Harald Reuter

Calmodulin supports both inactivation and facilitation of L-type calcium channels

L-type Ca2+ channels support Ca2+ entry into cells, which triggers cardiac contraction, controls hormone secretion from endocrine cells and initiates transcriptional events that support learning and memory. These channels are examples of molecular signal-transduction units that regulate themselves through their own activity. Among the many types of voltage-gated Ca2+ channel, L-type Ca2+ channels particularly display inactivation and facilitation, both of which are closely linked to the earlier entry of Ca2+ ions. Both forms of autoregulation have a significant impact on the amount of Ca2+ that enters the cell during repetitive activity, with major consequences downstream. Despite extensivebiophysical analysis, the molecular basis of autoregulation remains unclear, although a putative Ca2+-binding EF-hand motif, and a nearby consensus calmodulin-binding isoleucine-glutamine (‘IQ’) motif, in the carboxy terminus of the α1C channel subunit have been implicated,. Here we show that calmodulin is a critical Ca2+ sensor for both inactivation and facilitation, and that the nature of the modulatory effect depends on residues within the IQ motif important for calmodulin binding. Replacement of the native isoleucine by alanine removed Ca2+-dependent inactivation and unmasked a strong facilitation; conversion of the same residue to glutamate eliminated both forms of autoregulation. These results indicate that the same calmodulin molecule may act as a Ca2+ sensor for both positive and negative modulation.

The kinetics of synaptic vesicle recycling measured at single presynaptic boutons

We used the fluorescent membrane probe FM 1-43 to label recycling synaptic vesicles within the presynaptic boutons of dissociated hippocampal neurons in culture. Quantitative time-lapse fluorescence imaging was employed in combination with rapid superfusion techniques to study the dynamics of synaptic vesicles within single boutons. This approach enabled us to measure exocytosis and to analyze the kinetics of endocytosis and the preparation of endocytosed vesicles for re-release (repriming). Our measurements indicate that under sustained membrane depolarization, endocytosis persists much longer than exocytosis, with a t1/2 approximately 60 s (approximately 24 degrees C); once internalized, vesicles become reavailable for exocytosis in approximately 30 s. Furthermore, we have shown that endocytosis is not dependent on membrane potential and, unlike exocytosis, that it is independent of extracellular Ca2+.

Brief Reviews Exchange of Calcium Ions in the Mammalian Myocardium : Mechanisms and Physiological Significance

• Calcium ions are essential for coupling between excitation of the cardiac cell membrane and activation of the contractile proteins (excitation-contraction coupling). This process can be summarized in the following way (1-3). During membrane depolarization, i.e., during the cardiac action potential, calcium (Ca) ions flow across the membrane into the cardiac muscle fiber. This influx directly and indirectly (possibly by a regenerative release of Ca from the sarcoplasmic reticulum) causes an increase in the concentration of free Ca ions in the myoplasm. In the relaxed muscle, this concentration is on the order of 10~M. During excitation, the free myoplasmic Ca concentration increases by one to two orders of magnitude, which permits binding of Ca ions to the protein troponin and ultimately leads to a contraction of the myofibrils. However, a transport system of the sarcoplasmic reticulum with a high affinity for Ca ions reduces the free myoplasmic Ca concentration again and therefore causes relaxation of the contractile proteins. Ca ions are then stored in the sarcoplasmic reticulum. To avoid an overload of the sarcoplasmic reticulum and the mitochondria with Ca, these ions are pumped out of the cell across the cell membrane (sarcolemma) apparently by a transport mechanism involving exchange of external sodium (Na) ions for internal Ca ions (Na-Ca exchange). Obviously, the sarcolemma plays an important role in the regulation of intracellular Ca concentration. Only recently, some information has become available about the mechanisms of inward Ca movement during the action potential and outward Ca transport (4-6). The following discussion summarizes the available information about the mechanisms of these Ca movements across the sarcolemma of cardiac cells. Also some basic relations between Ca movements and contractile activation are presented.

Imaging of cytosolic Ca2+ transients arising from Ca2+ stores and Ca2+ channels in sympathetic neurons.

Changes in cytosolic free Ca2+ concentration [( Ca2+]i) due to Ca2+ entry or Ca2+ release from internal stores were spatially resolved by digital imaging with the Ca2+ indicator fura-2 in frog sympathetic neurons. Electrical stimulation evoked a rise in [Ca2+]i spreading radially from the periphery to the center of the soma. Elevated [K+]o also increased [Ca2+]i, but only in the presence of external Ca2+, indicating that Ca2+ influx through Ca2+ channels is the primary event in the depolarization response. Ca2+ release or uptake from caffeine-sensitive internal stores was able to amplify or attenuate the effects of Ca2+ influx, to generate continued oscillations in [Ca2+]i, and to persistently elevate [Ca2+]i above basal levels after the stores had been Ca2(+)-loaded.

Cellular calcium and cardiac cell death

Claude Bernard recognized over a century ago that an extracellular environment of constant chemical composition was prerequisite for life outside of the stable aquatic environment of the sea. This concept of constancy of composition extends also to the intracellular environment, which can vary only within rather narrow limits that are set by the conditions essential for cell viability. A major role for the sarcolemma, which separates the intracellular and extracellular environment, is to maintain the intracellular environment within these limits. Nevertheless, it is important to note that small variations in this intracellular composition have important implications for cell function. For example, small fluctuations of intracellular free ionized calcium (Ca2+) concentration determine both the contractile and the energetic state of the heart.

Optical detection of a quantal presynaptic membrane turnover

Exploration of the mechanisms and plasticity of synaptic transmission has been hindered by the lack of a method to measure single vesicle turnover directly in individual presynaptic boutons at isolated nerve terminals. Although postsynaptic electrical recordings have provided a wealth of invaluable basic information about quantal presynaptic processes, this approach has often proved difficult to apply at most central nervous system synapses. Here we describe the direct optical detection of single quantal events in individual presynaptic boutons of cultured hippocampal neurons. Using the fluorescent dye FM 1-43 as a tracer for presynaptic endocytosis, we have characterized both evoked and spontaneous components of presynaptic function at the level of individual quanta. Our results are consistent with quantal interpretations of previous electrophysiological analyses and provide new information about the unitary membrane recycling event and its coupling to individual action potential stimuli, about spontaneous vesicle turnover at individual boutons, and about the numbers of vesicles recycling at individual boutons.

Molecular Structures Involved in L-type Calcium Channel Inactivation ROLE OF THE CARBOXYL-TERMINAL REGION ENCODED BY EXONS 40-42 IN α1C SUBUNIT IN THE KINETICS AND Ca2+ DEPENDENCE OF INACTIVATION

The pore-forming α1C subunit is the principal component of the voltage-sensitive L-type Ca2+ channel. It has a long cytoplasmic carboxyl-terminal tail playing a critical role in channel gating. The expression of α1C subunits is characterized by alternative splicing, which generates its multiple isoforms. cDNA cloning points to a diversity of human hippocampus α1C transcripts in the region of exons 40-43 that encode a part of the 662-amino acid carboxyl terminus. We compared electrophysiological properties of the well defined 2138-amino acid α1C,77 channel isoform with two splice variants, α1C,72 and α1C,86. They contain alterations in the carboxyl terminus due to alternative splicing of exons 40-42. The 2157-amino acid α1C,72 isoform contains an insertion of 19 amino acids at position 1575. The 2139-amino acid α1C,86 has 80 amino acids replaced in positions 1572-1651 of α1C,77 by a non-identical sequence of 81 amino acids. When expressed in Xenopus oocytes, all three splice variants retained high sensitivity toward dihydropyridine blockers but showed large differences in gating properties. Unlike α1C,77 and α1C,72, Ba2+ currents (IBa) through α1C,86 inactivated 8-10 times faster at +20 mV, and its inactivation rate was strongly voltage-dependent. Compared to α1C,77, the inactivation curves of IBa through α1C,86 and α1C,72 channels were shifted toward more negative voltages by 11 and 6 mV, respectively. Unlike α1C,77 and α1C,72, the α1C,86 channel lacks a Ca2+-dependent component of inactivation. Thus the segment 1572-1651 of the cytoplasmic tail of α1C is critical for the kinetics as well as for the Ca2+ and voltage dependence of L-type Ca2+ channel gating.

Diversity and function of presynaptic calcium channels in the brain

Calcium channels in presynaptic nerve terminals are essential for neurotransmitter release, and current research has provided evidence for the involvement of a multitude of Ca2+ channel types. The diversity of Ca2+ channel structure and distribution in the brain suggests specific functional roles. Modulation by interaction with other proteins and/or by phosphorylation/dephosphorylation reactions enhances the regulatory impact of these channels on brain function.

LOCALIZATION AND FUNCTIONAL SIGNIFICANCE OF THE NA+/CA2+ EXCHANGER IN PRESYNAPTIC BOUTONS OF HIPPOCAMPAL CELLS IN CULTURE

Immunocytochemical evidence for localized distribution of the Na+/Ca2+ exchange protein in nerve terminals of cultured hippocampal cells is presented together with results on the functional relevance of the exchanger in the control of [Ca2+]i and of synaptic vesicle recycling. The monoclonal antibody R3F1, directed against an epitope on the intracellular loop of the protein, revealed higher densities of expression in synaptic regions than in other parts of the neurons. Removal of extracellular Na+ produced enhanced and prolonged elevation of [Ca2+]i in nerve terminals during and after electrical stimulation of the cells. Correspondingly, initial rates of exocytosis, measured by fluorescence changes of FM 1-43 during stimulation, were faster in LiCl-containing solution than in NaCl-containing solution. By contrast, endocytosis at 20 s was the same in both solutions.

Measurements of exocytosis from single presynaptic nerve terminals reveal heterogeneous inhibition by Ca2+-channel blockers

The effect of various Ca(2+)-channel blockers on exocytosis has been studied at the level of single presynaptic terminals in rat hippocampal cell cultures. The fluorescence change of the styryl dye FM 1-43 has been used as a measure of exocytosis during electrical stimulation. omega-Conotoxin GVIA (2-10 microM) completely inhibited exocytosis in approximately 45% of the boutons in the field of view, while in approximately 55% exocytosis was inhibited incompletely (by 38%). This heterogeneity in response of presynaptic boutons was not seen with isradipine (5 microM) or omega-agatoxin IVA (80 nM), which inhibited exocytosis by 23% and 17%, respectively. However, it was observed with a combination of all three blockers. Pre- and postsynaptic events could be separated in single synapses by measuring FM1-43 release and NMDA-induced changes in the intracellular Ca2+ concentration independently.