Resonance between beams of x-ray waves and electronic transitions from bound atomic orbitals leads to a phenomenon known as anomalous scattering. This effect can be exploited in x-ray crystallographic studies on biological macromolecules by making diffraction measurements at selected wavelengths associated with a particular resonant transition. In this manner the problem of determining the three-dimensional structure of thousands of atoms is reduced to that of initially solving for a few anomalous scattering centers that can then be used as a reference for developing the entire structure. This method of multiwavelength anomalous diffraction has now been applied in a number of structure determinations. Optimal experiments require appropriate synchrotron instrumentation, careful experimental design, and sophisticated analytical procedures. There are rich opportunities for future applications.

https://science.sciencemag.org/content/sci/254/5028/51.full.pdf

Determination of Macromolecular Structures from Anomalous Diffraction of Synchrotron Radiation

Hyperpolarization-activated cation currents, termed If, Ih, or Iq, were initially discovered in heart and nerve cells over 20 years ago. These currents contribute to a wide range of physiological functions, including cardiac and neuronal pacemaker activity, the setting of resting potentials, input conductance and length constants, and dendritic integration. The hyperpolarization-activated, cation nonselective (HCN) gene family encodes the channels that underlie Ih. Here we review the relation between the biophysical properties of recombinant HCN channels and the pattern of HCN mRNA expression with the properties of native Ih in neurons and cardiac muscle. Moreover, we consider selected examples of the expanding physiological functions of Ih with a view toward understanding how the properties of HCN channels contribute to these diverse functional roles.

Hyperpolarization-Activated Cation Currents: From Molecules to Physiological Function

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels comprise a small subfamily of proteins within the superfamily of pore-loop cation channels. In mammals, the HCN channel family com...

Hyperpolarization-activated cation channels: from genes to function.

Ion channels on the surface membrane of sinoatrial nodal pacemaker cells (SANCs) are the proximal cause of an action potential. Each individual channel type has been thoroughly characterized under voltage clamp, and the ensemble of the ion channel currents reconstructed in silico generates rhythmic action potentials. Thus, this ensemble can be envisioned as a surface "membrane clock" (M clock). Localized subsarcolemmal Ca(2+) releases are generated by the sarcoplasmic reticulum via ryanodine receptors during late diastolic depolarization and are referred to as an intracellular "Ca(2+) clock," because their spontaneous occurrence is periodic during voltage clamp or in detergent-permeabilized SANCs, and in silico as well. In spontaneously firing SANCs, the M and Ca(2+) clocks do not operate in isolation but work together via numerous interactions modulated by membrane voltage, subsarcolemmal Ca(2+), and protein kinase A and CaMKII-dependent protein phosphorylation. Through these interactions, the 2 subsystem clocks become mutually entrained to form a robust, stable, coupled-clock system that drives normal cardiac pacemaker cell automaticity. G protein-coupled receptors signaling creates pacemaker flexibility, ie, effects changes in the rhythmic action potential firing rate, by impacting on these very same factors that regulate robust basal coupled-clock system function. This review examines evidence that forms the basis of this coupled-clock system concept in cardiac SANCs.

A Coupled SYSTEM of Intracellular Ca2+ Clocks and Surface Membrane Voltage Clocks Controls the Timekeeping Mechanism of the Heart’s Pacemaker

The heart automaticity is a fundamental physiological function in higher organisms. The spontaneous activity is initiated by specialized populations of cardiac cells generating periodical electrica...

Genesis and Regulation of the Heart Automaticity

1
Myocytes were dissociated from the sinoatrial (SA) node of rat heart using a new enzymatic dissociation technique. Only a small number of isolated SA node myocytes showed regular rhythmic contractions and spontaneous action potentials, and these were used in the present study.

2
The spontaneous action potential was resistant to TTX, and the action potential parameters were similar to those of rabbit and guinea-pig pacemaker cells. Major time- and voltage-dependent currents were the delayed rectifier K+ current IKr, the L-type Ca2+ current ICa,L and the sodium current INa. The hyperpolarization-activated cation current (If) was recorded from ≈50 % of the cells with hyperpolarization beyond -90 mV.

3
The instantaneous current jump at the onset of a hyperpolarizing pulse showed inward rectification and was largely blocked by Ba2+. This Ba2+-sensitive current corresponded well to the inward rectifier K+ current (IK1), although it was much smaller in amplitude than in the ventricle.

4
A sustained inward current was activated on depolarization from -80 mV to the voltage range of slow diastolic depolarization. The current was blocked by nicardipine, enlarged by isoprenaline and was insensitive to removal of external Ca2+. These characteristics were similar to the sustained inward current, Ist, previously described in the rabbit and guinea-pig SA node cells.

5
The role of Ist was considered by constructing empirical equations, which were applied to the experimental record of the action potential. It is demonstrated that the voltage-dependent activation of Ist constitutes a positive feedback loop with the depolarization of the membrane.

The sustained inward current and inward rectifier K+ current in pacemaker cells dissociated from rat sinoatrial node

—Several time- and voltage-dependent ionic currents have been identified in cardiac pacemaker cells, including Na+ current, L- and T-type Ca2+ currents, hyperpolarization-activated cation current, and various types of delayed rectifier K+ currents. Mathematical models have demonstrated that spontaneous action potentials can be reconstructed by incorporating these currents, but relative contributions of individual currents vary widely between different models. In 1995, the presence of a novel inward current that was activated by depolarization to the potential range of the slow diastolic depolarization in rabbit sinoatrial (SA) node cells was reported. Because the current showed little inactivation during depolarizing pulses, it was called the sustained inward current ( I st). A similar current is also found in SA node cells of the guinea pig and rat and in subsidiary pacemaker atrioventricular node cells. Recently, single-channel analysis has revealed a nicardipine-sensitive, 13-pS Na+ current, which is activated by depolarization to the diastolic potential range in guinea pig SA node cells. This channel differs from rapid voltage-gated Na+ or L-type Ca2+ channels both in unitary conductance and gating kinetics. Because I st was observed only in spontaneously beating SA node cells, ie, it was absent in quiescent cells dissociated from the same SA or atrioventricular node, an important role of I st for generation of intrinsic cardiac automaticity was suggested. (Circ Res. 2000;87:88-91.)

Sustained Inward Current During Pacemaker Depolarization in Mammalian Sinoatrial Node Cells

A New Cytochrome in Neutrophilic Granules of Rabbit Leucocyte

GIUDITTA and STRECKER (1959) reported the occurrence in rat cerebral cortex of an antimycinand amylobarbitone-insensitive mechanism catalysing the oxidation of reduced pyridine nucleotides by cytochrome c. According to these investigators, the intracellular distribution of this oxidative activity paralleled that of several enzymes known to be localized in microsomes or the endoplasmic reticulum. However, little is as yet known of the enzyme components involved in this activity in brain microsomes. The localization of a powerful NADH-cytochrome c reductase activity in liver microsomes has been known for some time (HOGEBOOM, 1949); this hepatic activity is also insensitive to antimycin A and other respiratory inhibitors. Extensive studies have now established that the NADH-cytochrome c reductase activity in liver microsomes is catalysed by a flavoprotein called NADH-cytochrome b, reductase (STRITTMATTER and VELICK, 1956) and a cytochrome designated as b, (STRITTMATTER and BALL, 1954), both of which have been highly purified and well characterized (cf. STRITTMATTER, 1962). Liver microsomes also contain a NADPH-cytochrome c reductase activity (DE DUVE, PRESSMAN, GIANETTO, WATTIAUX and APPELMANS, 1955). Recent work has shown that this activity is caused by a single microsomal flavoprotein called NADPH-cytochrome c reductase (WILLIAMS and KAMIN, 1962; PHILLIPS and LANCDON, 1962). This flavoprotein has further been shown to act as an NADPH oxidase in the presence of certain naphthoquinones as cofactor (SATO, NISHIBAYASHI and OMURA, 1962; NISHIBAYASHI et al., 1963; SATO et al., 1964). Since there was apparent similarity between the reduced pyridine nucleotide oxidizing systems in brain and liver microsomes, it seemed of interest to search for cytochrome b, and other electron transfer components in brain microsomes. The present paper reports the finding that brain microsomes actually contain cytochrome b,. Several electron transfer activities of brain microsomes and their relations to cytochrome b, are also described. MATERIALS A N D M E T H O D S Nerve tissues. Adult albino rabbits were bled from the carotid artery under urethane narcosis, and the brain was perfused in sifu with the medium to be used for tissue fractionation. The brain was then excised and homogenized with isotonic (1.15 %) KCl or 0.32 M-sucrose in a glass homogenizer equipped with a Teflon pestle. The homogenate was subjected to centrifugal fractionation as described below. For the study of the regional distribution of brain cytochrome b,, fresh bovine brains were obtained from a slaughter house and brought to the laboratory in the cold.

Yasuko Shinagawa

Papers

The sustained inward current and inward rectifier K+ current in pacemaker cells dissociated from rat sinoatrial node

Sustained Inward Current During Pacemaker Depolarization in Mammalian Sinoatrial Node Cells

A New Cytochrome in Neutrophilic Granules of Rabbit Leucocyte

Cytochrome b5 and related oxidative activities in mammalian brain microsomes.

Molecular orbital studies of the radicals of hydroxybenzenes and epinephrine