The purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline–cobalt/H+ and Na+(K+)/H+ exchange

Recent work has suggested that the chromosomally encoded TetA(L) transporter of Bacillus subtilis, for which no physiological function had been shown earlier, not only confers resistance to low concentrations of tetracycline but is also a multifunctional antiporter protein that has dominant roles in both Na⁺- and K⁺-dependent pH homeostasis and in Na⁺ resistance during growth at alkaline pH. To rigorously test this hypothesis, TetA(L) has been purified with a hexahistidine tag at its C terminus and reconstituted into proteoliposomes. The TetA(L)–hexahistidine proteoliposomes exhibit high activities of tetracycline–cobalt/H⁺, Na⁺/H⁺, and K⁺/H⁺ antiport in an assay in which an outwardly directed proton gradient is artificially imposed and solute uptake is monitored. Tetracycline uptake depends on the presence of cobalt and vice versa, with the cosubstrates being transported in a 1:1 ratio. Evidence for the electrogenicity of both tetracycline–cobalt/H⁺ and Na⁺/H⁺ antiports is presented. K⁺ and Li⁺ inhibit Na⁺ uptake, but there is little cross-inhibition between Na⁺ and tetracycline–cobalt uptake activities. The results strongly support the conclusion that TetA(L) is a multifunctional antiporter. They expand the roster of such porters to encompass one with a complex organic substrate and monovalent cation substrates that may have distinct binding domains, and provide the first functional reconstitution of a member of the 14-transmembrane segment transporter family.     

Cloning and characterization of a potassium-coupled amino acid transporter

Active solute uptake in bacteria, fungi, plants, and animals is known to be mediated by cotransporters that are driven by Na⁺ or H⁺ gradients. The present work extends the Na⁺ and H⁺ dogma by including the H⁺ and K⁺ paradigm. Lepidopteran insect larvae have a high K⁺ and a low Na⁺ content, and their midgut cells lack Na⁺/K⁺ ATPase. Instead, an H⁺ translocating, vacuolar-type ATPase generates a voltage of approximately −240 mV across the apical plasma membrane of so-called goblet cells, which drives H⁺ back into the cells in exchange for K⁺, resulting in net K⁺ secretion into the lumen. The resulting inwardly directed K⁺ electrochemical gradient serves as a driving force for active amino acid uptake into adjacent columnar cells. By using expression cloning with Xenopus laevis oocytes, we have isolated a cDNA that encodes a K⁺-coupled amino acid transporter (KAAT1). We have cloned this protein from a larval lepidopteran midgut (Manduca sexta) cDNA library. KAAT1 is expressed in absorptive columnar cells of the midgut and in labial glands. When expressed in Xenopus oocytes, KAAT1 induced electrogenic transport of neutral amino acids but excludes α-(methylamino)isobutyric acid and charged amino acids resembling the mammalian system B. K⁺, Na⁺, and to a lesser extent Li⁺ were accepted as cotransported ions, but K⁺ is the principal cation, by far, in living caterpillars. Moreover, uptake was Cl⁻-dependent, and the K⁺/Na⁺ selectivity increased with hyperpolarization of oocytes, reflecting the increased K⁺/Na⁺ selectivity with hyperpolarization observed in midgut tissue. KAAT1 has 634 amino acid residues with 12 putative membrane spanning domains and shows a low level of identity with members of the Na⁺ and Cl⁻-coupled neurotransmitter transporter family.     

Does the colonic H,K-ATPase also act as an Na,K-ATPase?

We previously have demonstrated that the colonic P-ATPase α subunit cDNA encodes an H,K-ATPase when expressed in Xenopus laevis oocytes. Besides its high level of amino acid homology (75%) with the Na,K-ATPase, the colonic H,K-ATPase also shares a common pharmacological profile with Na,K-ATPase, because both are ouabain-sensitive and Sch 28080-insensitive. These features raise the possibility that an unrecognized property of the colonic H,K-ATPase would be Na⁺ translocation. To test this hypothesis, ion-selective microelectrodes were used to measure the intracellular Na⁺ activity of X. laevis oocytes expressing various combinations of P-ATPase subunits. The results show that expression in oocytes of the colonic H,K-ATPase affects intracellular Na⁺ homeostasis in a way similar to the expression of the Bufo marinus Na,K-ATPase; intracellular Na⁺ activity is lower in oocytes expressing the colonic H,K-ATPase or the B. marinus Na,K-ATPase than in oocytes expressing the gastric H,K-ATPase or a β subunit alone. In oocytes expressing the colonic H,K-ATPase, the decrease in intracellular Na⁺ activity persists when diffusive Na⁺ influx is enhanced by functional expression of the amiloride-sensitive epithelial Na⁺ channel, suggesting that the decrease is related to increased active Na⁺ efflux. The Na⁺ decrease depends on the presence of K⁺ in the external medium and is inhibited by 2 mM ouabain, a concentration that inhibits the colonic H,K-ATPase. These data are consistent with the hypothesis that the colonic H,K-ATPase may transport Na⁺, acting as an (Na,H),K-ATPase. Despite its molecular and functional characterization, the physiological role of the colonic (Na,H),K-ATPase in colonic and renal ion homeostasis remains to be elucidated.     

A regenerative link in the ionic fluxes through the weaver potassium channel underlies the pathophysiology of the mutation

The homozygous weaver mouse displays neuronal degeneration in several brain regions. Previous experiments in heterologous expression systems showed that the G protein-gated inward rectifier K⁺ channel (GIRK2) bearing the weaver pore-region GYG-to-SYG mutation (i) is not activated by G_βγ subunits, but instead shows constitutive activation, and (ii) is no longer a K⁺-selective channel but conducts Na⁺ as well. The present experiments on weaverGIRK2 (wvGIRK2) expressed in Xenopus oocytes show that the level of constitutive activation depends on intracellular Na⁺ concentration. In particular, manipulations that decrease intracellular Na⁺ produce a component of Na⁺-permeable current activated via a G protein pathway. Therefore, constitutive activation may not arise because the weaver mutation directly alters the gating transitions of the channel protein. Instead, there may be a regenerative cycle of Na⁺ influx through the wvGIRK2 channel, leading to additional Na⁺ activation. We also show that the wvGIRK2 channel is permeable to Ca²⁺, providing an additional mechanism for the degeneration that characterizes the weaver phenotype. We further demonstrate that the GIRK4 channel bearing the analogous weaver mutation has properties similar to those of the wvGIRK2 channel, providing a glimpse of the selective pressures that have maintained the GYG sequence in nearly all known K⁺ channels.     

Molecular and functional characterization of a novel low-affinity cation transporter (LCT1) in higher plants

The transport of cations across membranes in higher plants plays an essential role in many physiological processes including mineral nutrition, cell expansion, and the transduction of environmental signals. In higher plants the coordinated expression of transport mechanisms is essential for specialized cellular processes and for adaptation to variable environmental conditions. To understand the molecular basis of cation transport in plant roots, a Triticum aestivum cDNA library was used to complement a yeast mutant deficient in potassium (K⁺) uptake. Two genes were cloned that complemented the mutant: HKT1 and a novel cDNA described in this report encoding a cation transporter, LCT1 (low-affinity cation transporter). Analysis of the secondary structure of LCT1 suggests that the protein contains 8–10 transmembrane helices and a hydrophilic amino terminus containing sequences enriched in Pro, Ser, Thr, and Glu (PEST). The transporter activity was assayed using radioactive isotopes in yeast cells expressing the cDNA. LCT1 mediated low-affinity uptake of the cations Rb⁺ and Na⁺, and possibly allowed Ca²⁺ but not Zn²⁺ uptake. LCT1 is expressed in low abundance in wheat roots and leaves. The precise functional role of this cation transporter is not known, although the competitive inhibition of cation uptake by Ca²⁺ has parallels to whole plant and molecular studies that have shown the important role of Ca²⁺ in reducing Na⁺ uptake and ameliorating Na⁺ toxicity. The structure of this higher plant ion transport protein is unique and contains PEST sequences.     

An Arabidopsis mutant that requires increased calcium for potassium nutrition and salt tolerance

Potassium (K⁺) nutrition and salt tolerance are key factors controlling plant productivity. However, the mechanisms by which plants regulate K⁺ nutrition and salt tolerance are poorly understood. We report here the identification of an Arabidopsis thaliana mutant, sos3 (salt-overly-sensitive 3), which is hypersensitive to Na⁺ and Li⁺ stresses. The mutation is recessive and is in a nuclear gene that maps to chromosome V. The sos3 mutation also renders the plant unable to grow on low K⁺. Surprisingly, increased extracellular Ca²⁺ suppresses the growth defect of sos3 plants on low K⁺ or 50 mM NaCl. In contrast, high concentrations of external Ca²⁺ do not rescue the growth of the salt-hypersensitive sos1 mutant on low K⁺ or 50 mM NaCl. Under NaCl stress, sos3 seedlings accumulated more Na⁺ and less K⁺ than the wild type. Increased external Ca²⁺ improved K⁺/Na⁺ selectivity of both sos3 and wild-type plants. However, this Ca²⁺ effect in sos3 is more than twice as much as that in the wild type. In addition to defining the first plant mutant with an altered calcium response, these results demonstrate that the SOS3 locus is essential for K⁺ nutrition, K⁺/Na⁺ selectivity, and salt tolerance in higher plants.     

The selectivity of the hair cell’s mechanoelectrical-transduction channel promotes Ca2+ flux at low Ca2+ concentrations

The mechanoelectrical-transduction channel of the hair cell is permeable to both monovalent and divalent cations. Because Ca²⁺ entering through the transduction channel serves as a feedback signal in the adaptation process that sets the channel’s open probability, an understanding of adaptation requires estimation of the magnitude of Ca²⁺ influx. To determine the Ca²⁺ current through the transduction channel, we measured extracellular receptor currents with transepithelial voltage-clamp recordings while the apical surface of a saccular macula was bathed with solutions containing various concentrations of K⁺, Na⁺, or Ca²⁺. For modest concentrations of a single permeant cation, Ca²⁺ carried much more receptor current than did either K⁺ or Na⁺. For higher cation concentrations, however, the flux of Na⁺ or K⁺ through the transduction channel exceeded that of Ca²⁺. For mixtures of Ca²⁺ and monovalent cations, the receptor current displayed an anomalous mole-fraction effect, which indicates that ions interact while traversing the channel’s pore. These results demonstrate not only that the hair cell’s transduction channel is selective for Ca²⁺ over monovalent cations but also that Ca²⁺ carries substantial current even at low Ca²⁺ concentrations. At physiological cation concentrations, Ca²⁺ flux through transduction channels can change the local Ca²⁺ concentration in stereocilia in a range relevant for the control of adaptation.     

Na+ pump low and high ouabain affinity α subunit isoforms are differently distributed in cells

Three isoforms (α1, α2, and α3) of the catalytic (α) subunit of the plasma membrane (PM) Na⁺ pump have been identified in the tissues of birds and mammals. These isoforms differ in their affinities for ions and for the Na⁺ pump inhibitor, ouabain. In the rat, α1 has an unusually low affinity for ouabain. The PM of most rat cells contains both low (α1) and high (α2 or α3) ouabain affinity isoforms, but precise localization of specific isoforms, and their functional significance, are unknown. We employed high resolution immunocytochemical techniques to localize α subunit isoforms in primary cultured rat astrocytes, neurons, and arterial myocytes. Isoform α1 was ubiquitously distributed over the surfaces of these cells. In contrast, high ouabain affinity isoforms (α2 in astrocytes, α3 in neurons and myocytes) were confined to a reticular distribution within the PM that paralleled underlying endoplasmic or sarcoplasmic reticulum. This distribution is identical to that of the PM Na/Ca exchanger. This raises the possibility that α1 may regulate bulk cytosolic Na⁺, whereas α2 and α3 may regulate Na⁺ and, indirectly, Ca²⁺ in a restricted cytosolic space between the PM and reticulum. The high ouabain affinity Na⁺ pumps may thereby modulate reticulum Ca²⁺ content and Ca²⁺ signaling.     

Cotransport of water by the Na+/glucose cotransporter

Water is transported across epithelial membranes in the absence of any hydrostatic or osmotic gradients. A prime example is the small intestine, where 10 liters of water are absorbed each day. Although water absorption is secondary to active solute transport, the coupling mechanism between solute and water flow is not understood. We have tested the hypothesis that water transport is directly linked to solute transport by cotransport proteins such as the brush border Na⁺/glucose cotransporter. The Na⁺/glucose cotransporter was expressed in Xenopus oocytes, and the changes in cell volume were measured under sugar-transporting and nontransporting conditions. We demonstrate that 260 water molecules are directly coupled to each sugar molecule transported and estimate that in the human intestine this accounts for 5 liters of water absorption per day. Other animal and plant cotransporters such as the Na⁺/Cl⁻/γ-aminobutyric acid, Na⁺/iodide and H⁺/amino acid transporters are also able to transport water and this suggests that cotransporters play an important role in water homeostasis.     

Point mutations in segment I-S6 render voltage-gated Na+ channels resistant to batrachotoxin

Batrachotoxin (BTX) is a steroidal alkaloid that causes Na⁺ channels to open persistently. This toxin has been used widely as a tool for studying Na⁺ channel gating processes and for estimating Na⁺ channel density. In this report we used point mutations to identify critical residues involved in BTX binding and to examine if such mutations affect channel gating. We show that a single asparagine→lysine substitution of the rat muscle Na⁺ channel α-subunit, μ1-N434K, renders the channel completely insensitive to 5 μM BTX when expressed in mammalian cells. This mutant channel nonetheless displays normal current kinetics with minimal changes in gating properties. Another substitution, μ1-N434A, yields a partial BTX-sensitive mutant. Unlike wild-type currents, the BTX-modified μ1-N434A currents continue to undergo fast and slow inactivation as if the inactivation processes remain functional. This finding implies that the μ1-N434 residue upon binding with BTX is critical for subsequent changes on gating; alanine at the μ1–434 position apparently diminishes the efficacy of BTX on eliminating Na⁺ channel inactivation. Mutants of two adjacent residues, μ1-I433K and μ1-L437K, also were found to exhibit the identical BTX-resistant phenotype. We propose that the μ1-I433, μ1-N434, and μ1-L437 residues in transmembrane segment I-S6 probably form a part of the BTX receptor.     

Glutamate transport in Rhodobacter sphaeroides is mediated by a novel binding protein-dependent secondary transport system

Growth of a glutamate transport-deficient mutant of Rhodobacter sphaeroides on glutamate as sole carbon and nitrogen source can be restored by the addition of millimolar amounts of Na⁺. Uptake of glutamate (K_t of 0.2 μM) by the mutant strictly requires Na⁺ (K_m of 25 mM) and is inhibited by ionophores that collapse the proton motive force (pmf). The activity is osmotic-shock-sensitive and can be restored in spheroplasts by the addition of osmotic shock fluid. Transport of glutamate is also observed in membrane vesicles when Na⁺, a proton motive force, and purified glutamate binding protein are present. Both transport and binding is highly specific for glutamate. The Na⁺-dependent glutamate transporter of Rb. sphaeroides is an example of a secondary transport system that requires a periplasmic binding protein and may define a new family of bacterial transport proteins.     

Salt restriction induces pseudohypoaldosteronism type 1 in mice expressing low levels of the β-subunit of the amiloride-sensitive epithelial sodium channel

The amiloride-sensitive epithelial sodium channel (ENaC) is a heteromultimer of three homologous subunits (α-, β-, and γ-subunits). To study the role of the β-subunit in vivo, we analyzed mice in which the βENaC gene locus was disrupted. These mice showed low levels of βENaC mRNA expression in kidney (≈1%), lung (≈1%), and colon (≈4%). In homozygous mutant βENaC mice, no βENaC protein could be detected with immunofluorescent staining. At birth, there was a small delay in lung-liquid clearance that paralleled diminished amiloride-sensitive Na⁺ absorption in tracheal explants. With normal salt intake, these mice showed a normal growth rate. However, in vivo, adult βENaC m/m mice exhibited a significantly reduced ENaC activity in colon and elevated plasma aldosterone levels, suggesting hypovolemia and pseudohypoaldosteronism type 1. This phenotype was clinically silent, as βENaC m/m mice showed no weight loss, normal plasma Na⁺ and K⁺ concentrations, normal blood pressure, and a compensated metabolic acidosis. On low-salt diets, βENaC-mutant mice developed clinical symptoms of an acute pseudohypoaldosteronism type 1 (weight loss, hyperkalemia, and decreased blood pressure), indicating that βENaC is required for Na⁺ conservation during salt deprivation.     

Crystal structure of troponin C in complex with troponin I fragment at 2.3-Å resolution

Troponin (Tn), the complex of three subunits (TnC, TnI, and TnT), plays a key role in Ca²⁺-dependent regulation of muscle contraction. To elucidate the interactions between the Tn subunits and the conformation of TnC in the Tn complex, we have determined the crystal structure of TnC (two Ca²⁺ bound state) in complex with the N-terminal fragment of TnI (TnI_1–47). The structure was solved by the single isomorphous replacement method in combination with multiple wavelength anomalous dispersion data. The refinement converged to a crystallographic R factor of 22.2% (R_free = 32.6%). The central, connecting α-helix observed in the structure of uncomplexed TnC (TnC_free) is unwound at the center (residues Ala-87, Lys-88, Gly-89, Lys-90, and Ser-91) and bent by 90°. As a result, TnC in the complex has a compact globular shape with direct interactions between the N- and C-terminal lobes, in contrast to the elongated dumb-bell shaped molecule of uncomplexed TnC. The 31-residue long TnI_1–47 α-helix stretches on the surface of TnC and stabilizes its compact conformation by multiple contacts with both TnC lobes. The amphiphilic C-end of the TnI_1–47 α-helix is bound in the hydrophobic pocket of the TnC C-lobe through 38 van der Waals interactions. The results indicate the major difference between Ca²⁺ receptors integrated with the other proteins (TnC in Tn) and isolated in the cytosol (calmodulin). The TnC/TnI_1–47 structure implies a mechanism of how Tn regulates the muscle contraction and suggests a unique α-helical regulatory TnI segment, which binds to the N-lobe of TnC in its Ca²⁺ bound conformation.     

The Arabidopsis thaliana proton transporters, AtNhx1 and Avp1, can function in cation detoxification in yeast

Overexpression of the Arabidopsis thaliana vacuolar H⁺-pyrophosphatase (AVP1) confers salt tolerance to the salt-sensitive ena1 mutant of Saccharomyces cerevisiae. Suppression of salt sensitivity requires two ion transporters, the Gef1 Cl⁻ channel and the Nhx1 Na⁺/H⁺ exchanger. These two proteins colocalize to the prevacuolar compartment of yeast and are thought to be required for optimal acidification of this compartment. Overexpression of AtNHX1, the plant homologue of the yeast Na⁺/H⁺ exchanger, suppresses some of the mutant phenotypes of the yeast nhx1 mutant. Moreover, the level of AtNHX1 mRNA in Arabidopsis is increased in the presence of NaCl. The regulation of AtNHX1 by NaCl and the ability of the plant gene to suppress the yeast nhx1 mutant suggest that the mechanism by which cations are detoxified in yeast and plants may be similar.     

Stabilization of ion selectivity filter by pore loop ion pairs in an inwardly rectifying potassium channel

Ion selectivity is critical for the biological functions of voltage-dependent cation channels and is achieved by specific ion binding to a pore region called the selectivity filter. In voltage-gated K⁺, Na⁺ and Ca²⁺ channels, the selectivity filter is formed by a short polypeptide loop (called the H5 or P region) between the fifth and sixth transmembrane segments, donated by each of the four subunits or internal homologous domains forming the channel. While mutagenesis studies on voltage-gated K⁺ channels have begun to shed light on the structural organization of this pore region, little is known about the physical and chemical interactions that maintain the structural stability of the selectivity filter. Here we show that in an inwardly rectifying K⁺ (IRK) channel, IRK1, short range interactions of an ion pair in the H5 pore loop are crucial for pore structure and ion permeation. The two residues, a glutamate and an arginine, appear to form exposed salt bridges in the tetrameric channel. Alteration or disruption of such ion pair interactions dramatically alters ion selectivity and permeation. Since this ion pair is conserved in all IRK channels, it may constitute a general mechanism for maintaining the stability of the pore structure in this channel superfamily.     

Sodium channel selectivity filter regulates antiarrhythmic drug binding

Local anesthetic antiarrhythmic drugs block Na⁺ channels and have important clinical uses. However, the molecular mechanism by which these drugs block the channel has not been established. The family of drugs is characterized by having an ionizable amino group and a hydrophobic tail. We hypothesized that the charged amino group of the drug may interact with charged residues in the channel’s selectivity filter. Mutation of the putative domain III selectivity filter residue of the adult rat skeletal muscle Na⁺ channel (μ1) K1237E increased resting lidocaine block, but no change was observed in block by neutral analogs of lidocaine. An intermediate effect on the lidocaine block resulted from K1237S and there was no effect from K1237R, implying an electrostatic effect of Lys. Mutation of the other selectivity residues, D400A (domain I), E755A (domain II), and A1529D (domain IV) allowed block by externally applied quaternary membrane-impermeant derivatives of lidocaine (QX314 and QX222) and accelerated recovery from block by internal QX314. Neo-saxitoxin and tetrodotoxin, which occlude the channel pore, reduced the amount of QX314 bound in D400A and A1529D, respectively. Block by outside QX314 in E755A was inhibited by mutation of residues in transmembrane segment S6 of domain IV that are thought to be part of an internal binding site. The results demonstrate that the Na⁺ channel selectivity filter is involved in interactions with the hydrophilic part of the drugs, and it normally limits extracellular access to and escape from their binding site just within the selectivity filter. Participation of the selectivity ring in antiarrhythmic drug binding and access locates this structure adjacent to the S6 segment.     

Two serine residues of the glutamate transporter GLT-1 are crucial for coupling the fluxes of sodium and the neurotransmitter

The neurotoxicity of glutamate in the central nervous system is restricted by several (Na⁺ + K⁺)-coupled transporters for this neurotransmitter. The astroglial transporter GLT-1 is the only subtype that exhibits high sensitivity to the nontransportable glutamate analogue dihydrokainate. A marked reduction in sensitivity to the blocker is observed when serine residues 440 and 443 are mutated to glycine and glutamine, which, respectively, occupy these positions in the other homologous glutamate transporters. They are located in the ascending limb of the recently identified pore-loop-like structure. Strikingly, mutation of serine-440 to glycine enables not only sodium but also lithium ions to drive net influx of acidic amino acids. Moreover, the efficiency of lithium as a driving ion for glutamate transport depends on the nature of the amino acid residue present at position 443. Mutant transporters containing single cysteines at the position of either serine residue become sensitive to positively as well as negatively charged methanethiosulfonate derivatives. In S440C transporters significant protection against this inhibition is provided both by transportable and nontransportable glutamate analogues, but not by sodium alone. Our observations indicate that the pore-loop-like structure plays a pivotal role in coupling ion and glutamate fluxes and suggest that it is close to the glutamate-binding site.     

Disruption of the β subunit of the epithelial Na+ channel in mice: Hyperkalemia and neonatal death associated with a pseudohypoaldosteronism phenotype

The epithelial Na⁺ channel (ENaC) is composed of three homologous subunits: α, β and γ. We used gene targeting to disrupt the β subunit gene of ENaC in mice. The βENaC-deficient mice showed normal prenatal development but died within 2 days after birth, most likely of hyperkalemia. In the −/− mice, we found an increased urine Na⁺ concentration despite hyponatremia and a decreased urine K⁺ concentration despite hyperkalemia. Moreover, serum aldosterone levels were increased. In contrast to αENaC-deficient mice, which die because of defective lung liquid clearance, neonatal βENaC deficient mice did not die of respiratory failure and showed only a small increase in wet lung weight that had little, if any, adverse physiologic consequence. The results indicate that, in vivo, the β subunit is required for ENaC function in the renal collecting duct, but, in contrast to the α subunit, the β subunit is not required for the transition from a liquid-filled to an air-filled lung. The phenotype of the βENaC-deficient mice is similar to that of humans with pseudohypoaldosteronism type 1 and may provide a useful model to study the pathogenesis and treatment of this disorder.     

Depletion of Ca2+ from the sarcoplasmic reticulum of cardiac muscle prompts phosphorylation of phospholamban to stimulate store refilling

Nonmuscle cells have almost ubiquitously evolved a mechanism to detect and prevent Ca²⁺ store depletion—store operated calcium entry. No such mechanism has, as yet, been reported in cardiac myocytes. However, it is conceivable that such a mechanism may play an important role in cardiac Ca²⁺ homeostasis to ensure the availability of sufficient stored Ca²⁺ to maintain normal excitation contraction coupling. We present data that confirms the presence of a mechanism that is able to monitor the Ca²⁺ load of the SR and initiate a signaling process to accelerate Ca²⁺ uptake by the SR when store depletion is detected. Depletion of SR Ca²⁺ activates a protein kinase, the principal SR substrate of which is phospholamban. Phosphorylation of this SR protein promotes Ca²⁺ pump activity and therefore store refilling. Furthermore, a protein kinase activity associated with the SR that is inhibited by Ca²⁺ ions has been identified. We have measured lumenal [Ca²⁺] by using a fluorescent Ca²⁺ indicator and found that by initiating Ca²⁺ uptake and increasing Ca²⁺ load, we can inhibit the protein kinase activity associated with the SR. This confirms that a protein kinase, that is regulated by lumenal [Ca²⁺], has been identified and represents part of a previously unidentified signalling cascade. This local feedback mechanism would allow the myocyte to detect and prevent SR Ca²⁺ load depletion.     

R-type Ca2+ currents evoke transmitter release at a rat central synapse

Voltage-dependent Ca²⁺ currents evoke synaptic transmitter release. Of six types of Ca²⁺ channels, L-, N-, P-, Q-, R-, and T-type, only N- and P/Q-type channels have been pharmacologically identified to mediate action-potential-evoked transmitter release in the mammalian central nervous system. We tested whether Ca²⁺ channels other than N- and P/Q-type control transmitter release in a calyx-type synapse of the rat medial nucleus of the trapezoid body. Simultaneous recordings of presynaptic Ca²⁺ influx and the excitatory postsynaptic current evoked by a single action potential were made at single synapses. The R-type channel, a high-voltage-activated Ca²⁺ channel resistant to L-, N-, and P/Q-type channel blockers, contributed 26% of the total Ca²⁺ influx during a presynaptic action potential. This Ca²⁺ current evoked transmitter release sufficiently large to initiate an action potential in the postsynaptic neuron. The R-type current controlled release with a lower efficacy than other types of Ca²⁺ currents. Activation of metabotropic glutamate receptors and γ-aminobutyric acid type B receptors inhibited the R-type current. Because a significant fraction of presynaptic Ca²⁺ channels remains unidentified in many other central synapses, the R-type current also could contribute to evoked transmitter release in these synapses.