) CFTR isoform, Cl- currents activated by PKA were sustained, whereas PKC-activated currents were transient, with initial activation followed by slow current decay in the continued presence of phorbol esters, the latter effect likely due to down-regulation of endogenous PKC activity. The specific PKA inhibitor, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), and various protein phosphatase inhibitors were used to determine whether the stimulatory effects of PKC are dependent upon the PKA phosphorylation state of cardiac CFTR channels. Intraoocyte injection of 1,2-bis(2-aminophenoxy)ethane-N,N, N,N-tetraacetic acid (BAPTA) or pretreatment of oocytes with BAPTA-acetoxymethyl-ester (BAPTA-AM) nearly completely prevented dephosphorylation of CFTR currents activated by cAMP, an effect consistent with inhibition of protein phosphatase 2C (PP2C) by chelation of intracellular Mg2+. PKC-induced stimulation of CFTR channels was prevented by inhibition of basal endogenous PKA activity, and phorbol esters failed to stimulate CFTR channels trapped into either the partially PKA phosphorylated (P1) or the fully PKA phosphorylated (P1P2) channel states. Site-directed mutagenesis of serines (S686 and S790) within two consensus PKC phosphorylation sites on the cardiac CFTR regulatory domain attentuated, but did not eliminate, the stimulatory effects of phorbol esters on mutant CFTR channels. The effects of PKC on cardiac CFTR Cl- channels are consistent with a simple model in which PKC phosphorylation of the R domain facilitates PKA-induced transitions from dephosphorylated (D) to partially (P1) phosphorylated and fully (P1P2) phosphorylated channel states.

Cystic fibrosis gene encodes a cAMP-dependent chloride channel in heart.

cAMP-dependent chloride channels in heart contribute to autonomic regulation of action potential duration and membrane potential and have been inferred to be due to cardiac expression of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. In this report, a cDNA from rabbit ventricle was isolated and sequenced, which encodes an exon 5 splice variant (exon 5-) of CFTR, with >90% identity to human CFTR cDNA present in epithelial cells. Expression of this cDNA in Xenopus oocytes gave rise to robust cAMP-activated chloride currents that were absent in control water-injected oocytes. Antisense oligodeoxynucleotides directed against CFTR significantly reduced the density of cAMP-dependent chloride currents in acutely cultured myocytes, thereby establishing a direct functional link between cardiac expression of CFTR protein and an endogenous chloride channel in native cardiac myocytes.

Unitary Cl- channels activated by cytoplasmic Ca2+ in canine ventricular myocytes.

Recent whole-cell studies have shown that Ca(2+)-activated Cl- currents contribute to the Ca(2+)-dependent 4-aminopyridine-insensitive component of the transient outward current and to the arrhythmogenic transient inward current in rabbit and canine cardiac cells. These Cl(-)-sensitive currents are activated by Ca2+ release from the sarcoplasmic reticulum and are inhibited by anion transport blockers; however, the unitary single channels responsible have yet to be identified. We used inside-out patches from canine ventricular myocytes and conditions under which the only likely permeant ion is Cl- to identify 4-aminopyridine-resistant unitary Ca(2+)-activated Cl- channels, Ca2+ applied to the cytoplasmic surface of membrane patches activated small-conductance (1.0 to 1.3 pS) channels. These channels were Cl- selective, with rectification properties that could be described by the Goldman-Hodgkin-Katz current equation. Channel activity exhibited time independence when cytoplasmic Ca2+ was held constant and was blocked by the anion transport blockers, DIDS and niflumic acid. Ca2+ (ranging from pCa > or = 6 to pCa 3) applied to the cytoplasmic surface of inside-out patches increased, in a dose-dependent manner, NPo, where N is the number of channels opened and Po is open probability. At negative membrane potentials (-60 to -130 mV), an estimate of the dependence of NPo on cytoplasmic Ca2+ yielded an apparent Kd of 150.2 mumol/L. At pCa 3, an average channel density of approximately equal to 3 microns-2 was estimated. Calculations based on these estimates of cytoplasmic Ca2+ sensitivity and channel current amplitude and density suggest that these small-conductance Cl- channels contribute significant whole-cell membrane current in response to changes in intracellular Ca2+ within the physiological range. We suggest that these small-conductance Ca(2+)-activated Cl- channels underlie the transient Ca(2+)-activated 4-aminopyridine-insensitive current, which contributes to phase-1 repolarization, and under conditions of Ca2+ overload, these channels may generate transient inward currents, contributing to the development of triggered cardiac arrhythmias.

CFTR chloride channels in human and simian heart.

OBJECTIVES: The cAMP-dependent Cl- conductance in heart is believed to be due to cardiac expression of the cystic fibrosis transmembrane conductance regulator (CFTR). While CFTR expressed in rabbit and guinea-pig heart (CFTRcardiac) is an alternatively spliced isoform of the epithelial gene product, little information is known regarding possible expression of CFTR in primate heart. In this study, we examined molecular expression of CFTR in human and simian atrium and ventricle and functional expression of cAMP-dependent Cl- currents in isolated human atrial and simian ventricular cells. METHODS: The reverse transcription polymerase chain reaction (RT-PCR) was performed on human and simian atrial and ventricular mRNA using primers designed to border regions of the CFTR gene product corresponding to transmembrane segments I-VI (TSI-VI), the first nucleotide binding domain (NBD1), transmembrane segments VII-XII (TSVII-XII), and the large cytoplasmic domain which includes the regulatory (R) domain and NBD1. Functional expression of CFTR Cl- channels in human atrial and simian ventricular myocytes was determined using whole-cell and giant inside-out patch-clamp techniques. RESULTS: Southern blot analysis of these RT-PCR products demonstrated expression of CFTR transcripts in human and simian atrial and ventricular tissue and revealed a novel pattern of expression compared to most animal species studies: both the exon 5 plus (unspliced) and exon 5 minus (spliced) CFTR transcripts are co-expressed in human and simian atrium and ventricle. Whole-cell experiments demonstrated a Cl- sensitive time-independent background conductance in both human atrial and simian ventricular myocytes that was activated by forskolin (FSK) and insensitive to 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). In inside-out patches utilizing the giant patch technique on human atrial myocytes, unitary Cl- sensitive channels resembling CFTR Cl- channels (approximately 14 pS conductance) were activated by the catalytic subunit of protein kinase A (PKA) in 3/12 patches examined. CONCLUSIONS: These results clearly demonstrate the molecular expression of CFTR Cl- channels and provide electrophysiological evidence consistent with functional expression of these channels in human atrial and simian ventricular myocardium.

Unitary chloride channels activated by protein kinase C in guinea pig ventricular myocytes.

Recent evidence suggests that protein kinase A (PKA)-activated Cl- channels in heart are encoded by an isoform of the epithelial cystic fibrosis transmembrane conductance regulator gene (CFTR). Macroscopic current measurements indicate that a similar time-independent Cl- conductance can be activated through a protein kinase C (PKC)-dependent pathway in guinea pig and feline ventricle. However, it is presently not clear whether PKC is activating the same population of channels as PKA or a separate class of Cl- channels. even though the regulatory (R) domain of CFTR is known to contain consensus phosphorylation sites for both PKA and PKC. In the present study we directly compare the properties of single Cl- channels activated by PKC and PKA in cell-attached patches of guinea pig ventricular myocytes. Pipette and bath solutions contained N-methyl-D-glucamine and Cs+ or tetraethylammonium as substitutes for Na+ and K+, respectively, and Cl- concentration in the patch pipette was either 150 mmol/L or 40 mmol/L. Bath application of phorbol 12,13-dibutyrate or phorbol 12-myristate 13-acetate (PDBu or PMA; 50 nmol/L), activators of PKC, resulted in the appearance of unitary Cl- channels with a mean conductance of 9.31 +/- 0.94 pS (n = 8) and 8.8 pS (n = 2), respectively, and reversal potentials were close to predicted ECl. Addition of staurosporine (500 nmol/L) reduced open probability (Po) of channels activated by PDBu. Bath application of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX, 500 mumol/L) resulted in the activation of Cl- channels with a conductance (mean 8.76 +/- 0.67 pS, n = 3) similar to those activated by PDBu.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular physiology of CFTR Cl- channels in heart.

Recent electrophysiological data suggests a number of similarities in the properties of cAMP-dependent Cl- channels in heart and cAMP-dependent Cl- channels encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene product in various epithelial cells. We tested the hypothesis that cAMP-dependent Cl- channels in heart may be due to cardiac expression of CFTR by amplification and sequencing of several regions of CFTR from myocardial tissue derived from various species and areas of the heart. Regions corresponding to the first nucleotide binding domain (NBD1), transmembrane segments I-VI (TS I-VI), transmembrane segments VII-XII (TS VII-XII), and the regulatory domain (R domain) were amplified and sequenced from rabbit ventricle (see Fig. 1). Comparison of the known amino acid sequence of human epithelial CFTR with the deduced sequence from rabbit heart indicated deletion of exon 5 in the first cytoplasmic loop of TS I-VI suggesting that CFTR is an alternatively spliced isoform in rabbit ventricle. Outside of the alternatively spliced region, the heart CFTR Cl- channel isoform displayed greater than 95% identity to human epithelial CFTR Cl- channels. We have also compared the molecular distribution of the CFTR gene product to the distribution of cAMP-dependent Cl- channels in native cardiac myocytes derived from various species and areas of the heart. Amplification of regions corresponding to NBD1, R domain, and TS VII-XII from atrium and ventricle of guinea pigs, rabbit, and dog hearts exhibited a distribution which closely matched the distribution of cAMP-dependent Cl- channels assessed using electrophysiological techniques.(ABSTRACT TRUNCATED AT 250 WORDS)