Clinical and vital sign changes associated with late-onset sepsis in very low birth weight infants at 3 NICUs.

BACKGROUND: In premature infants, clinical changes frequently occur due to sepsis or non-infectious conditions, and distinguishing between these is challenging. Baseline risk factors, vital signs, and clinical signs guide decisions to culture and start antibiotics. We sought to compare heart rate (HR) and oxygenation (SpO2) patterns as well as baseline variables and clinical signs prompting sepsis work-ups ultimately determined to be late-onset sepsis (LOS) and sepsis ruled out (SRO). METHODS: At three NICUs, we reviewed records of very low birth weight (VLBW) infants around their first sepsis work-up diagnosed as LOS or SRO. Clinical signs prompting the evaluation were determined from clinician documentation. HR-SpO2 data, when available, were analyzed for mean, standard deviation, skewness, kurtosis, and cross-correlation. We used LASSO and logistic regression to assess variable importance and associations with LOS compared to SRO. RESULTS: We analyzed sepsis work-ups in 408 infants (173 LOS, 235 SRO). Compared to infants with SRO, those with LOS were of lower GA and BW, and more likely to have a central catheter and mechanical ventilation. Clinical signs cited more often in LOS included hypotension, acidosis, abdominal distension, lethargy, oliguria, and abnormal CBC or CRP(p < 0.05). HR-SpO2 data were available in 266 events. Cross-correlation HR-SpO2 before the event was associated with LOS after adjusting for GA, BW, and postnatal age. A model combining baseline, clinical and HR-SpO2 variables had AUC 0.821. CONCLUSION: In VLBW infants at 3-NICUs, we describe the baseline, clinical, and HR-SpO2 variables associated with LOS versus SRO.

Blood pressure ranges via non-invasive and invasive monitoring techniques in premature neonates using high resolution physiologic data.

BACKGROUND: There are limited evidence-based published blood pressure ranges for premature neonates. The aim of the study was to determine blood pressure ranges in a large cohort of premature neonates based on gestational and post-menstrual age. METHODS: Retrospective observational study of premature neonates admitted to the neonatal intensive care unit at our institution between January 2009 and October 2015. We stratified data by gestational and post-menstrual age groups as well as by method of blood pressure measurement (non-invasive vs. invasive). RESULTS: Over two billion blood pressure values in 1708 neonates were analyzed to generate heat maps and establish percentile-based reference ranges. The median gestational age of the cohort was 31 weeks (IQR 28-33 weeks). We found moderate correlation (r = 0.57) between simultaneously obtained non-invasive and invasive blood pressure measurements. CONCLUSIONS: Our results can serve as a reference during the bedside assessment of the critically-ill neonate.

Early Pulse Oximetry Data Improves Prediction of Death and Adverse Outcomes in a Two-Center Cohort of Very Low Birth Weight Infants.

BACKGROUND: We previously showed, in a single-center study, that early heart rate (HR) characteristics predicted later adverse outcomes in very low birth weight (VLBW) infants. We sought to improve predictive models by adding oxygenation data and testing in a second neonatal intensive care unit (NICU). METHODS: HR and oxygen saturation (SpO2) from the first 12 hours and first 7 days after birth were analyzed for 778 VLBW infants at two NICUs. Using multivariate logistic regression, clinical predictive scores were developed for death, severe intraventricular hemorrhage (sIVH), bronchopulmonary dysplasia (BPD), treated retinopathy of prematurity (tROP), late-onset septicemia (LOS), and necrotizing enterocolitis (NEC). Ten HR-SpO2 measures were analyzed, with first 12 hours data used for predicting death or sIVH and first 7 days for the other outcomes. HR-SpO2 models were combined with clinical models to develop a pulse oximetry predictive score (POPS). Net reclassification improvement (NRI) compared performance of POPS with the clinical predictive score. RESULTS: Models using clinical or pulse oximetry variables alone performed well for each outcome. POPS performed better than clinical variables for predicting death, sIVH, and BPD (NRI > 0.5, p < 0.01), but not tROP, LOS, or NEC. CONCLUSION: Analysis of early HR-SpO2 characteristics adds to clinical risk factors to predict later adverse outcomes in VLBW infants.

Abnormal heart rate characteristics are associated with abnormal neuroimaging and outcomes in extremely low birth weight infants.

OBJECTIVE: Brain injury in preterm infants may lead to an inflammatory response and central nervous system dysfunction reflected by abnormal heart rate characteristics (HRC). We hypothesized that a continuously monitored HRC index reflecting reduced HR variability and decelerations correlates with abnormal neuroimaging and outcomes in extremely low birth weight infants (ELBW). STUDY DESIGN: We analyzed the average HRC index within 28 days after birth (aHRC28) and head ultrasound (HUS) in 384 ELBW infants. In 50 infants with brain magnetic resonance imaging (MRI) and 70 infants with Bayley neurodevelopmental testing at 1 year of age, we analyzed the relationship between aHRC28, MRI abnormalities and low Bayley scores. RESULT: aHRC28 was higher in infants with severe HUS abnormalities (2.65±1.27 for Grade III-IV intraventricular hemorrhage (IVH) or cystic periventricular leukomalacia (cPVL) versus 1.72±0.95 for normal or Grade I-II IVH, P<0.001). Higher aHRC28 was also associated with white matter damage on MRI and death or Bayley motor or mental developmental index <70. Associations persisted after adjusting for gestational age, birth weight and septicemia. For every one point increase in aHRC28, the odds ratio of death or Bayley score <70 was 2.45 (95% CI 1.46, 4.05, P<0.001). CONCLUSION: A continuously monitored HRC index provides an objective, noninvasive measure associated with abnormal brain imaging and adverse neurologic outcomes in ELBW infants.

Application of dynamical analyses of heart rate to rhythm classification and prognosis.

We have developed numerical approaches to dynamical analysis of heart rates, measured as interbeat or RR, intervals, based on entropy and fluctuation analyses in a large data base of consecutive Holter monitor recordings. In Part I, we present a RR interval-based classifier that distinguishes normal sinus rhythm (NSR), atrial fibrillation (AF) and sinus rhythm with ectopy with an accuracy of 99%, 81% and 77%respectively, using 10-minute segments. In Part II, we present 2-year mortality estimation based on the entropy calculations. The major finding is that normal dynamics identify a very low risk group. Taken together, these results point to automated analysis of heart rate time series with important clinical applications.

Abnormal heart rate characteristics before clinical diagnosis of necrotizing enterocolitis.

OBJECTIVE: Earlier diagnosis and treatment of necrotizing enterocolitis (NEC) in preterm infants, before clinical deterioration, might improve outcomes. A monitor that measures abnormal heart rate characteristics (HRC) of decreased variability and transient decelerations was developed as an early warning system for sepsis. As NEC shares pathophysiologic features with sepsis, we tested the hypothesis that abnormal HRC occur before clinical diagnosis of NEC. STUDY DESIGN: Retrospective review of Bells stage II to III NEC cases among infants <34 weeks gestation enrolled in a prospective randomized clinical trial of HRC monitoring at three neonatal intensive care units. RESULT: Of 97 infants with NEC and HRC data, 33 underwent surgical intervention within 1 week of diagnosis. The baseline HRC index from 1 to 3 days before diagnosis was higher in patients who developed surgical vs medical NEC (2.06±1.98 vs 1.22±1.10, P=0.009). The HRC index increased significantly 16 h before the clinical diagnosis of surgical NEC and 6 h before medical NEC. At the time of clinical diagnosis, the HRC index was higher in patients with surgical vs medical NEC (3.3±2.2 vs 1.9±1.7, P<0.001). CONCLUSION: Abnormal HRC occur before clinical diagnosis of NEC, suggesting that continuous HRC monitoring may facilitate earlier detection and treatment.

Heart rate characteristics and neurodevelopmental outcome in very low birth weight infants.

BACKGROUND: Sepsis in very low birth weight (VLBW) infants has been associated with an increased risk of adverse developmental outcome. We have identified abnormal heart rate characteristics (HRCs) that are predictive of impending sepsis, and we have developed a summary measure of an infant's abnormal HRCs during the neonatal hospitalization that we refer to as the cumulative HRC score (cHRC). OBJECTIVE: In this study, we tested the hypothesis that increasing cHRC is associated with an increasing risk of adverse neurodevelopmental outcome in VLBW infants. METHOD: Data were collected on 65 VLBW infants whose HRCs were monitored while in the neonatal intensive care unit and who were examined at 12 to 18 months adjusted age. Using the Bayley Scale of Infant Development-II, we identified delays in early cognitive function (i.e., Mental Developmental Index <70) and psychomotor development (i.e., Psychomotor Developmental Index <70). Cerebral palsy (CP) was diagnosed using a standard neurological examination. RESULT: Increasing cHRC score was associated with an increased risk of CP (odds ratio per 1 standard deviation increase in cHRC: 2.6, 95% confidence limits: 1.42, 5.1) and delayed early cognitive development [odds ratio: 2.3 (1.3; 4.3)]. These associations remain statistically significant when adjusted for major cranial ultrasound abnormality. There was an association of increasing cHRC and delayed psychomotor development, which did not reach statistical significance [odds ratio: 1.7 (1.0, 3.0)]. CONCLUSION: Among VLBW infants, the cumulative frequency of abnormal HRCs, which can be assessed non-invasively in the neonatal intensive care unit, is associated with an increased risk of adverse neurodevelopmental outcome.

Comparison and clinical application of frequency domain methods in analysis of neonatal heart rate time series.

The frequency content of the heart rate (HR) series contains information regarding the state of the autonomic nervous system. Of particular importance is respiratory sinus arrhythmia (RSA), the high-frequency fluctuation in HR attributable to respiration. The unevenly sampled nature of heart rate data, however, presents a problem for the discrete Fourier transform. Interpolation of the HR series allows even sampling, but filters high-frequency content. The Lomb periodogram (LP) is a regression-based method that addresses these issues. To evaluate the efficacy of the LP and Fourier techniques in detecting RSA, we compared the spectrum of intervals, the spectrum of HR samples, and the LP of simulated and clinical neonatal time series. We found the LP was superior to the spectrum of intervals and the spectrum of HR samples in analysis near the critical frequency of one half the average sampling rate. Applying the LP to clinical data, we found (1) evidence of stochastic resonance, an enhancement of periodicity with the addition of small amounts of noise, and (2) reduced power at all frequencies prior to clinical diagnosis of neonatal sepsis.

Frequency domain algorithm for quantifying atrial fibrillation organization to increase defibrillation efficacy.

We hypothesized that frequency domain analysis of an interatrial atrial fibrillation (AF) electrogram would show a correlation of the variance of the signal and the amplitude of harmonic peaks with the periodicity and morphology (organization) of the AF signal and defibrillation efficacy. We sought to develop an algorithm that would provide a high-resolution measurement of the changes in the spatiotemporal organization of AF. AF was initiated with burst atrial pacing in ten dogs. The atrial defibrillation threshold (ADFT50) was determined, and defibrillation was repeated at the ADFT50. Bipolar electrograms from the shocking electrodes were acquired immediately preshock, digitally filtered, and a FFT was performed. The organization index (OI) was calculated as the ratio of the area under the first four harmonic peaks to the total area of the spectrum. For a 4-s window, the mean OI was 0.505 +/- 0.087 for successful shocks, versus 0.352 +/- 0.068 for unsuccessful shocks (p < 0.001). Receiver operator characteristic (ROC) curve analysis was used to determine the optimal sampling window for predicting successful shocks. The area of the ROC curve was 0.8 for a 1-s window, and improved to 0.9 for a 4-s window. We conclude that the spectrum of an AF signal contains information relating to its organization, and can be used in predicting a successful defibrillation.

Skeletal muscle Na currents in mice heterozygous for Six5 deficiency.

Myotonic dystrophy results from a trinucleotide repeat expansion between the myotonic dystrophy protein kinase gene (Dmpk), which encodes a serine-threonine protein kinase, and the Six5 gene, which encodes a homeodomain protein. The disease is characterized by late bursts of skeletal muscle Na channel openings, and this is recapitulated in Dmpk -/- and Dmpk +/- murine skeletal muscle. To test whether deficiency of the nearby Six5 gene also affected Na channel gating in murine skeletal muscle, we measured Na currents from cell-attached patches in Six5 +/- mice and age-matched wild-type and Dmpk +/- mice. Late bursts of Na channel activity were defined as an opening probability >10% measured from 10 to 110 ms after depolarization. There was no significant difference in the occurrence of late Na channel bursts in wild-type and Six5 +/- muscle, whereas in Dmpk +/- muscle there was greater than fivefold increase in late bursts (P < 0.001). Compared with wild-type mice, Na current amplitude was unchanged in Six5 +/- muscle, whereas in Dmpk +/- muscle it was 36% reduced (P < 0.05). Thus, since Six5 +/- mice do not exhibit the Na channel gating abnormality of Dmpk deficiency, we conclude that Six5 deficiency does not contribute to the Na channel gating abnormality seen in dystrophia myotonica patients.

Assessment of global atrial fibrillation organization to optimize timing of atrial defibrillation.

BACKGROUND: We hypothesized that frequency domain analysis of a wide bipolar interatrial electrogram describes the global organization of atrial fibrillation (AF) and should vary over time. By timing shocks to periods of high organization of AF, cardioversion efficacy should improve. METHODS AND RESULTS: A total of 15 dogs (weight, 28.2+/-3.4 kg) were rapidly paced for 48 to 72 hours to induce AF. Coil electrodes with a surface area of 1.80 cm(2) were then placed in the left and right atria to form a wide bipole. Wide bipolar electrograms were digitally filtered, and a fast Fourier transform was performed over a sliding 2-s window every 0.5 s. The organization index (OI) was calculated as the ratio of the area of the dominant peak and its harmonics to the total area of the magnitude spectrum. The atrial defibrillation threshold (ADFT(50)) was determined using a 3-ms/3-ms biphasic shock and an up-down-up protocol. Additional shocks with higher and lower energies were delivered in a random sequence to develop a distribution curve. The OI varied over time, with a mean of 0.42+/-0.03, a maximum of 0.65+/-0.07, and a minimum of 0.20+/-0.06. The OI changed rapidly, with durations of high organization (OI>0.5) ranging from 1 to 5 s. The ADFT(50) for QRS complex-synchronized shocks was 183+/-56 V, versus 142+/-49 V for shocks synchronized to an OI>0.5 (P<0.001). The distribution curve shifted leftward when shocks were synchronized to an OI>0.5. CONCLUSIONS: AF signals show a high degree of variability. Shock efficacy is increased when shocks are delivered during periods of high AF organization as determined by the OI method.

Gene structure and expression of phospholemman in mouse.

Phospholemman (PLM) is a small transmembrane cardiac protein that is the major sarcolemmal substrate for phosphorylation in response to adrenergic stimulation. PLM likely plays a role in muscle contractility and cell volume regulation through its function as a channel or a channel regulator. We are the first to describe the structure of the PLM gene and to demonstrate PLM cDNA splice variants. We cloned the murine PLM cDNA and used it as a probe to isolate the gene from a 129/SvJ genomic library. The gene contains seven introns and eight exons. The coding sequence is interrupted by five introns; the 5' untranslated region by two. Using rapid amplification of 5' cDNA ends we identified transcription start sites and four splice variants of the 5' untranslated domain. There was no TATA box or CAAT box in the putative promoter regions. The gene has several stretches of dinucleotide repeats. The 3' untranslated domains of mouse PLM cDNA clones show sequence differences not accounted for by alternative splicing. Mouse PLM shares 93, 83 and 80% amino acid identity with rat, dog, and human PLMs, respectively. Tissue expression of murine PLM parallels that in other species, being highest in heart, skeletal muscle, and liver.

Toward the early diagnosis of neonatal sepsis and sepsis-like illness using novel heart rate analysis.

BACKGROUND AND OBJECTIVE: Abrupt clinical deterioration because of sepsis is a major cause of morbidity and mortality in neonates, and earlier diagnosis should improve therapy of this potentially catastrophic illness. In practice, clinical signs and laboratory data have not been perceived as sensitive or specific for early stages of sepsis. Because heart rate characteristics (HRC) are abnormal during fetal distress and neonatal illness, we hypothesized that abnormal HRC might precede the clinical diagnosis of neonatal sepsis, adding independent information to standard clinical parameters. METHODS: In the neonatal intensive care unit at the University of Virginia, we prospectively studied infants admitted from August 1995 to April 1999 who were at risk for developing sepsis. Infants in the sepsis (culture-positive) and sepsis-like illness (culture-negative) groups had an abrupt clinical deterioration that raised clinical suspicion of infection and prompted physicians to obtain blood cultures and start antibiotic therapy. Infants without sepsis raised no clinical suspicion of illness and had no cultures obtained. We measured novel characteristics-moments and percentiles-of normalized heart rate (HR) time series for 5 days before and 3 days after sepsis, sepsis-like illness, or a random time in controls. We also calculated the Score for Neonatal Acute Physiology (SNAP) and the Neonatal Therapeutic Intervention Scoring System (NTISS) as clinical scores of the severity of illness. RESULTS: There were 46 episodes of culture-positive sepsis in 40 patients and 27 episodes of culture-negative sepsis-like illness in 23 patients. We analyzed 29 control periods in 26 patients. Infants with sepsis and sepsis-like illness had lower birth weights and gestational ages and higher SNAP and NTISS scores than did infants without sepsis. The most important new finding was that the infants in the sepsis and sepsis-like illness groups had increasingly abnormal HRC for up to 24 hours preceding their abrupt clinical deterioration. The abnormal HRC were reduced baseline variability and short-lived decelerations in HR. These abnormalities led to significant changes in HRC measures, for example, the third moment (skewness:.59 +/-.10 for sepsis and.51 +/-. 12 for sepsis-like illness, compared with -.10 +/-.13 for control over the 6 hours before abrupt deterioration). Culture-positive and culture-negative patients had similar HRC and clinical scores, including a significant rise in SNAP in the 24 hours before the event. Multivariable logistic regression analysis showed that HRC and clinical scores independently added information in distinguishing infants with sepsis and sepsis-like illness from control patients in the 24 hours before abrupt deterioration. CONCLUSIONS: Newborn infants who had abrupt clinical deterioration as a result of sepsis and sepsis-like illness had abnormal HRC and SNAP that worsened over 24 hours before the clinical suspicion of sepsis. A strategy for monitoring these parameters in infants at risk for sepsis and sepsis-like illness might lead to earlier diagnosis and more effective therapy.heart rate variability, neonatal sepsis, Score for Neonatal Acute Physiology, Neonatal Therapeutic Intervention Scoring System, newborn.

Contributions of charged residues in a cytoplasmic linking region to Na channel gating.

Na channels inactivate quickly after opening, and the very highly positively charged cytoplasmic linking region between homologous domains III and IV of the channel molecule acts as the inactivation gate. To test the hypothesis that the charged residues in the domain III to domain IV linker have a role in channel function, we measured currents through wild-type and two mutant skeletal muscle Na channels expressed in Xenopus oocytes, each lacking two or three charged residues in the inactivation gate. Microscopic current measures showed that removing charges hastened activation and inactivation. Macroscopic current measures showed that removing charges altered the voltage dependence of inactivation, suggesting less coupling of the inactivation and activation processes. Reduced intracellular ionic strength shifted the midpoint of equilibrium activation gating to a greater extent, and shifted the midpoint of equilibrium inactivation gating to a lesser extent in the mutant channels. The results allow the possibility that an electrostatic mechanism contributes to the role of charged residues in Na channel inactivation gating.

Skeletal muscle sodium channel gating in mice deficient in myotonic dystrophy protein kinase.

Myotonic dystrophy, a progressive autosomal dominant disorder, is associated with an expansion of a CTG repeat tract located in the 3'-untranslated region of a serine/threonine protein kinase, DMPK. DMPK modulates skeletal muscle Na channels in vitro, and thus we hypothesized that mice deficient in DMPK would have altered muscle Na channel gating. We measured macroscopic and single channel Na currents from cell-attached patches of skeletal myocytes from mice heterozygous (DMPK(+/-)) and homozygous (DMPK(-/-)) for DMPK loss. In DMPK(-/-) myocytes, Na current amplitude was reduced because of reduced channel number. Single channel recordings revealed Na channel reopenings, similar to the gating abnormality of human myotonic muscular dystrophy (DM), which resulted in a plateau of Na current. The gating abnormality deteriorated with increasing age. In DMPK(+/-) muscle there was reduced Na current amplitude and increased Na channel reopenings identical to those in DMPK(-/-) muscle. Thus, these mouse models of complete and partial DMPK deficiency reproduce the Na channel abnormality of the human disease, providing direct evidence that DMPK deficiency underlies the Na channel abnormality in DM.

Physiological time-series analysis using approximate entropy and sample entropy.

Entropy, as it relates to dynamical systems, is the rate of information production. Methods for estimation of the entropy of a system represented by a time series are not, however, well suited to analysis of the short and noisy data sets encountered in cardiovascular and other biological studies. Pincus introduced approximate entropy (ApEn), a set of measures of system complexity closely related to entropy, which is easily applied to clinical cardiovascular and other time series. ApEn statistics, however, lead to inconsistent results. We have developed a new and related complexity measure, sample entropy (SampEn), and have compared ApEn and SampEn by using them to analyze sets of random numbers with known probabilistic character. We have also evaluated cross-ApEn and cross-SampEn, which use cardiovascular data sets to measure the similarity of two distinct time series. SampEn agreed with theory much more closely than ApEn over a broad range of conditions. The improved accuracy of SampEn statistics should make them useful in the study of experimental clinical cardiovascular and other biological time series.

Phospholemman is a substrate for myotonic dystrophy protein kinase.

The genetic abnormality in myotonic muscular dystrophy, multiple CTG repeats lie upstream of a gene that encodes a novel protein kinase, myotonic dystrophy protein kinase (DMPK). Phospholemman (PLM), a major membrane substrate for phosphorylation by protein kinases A and C, induces Cl currents (I(Cl(PLM))) when expressed in Xenopus oocytes. To test the idea that PLM is a substrate for DMPK, we measured in vitro phosphorylation of purified PLM by DMPK. To assess the functional effects of PLM phosphorylation we compared I(Cl(PLM)) in Xenopus oocytes expressing PLM alone to currents in oocytes co-expressing DMPK, and examined the effect of DMPK on oocyte membrane PLM expression. We found that PLM is indeed a good substrate for DMPK in vitro. Co-expression of DMPK with PLM in oocytes resulted in a reduction in I(Cl(PLM)). This was most likely a specific effect of phosphorylation of PLM by DMPK, as the effect was not present in oocytes expressing a phos(-) PLM mutant in which all potential phosphorylation had been disabled by Ser --> Ala substitution. The biophysical characteristics of I(Cl(PLM)) were not changed by DMPK or by the phos(-) mutation. Co-expression of DMPK reduced the expression of PLM in oocyte membranes, suggesting a possible mechanism for the observed reduction in I(Cl(PLM)) amplitude. These data show that PLM is a substrate for phosphorylation by DMPK and provide functional evidence for modulation of PLM function by phosphorylation.

Modulation of Xenopus oocyte-expressed phospholemman-induced ion currents by co-expression of protein kinases.

Phospholemman (PLM), the major sarcolemmal substrate for phosphorylation by cAMP-dependent kinase (PKA) protein kinase C (PKC) and NIMA kinase in muscle, induces hyperpolarization-activated anion currents in Xenopus oocytes, most probably by enhancing endogenous oocyte currents. PLM peptides from the cytoplasmic tail are phosphorylated by PKA at S68, by NIMA kinase at S63, and by PKC at both S63 and S68. We have confirmed the phosphorylation sites in the intact protein, and we have investigated the role of phosphorylation in the regulatory activity of PLM using oocyte expression experiments. We found: (1) the cytoplasmic domain is not essential for inducing currents in oocytes; (2) co-expression of PKA increased the amplitude of oocyte currents and the amount of PLM in the oocyte membrane largely, but not exclusively, through phosphorylation of S68; (3) co-expression of PKA had no effect on a PLM mutant in which all putative phosphorylation sites had been inactivated by serine to alanine mutation (SSST 62, 63, 68, 69 AAAA); (4) co-expression of PKC had no effect in this system; (5) co-expression of NIMA kinase increased current amplitude and membrane protein level, but did not require PLM phosphorylation. These findings point to a role for phosphorylation in the function of PLM.

Protein kinase C co-expression and the effects of halothane on rat skeletal muscle sodium channels.

1. Voltage-gated Na channels, which are potential targets for general anaesthetics, are substrates for PKC, which phosphorylates a conserved site in the channel inactivation gate. We investigated the idea that PKC modulates the effect of volatile anaesthetics on Na channels via phosphorylation of this inactivation gate site. 2. Na currents through rat skeletal muscle Na channel alpha-subunits expressed in Xenopus oocytes were measured by two-microelectrode voltage clamp in the presence of the volatile anaesthetic agent halothane (2-bromo-2-chloro-1,1,1-trifluroethane). PKC activity was modulated by co-expression of a constitutively active PKC alpha-isozyme. 3. Halothane (0.4 mM) had no effect on Na currents. With co-expression of PKC, however, halothane dose-dependently enhanced the rate of Na current decay and caused a small, but statistically significant reduction in Na current amplitude. 4. The enhancement of Na current decay was absent in a Na channel mutant in which the inactivation gate phosphorylation site was disabled. Effects of halothane on amplitude were independent of this mutation. 5. Co-expression of a PKC alpha-isozyme permits an effect of halothane to hasten current decay and reduce current amplitude, at least in part through interaction with the inactivation gate phosphorylation site. We speculate that the interaction between halothane and Na channels is direct, and facilitated by PKC activity and by phosphorylation of a site in the channel inactivation gate.

Ion currents through mutant phospholemman channel molecules.

Phospholemman (PLM), a small membrane protein with a single transmembrane domain, is a major substrate for protein kinases in muscle. In lipid bilayers, PLM forms ion channels with two unusual features--selectivity for the zwitterion taurine, and switching among cation- and anion-selective conformations. To investigate the molecular determinants of channel behavior, we measured ion currents through (1) PLM channels with point mutations in the extracellular and cytoplasmic domains, (2) chimeric PLM channels with the transmembrane domain of IsK, a K channel subunit, and (3) truncated PLM channels lacking the cytoplasmic domain. Truncated channels and those with a mutation at the cytoplasmic face had altered selectivity. Channels with a point mutation near the extracellular face were non-selective. Switching among channel conformations was less frequent in truncated channels and in channels with point mutations in either the extracellular or cytoplasmic domain. Chimeric channels with a different transmembrane domain had only a small reduction in conductance. We conclude that both the extracellular and cytoplasmic domains play roles in channel selectivity and conformational changes. Cation-binding sites appear to lie in the cytoplasmic domain, and an anion-binding site may reside in the extracellular domain. The cytoplasmic domain may function as a "ball and chain" to regulate channel selectivity.