Brett A. Adams

Localization in the II-III Loop of the Dihydropyridine Receptor of a Sequence Critical for Excitation-Contraction Coupling

Skeletal and cardiac muscles express distinct isoforms of the dihydropyridine receptor (DHPR), a type of voltage-gated Ca2+ channel that is important for excitation-contraction (EC) coupling. However, entry of Ca2+ through the channel is not required for skeletal muscle-type EC coupling. Previous work (Tanabe, T., Beam, K. G., Adams, B. A., Niidome, T., and Numa, S. (1990) Nature346, 567–569) revealed that the loop between repeats II and III (II-III loop) is an important determinant of skeletal-type EC coupling. In the present study we have further dissected the regions of the II-III loop critical for skeletal-type EC coupling by expression of cDNA constructs in dysgenic myotubes. Because Ser687 of the skeletal II-III loop has been reported to be rapidly phosphorylatedin vitro, we substituted this serine with alanine, the corresponding cardiac residue. This alanine-substituted skeletal DHPR retained the ability to mediate skeletal-type EC coupling. Weak skeletal-type EC coupling was produced by a chimeric DHPR, which was entirely cardiac except for a small amount of skeletal sequence (residues 725–742) in the II-III loop. Skeletal-type coupling was stronger when both residues 725–742 and adjacent residues were skeletal (e.g. a chimera containing skeletal residues 711–765). However, residues 725–742 appeared to be critical because skeletal-type coupling was not produced either by a chimera with skeletal residues 711–732 or by one with skeletal residues 734–765.

Physiological Consequences of Thermoregulation in a Tropical Lizard (Ameiva festiva)

Ameiva festiva, a teiid lizard from Costa Rica, alternately basks in the sun at the edge of forests and then forages in the shade of the forest. We used this natural analog of behavior in a laboratory shuttle-box to examine the effect of thermoregulatory behavior on physiological and ecological performances of lizards in nature. We observed body temperatures (by radiotelemetry) and locomotor behavior in the field and measured the thermal dependence of sprint speed, stamina, and aerobic scope in the laboratory. The mean upper and lower threshold temperatures for shuttling in the field were 39.4 ± 0.97 C (95% confidence interval) and 34.5 ± 1.11 C (95% confidence interval), respectively, and corresponded to very high levels of sprint speed (90% of maximum speed), stamina, and aerobic scope. Nevertheless, the lizards apparently rarely use these high levels of locomotor capacity. Although these lizards are active foragers, their speed and duration of movement in the field fall far below the levels of speed and stamina that they achieved in the lab when measured at temperatures that they regularly experienced in the field. The only time that we observed an individual use its (apparently) full locomotor capacities was in a single high-speed, long-distance attempt at escape from a predator. Thus the locomotor capacities of Ameiva festiva may be analogous to the principle of excessive construction (sensu Gans), whereby the phenotypic capacities of animals are not shaped by routine activities but instead by rare events that may be critical to an animals survival.

Muscular dysgenesis in mice: a model system for studying excitation-contraction coupling.

Muscular dysgenesis (mdg) is a lethal autosomal, recessive mutation of mice. Skeletal muscle from dysgenic mice is paralyzed due to the failure of excitation‐contraction (E‐C) coupling. Considerable evidence indicates that this failure results from the absence of a specific gene product, the α1 subunit of the skeletal muscle receptor for dihydropyridine calcium channel modifiers. This dihydropyridine receptor is hypothesized to function in E‐C coupling of normal skeletal muscle as the voltage sensor that triggers calcium release from the sarcoplasmic reticulum and thereby causes contraction. The skeletal muscle dihydropyridine receptor is also postulated to function as the ion channel responsible for a slowly activating, dihydropyridine‐sensitive calcium current (Islow). Dysgenic skeletal muscle lacks Islow but expresses, at low levels, a distinctly different dihydropyridine‐sensitive calcium current (Idys). The channel protein underlying Idys is incapable of serving as a voltage sensor for E‐C coupling. Studies using dysgenic skeletal muscle have provided significant insight into the role of dihydropyridine receptors in E‐C coupling.— Adams, B. A.; Beam, K. G. Muscular dysgenesis: a model system for studying excitation‐contraction coupling. FASEB J. 4: 2809‐2816; 1990.

RGS2 blocks slow muscarinic inhibition of N‐type Ca2+ channels reconstituted in a human cell line

1 Native N‐type Ca2+ channels undergo sustained inhibition through a slowly activating pathway linked to M1 muscarinic acetylcholine receptors and Gαq/11 proteins. Little is known concerning the regulation of this slow inhibitory pathway. We have reconstituted slow muscarinic inhibition of N‐type channels in HEK293 cells (a human embryonic kidney cell line) by coexpressing cloned α1B (CaV2.2) Ca2+ channel subunits and M1 receptors. Expressed Ca2+ currents were recorded using standard whole‐cell, ruptured‐patch techniques. 2 Rapid application of carbachol produced two kinetically distinct components of Ca2+ channel inhibition. The fast component of inhibition had a time constant of < 1 s, whereas the slow component had a time constant of 5‐40 s. Neither component of inhibition was reduced by pertussis toxin (PTX) or staurosporine. 3 The fast component of inhibition was selectively blocked by the Gβγ‐binding region of β‐adrenergic receptor kinase 1, suggesting that fast inhibition is mediated by Gβγ released from Gαq/11. 4 The slow component of inhibition was selectively blocked by regulator of G protein signalling 2 (RGS2), which preferentially interacts with Gαq/11 proteins. RGS2 also attenuated channel inhibition produced by intracellular dialysis with non‐hydrolysable GTPγS. Together these results suggest that RGS2 selectively blocked slow inhibition by functioning as an effector antagonist, rather than as a GTPase‐accelerating protein (GAP). 5 These experiments demonstrate that slow muscarinic inhibition of N‐type Ca2+ channels can be reconstituted in non‐neuronal cells, and that RGS2 can selectively block slow muscarinic inhibition while leaving fast muscarinic inhibition intact. These results identify RGS2 as a potential physiological regulator of the slow muscarinic pathway.

The monomeric G proteins AGS1 and Rhes selectively influence Gαi-dependent signaling to modulate N-type (CaV2.2) calcium channels

Activator of G protein Signaling 1 (AGS1) and Ras homologue enriched in striatum (Rhes) define a new group of Ras-like monomeric G proteins whose signaling properties and physiological roles are just beginning to be understood. Previous results suggest that AGS1 and Rhes exhibit distinct preferences for heterotrimeric G proteins, with AGS1 selectively influencing Galphai and Rhes selectively influencing Galphas. Here, we demonstrate that AGS1 and Rhes trigger nearly identical modulation of N-type Ca(2+) channels (Ca(V)2.2) by selectively altering Galphai-dependent signaling. Whole-cell currents were recorded from HEK293 cells expressing Ca(V)2.2 and Galphai- or Galphas-coupled receptors. AGS1 and Rhes reduced basal current densities and triggered tonic voltage-dependent (VD) inhibition of Ca(V)2.2. Additionally, each protein attenuated agonist-initiated channel inhibition through Galphai-coupled receptors without reducing channel inhibition through a Galphas-coupled receptor. The above effects of AGS1 and Rhes were blocked by pertussis toxin (PTX) or by expression of a Gbetagamma-sequestering peptide (masGRK3ct). Transfection with HRas, KRas2, Rap1A-G12V, Rap2B, Rheb2, or Gem failed to duplicate the effects of AGS1 and Rhes on Ca(V)2.2. Our data provide the first demonstration that AGS1 and Rhes exhibit similar if not identical signaling properties since both trigger tonic Gbetagamma signaling and both attenuate receptor-initiated signaling by the Gbetagamma subunits of PTX-sensitive G proteins. These results are consistent with the possibility that AGS1 and Rhes modulate Ca(2+) influx through Ca(V)2.2 channels under more physiological conditions and thereby influence Ca(2+)-dependent events such as neurosecretion.

Reconstituted Slow Muscarinic Inhibition of Neuronal (CaV1.2c) L-Type Ca2+ Channels

Ca(2+) influx through L-type channels is critical for numerous physiological functions. Relatively little is known about modulation of neuronal L-type Ca(2+) channels. We studied modulation of neuronal Ca(V)1.2c channels heterologously expressed in HEK293 cells with each of the known muscarinic acetylcholine receptor subtypes. Galphaq/11-coupled M1, M3, and M5 receptors each produced robust inhibition of Ca(V)1.2c, whereas Galphai/o-coupled M2 and M4 receptors were ineffective. Channel inhibition through M1 receptors was studied in detail and was found to be kinetically slow, voltage-independent, and pertussis toxin-insensitive. Slow inhibition of Ca(V)1.2c was blocked by coexpressing RGS2 or RGS3T or by intracellular dialysis with antibodies directed against Galphaq/11. In contrast, inhibition was not reduced by coexpressing betaARK1ct or Galphat. These results indicate that slow inhibition required signaling by Galphaq/11, but not Gbetagamma, subunits. Slow inhibition did not require Ca(2+) transients or Ca(2+) influx through Ca(V)1.2c channels. Additionally, slow inhibition was insensitive to pharmacological inhibitors of phospholipases, protein kinases, and protein phosphatases. Intracellular BAPTA prevented slow inhibition via a mechanism other than Ca(2+) chelation. The cardiac splice-variant of Ca(V)1.2 (Ca(V)1.2a) and a splice-variant of the neuronal/neuroendocrine Ca(V)1.3 channel also appeared to undergo slow muscarinic inhibition. Thus, slow muscarinic inhibition may be a general characteristic of L-type channels having widespread physiological significance.

Neurokinin 1 receptors trigger overlapping stimulation and inhibition of CaV2.3 (R-type) calcium channels.

Neurokinin (NK) 1 receptors and CaV2.3 calcium channels are both expressed in nociceptive neurons, and mice lacking either protein display altered responses to noxious stimuli. Here, we examined modulation of CaV2.3 through NK1 receptors expressed in human embryonic kidney 293 cells. We find that NK1 receptors generate complex modulation of CaV2.3. In particular, weak activation of these receptors evokes mainly stimulation of CaV2.3, whereas strong receptor activation elicits profound inhibition that overlaps with channel stimulation. Unlike R-type channels encoded by CaV2.3, L-type (CaV1.3), N-type (CaV2.2), and P/Q-type (CaV2.1) channels are inhibited, but not stimulated, through NK1 receptors. Pharmacological experiments show that protein kinase C (PKC) mediates stimulation of CaV2.3 through NK1 receptors. The signaling mechanisms underlying inhibition were explored by expressing proteins that buffer either Gαq/11 (regulator of G protein signaling protein 3T and carboxyl-terminal region of phospholipase C-β1) or Gβγ subunits (transducin and the carboxyl-terminal region of bovine G-protein-coupled receptor kinase). A fast component of inhibition was attenuated by buffering Gβγ, whereas a slow component of inhibition was reduced by buffering Gαq/11. When both Gβγ and Gαq/11 were simultaneously buffered in the same cells, inhibition was virtually eliminated, but receptor activation still triggered substantial stimulation of CaV2.3. We also report that NK1 receptors accelerate the inactivation kinetics of CaV2.3 currents. Altogether, our results indicate that NK1 receptors modulate CaV2.3 using three different signaling mechanisms: a fast inhibition mediated by Gβγ, a slow inhibition mediated by Gαq/11, and a slow stimulation mediated by PKC. This new information concerning R-type calcium channels and NK1 receptors may help in understanding nociception, synaptic plasticity, and other physiological processes.

Temperature and synaptic efficacy in frog skeletal muscle.

1. Intracellular recording and voltage‐clamp techniques were used to measure synaptic efficacy and the safety factor for neuromuscular transmission in frog skeletal muscle. All measurements were made in normal Ringer solution, in the absence of presynaptic or postsynaptic blocking agents. 2. Over a broad temperature range (10‐30 degrees C), a small percentage of sartorius fibres (about 6%) could be found which produced only subthreshold end‐plate potentials and no action potential in response to single, supramaximal nerve shock. At lower temperatures the proportion of such fibres increased; 42% of the fibres had subthreshold transmission at 5 degrees C, and 59% were subthreshold at 2.5 degrees C. 3. Threshold current, measured by intracellularly injecting short pulses of depolarizing current at end‐plate regions, was independent of temperature between 2.5 and 20 degrees C. Thus, the reduced synaptic efficacy observed at low temperatures was not due to decreased electrical excitability of the postsynaptic membrane. 4. The amplitude of evoked end‐plate currents (EPCs) decreased with cooling. At temperatures below 10 degrees C, the evoked EPCs at many end‐plates were too small to initiate action potentials. The decline in EPC amplitude was due to three factors: a decrease in the amplitude of single quantum currents (MEPCs), an increase in the temporal dispersion of transmitter release, and (below 5 degrees C) a decrease in quantal content. 5. The safety factor for neuromuscular transmission decreased dramatically as temperature was lowered. At 30 degrees C average safety factor was large and positive (540 nA), but at 2.5 degrees C it was negative (‐78 nA). 6. The quantal content of evoked transmitter release was independent of temperature change between 5 and 30 degrees C, the average value over this range being 180. However, at temperatures below 5 degrees C, quantal content fell off sharply (average value = 37). 7. The thermal independence of transmitter release may be an important mechanism in allowing poikilothermic animals to maintain physiological function over a wide range of body temperatures.

CA2+ CURRENT ACTIVATION RATE CORRELATES WITH ALPHA 1 SUBUNIT DENSITY

We report here that L-type Ca2+ channels activate rapidly in myotubes expressing current at high density and slowly in myotubes expressing current at low density. Partial block of the current in individual cells does not slow activation, indicating that Ca2+ influx does not link activation rate to current density. Activation rate is positively correlated with the density of gating charge (Qmax) associated with the L-type Ca2+ channels. The range of values for Qmax, and the relationship between activation rate and Qmax, are similar for myotubes expressing native or recombinant L-type Ca2+ channels, whereas peak Ca2+ current density is approximately 3-fold higher for native channels. Taken together, these results suggest that Ca2+ channel density can govern activation kinetics. Our findings have important important implications for studies of ion channel function because they suggest that biophysical properties can be significantly influenced by channel density, both in heterologous expression systems and in native tissues.

Phosphorylation-Dependent Regulation of Voltage-Gated Ca2+ Channels

Overview N eurotransmitters, hormones, growth factors and extracellular matrix proteins bind to cell membrane receptors that, in turn, activate intracellular signaling cascades. Often, these cascades involve signaling by protein or lipid kinases and protein or lipid phosphatases. Protein kinases add phosphate groups to serine, threonine or tyrosine residues within proteins, and lipid kinases add phosphates to the inositol rings of certain lipids; phosphatases reverse this process. Phosphorylation or dephosphorylation can switch-on** or switch-ofF* protein or lipid activity. It has long been known that voltage-gated Câ * channels are regulated through signaling events involving phosphorylation. In this chapter, we summarize recent findings in this field. We have attempted to minimize overlap with other recent reviews. Abbreviations AC= adenylyl cyclase; Akt/PKB= protein kinase B; AKAP= A kinase anchoring protein; p2AR= beta 2 adrenergic receptor; CaM= calmodulin; CaMKII= Ca^*/calmodulin-dependent protein kinase II; GPCR= G-protein-coupled receptor, NCS-1= neuronal calcium sensor-1; nNOS= neuronal nitric oxide synthase; PDE= phosphodiesterase; PI3K= phosphoinositide 3-kinase; PIP2= phosphatidylinositol 4,5-bisphosphate; PIP3= phosphatidylinositol 3,4,5-trisphosphate; PKA= protein kinase A; PKC= protein kinase C; PKG= protein kinase G; PLC= phospholipase C; PP2A= protein phosphatase 2A; RTK= receptor tyrosine kinase; sGC= soluble guanylyl cyclase.