Conformational transitions in the Ca2+ + Mg2+-activated ATPase and the binding of Ca2+ ions.

We have studied the fluorescence of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum labelled with fluorescein isothiocyanate. The change in intensity of fluorescein fluorescence caused by addition of Ca2+ to the labelled ATPase can be interpreted in terms of a two-conformation model for the ATPase, one conformation (E1) having a high affinity for Ca2+, the other (E2) a low affinity. Effects of Ca2+ as a function of pH allow an estimate of the effect of pH on the E1/E2 ratio, consistent with kinetic studies. A model is presented for binding of Ca2+ to the ATPase as a function of pH that is consistent both with the data on the E1/E2 equilibrium and with literature data on Ca2+ binding.     

Regulation of cardiac L-type Ca2+ current in Na+-Ca2+ exchanger knockout mice: functional coupling of the Ca2+ channel and the Na+-Ca2+ exchanger

L-type Ca2+ current (I(Ca)) is reduced in myocytes from cardiac-specific Na+-Ca2+ exchanger (NCX) knockout (KO) mice. This is an important adaptation to prevent Ca2+ overload in the absence of NCX. However, Ca2+ channel expression is unchanged, suggesting that regulatory processes reduce I(Ca). We tested the hypothesis that an elevation in local Ca2+ reduces I(Ca) in KO myocytes. In patch-clamped myocytes from NCX KO mice, peak I(Ca) was reduced by 50%, and inactivation kinetics were accelerated as compared to wild-type (WT) myocytes. To assess the effects of cytosolic Ca2+ concentration on I(Ca), we used Ba2+ instead of Ca2+ as the charge carrier and simultaneously depleted sarcoplasmic reticular Ca2+ with thapsigargin and ryanodine. Under these conditions, we observed no significant difference in Ba2+ current between WT and KO myocytes. Also, dialysis with the fast Ca2+ chelator BAPTA eliminated differences in both I(Ca) amplitude and decay kinetics between KO and WT myocytes. We conclude that, in NCX KO myocytes, Ca2+-dependent inactivation of I(Ca) reduces I(Ca) amplitude and accelerates current decay kinetics. We hypothesize that the elevated subsarcolemmal Ca2+ that results from the absence of NCX activity inactivates some L-type Ca2+ channels. Modulation of subsarcolemmal Ca2+ by the Na+-Ca2+ exchanger may be an important regulator of excitation-contraction coupling.     

Residues contributing to the Ca2+ and K+ binding pocket of the NCKX2 Na+/Ca2+-K+ exchanger

The Na(+)/Ca(2+)-K(+) exchanger (NCKX) extrudes Ca(2+) from cells utilizing both the inward Na(+) gradient and the outward K(+) gradient. NCKX is thought to operate by a consecutive mechanism in which a cation binding pocket accommodates both Ca(2+) and K(+) and alternates between inward and outward facing conformations. Here we developed a simple fluorometric method to analyze changes in K(+) and Ca(2+) dependences of mutant NCKX2 proteins in which candidate residues within membrane-spanning domains were substituted. The largest shifts in both K(+) and Ca(2+) dependences compared with wild-type NCKX2 were observed for the charge-conservative substitutions of Glu(188) and Asp(548), whereas the size-conservative substitutions resulted in nonfunctional proteins. Substitution of several other residues including two proline residues (Pro(187) and Pro(547)), three additional acidic residues (Asp(258), Glu(265), Glu(533)), and two hydroxyl-containing residues (Ser(185) and Ser(545)) showed smaller shifts, but shifts in Ca(2+) dependence were invariably accompanied by shifts in K(+) dependence. We conclude that Glu(188) and Asp(548) are the central residues of a single cation binding pocket that can accommodate both K(+) and Ca(2+). Furthermore, a single set of residues lines a transport pathway for both K(+) and Ca(2+).     

The Ca 2+ pumps and the Na + / Ca 2+ exchangers

The Ca 2+ ATPases or Ca 2+ pumps transport Ca 2+ ions out of the cytosol, by using the energy stored in ATP. The Na + / Ca 2+ exchanger uses the chemical energy of the Na + gradient (the Na + concentration is much higher outside than inside the cell) to remove Ca 2+ from the cytosol. Ca 2+ pumps are found in the plasma membrane and in the endoplasmic reticulum of the cells. The pumps are probably present in the membrane of other organelles, but little experimental information is available on this matter. The Na + / Ca 2+ exchangers are located on the plasma membrane. A Na + / Ca 2+ exchanger was found in the mitochondria, but very little is known on its structure and sequence. These transporters control the Ca 2+ concentration in the cytosol and are vital to prevent Ca 2+ overload of the cells. Their activity is controlled by different mechanisms, that are still under investigation. A number of the possible isoforms for both types of proteins has been detected.© Kluwer Academic Publishers     

Ca2+-calmodulin-dependent facilitation and Ca2+ inactivation of Ca2+ release-activated Ca2+ channels

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.     

How Does Regulatory Ca2+ Regulate the Na+-Ca2+ Exchanger?

Spatial and temporal regulation of intracellular Ca²⁺ concentrations is a fundamental requirement for life. The mammalian cardiac Na⁺-Ca²⁺ exchanger serves as the main mechanism for Ca²⁺ efflux after heart contraction. Exchange activity is highly regulated by intracellular Ca²⁺, which binds two regulatory domains (CBD1 and CBD2) and triggers the full activity of the exchanger. We solved the X-ray crystallographic structure of CBD2 in the presence and absence of Ca²⁺. Together with mutational analysis of the Ca²⁺ binding sites, this study reveals the crucial role of one of the two bound Ca²⁺ ions and helps propose hypotheses on the mechanism of regulation of the exchanger.     

Ca2+ and Mg2+ Influence the Thermodynamics of Peptide-Membrane Interactions

Accurate quantitative estimates of protein-membrane interactions are critical to studies of membrane proteins. Here, we demonstrate that thermodynamic analyses based on current hydropathy scales do not account for the significant and experimentally determined effects that Ca²⁺ or Mg²⁺ have on protein-membrane interactions. We examined distinct modes of interaction (interfacial partitioning and folding and transmembrane insertion) by studying three highly divergent peptides: Bid-BH3 (derived from apoptotic regulator Bid), peripherin-2-derived prph2-CTER, and the cancer-targeting pH-Low-Insertion-Peptide (pHLIP). Fluorescence experiments demonstrate that adding 1–2 mM of divalent cations led to a substantially more favorable bilayer partitioning and insertion, with free energy differences of 5–15 kcal/mol.     

A model for the phosphorylation of the Ca2+ + Mg2+-activated ATPase by phosphate.

We have shown that changes in fluorescence intensity for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum labelled with fluorescein isothiocyanate following the addition of Ca2+ can give the ratio of the two conformations (E1 and E2) of the ATPase. We show that the fluorescence response to Ca2+ is unaffected by Mg2+, phosphate or K+, implying that these ions bind equally well to the E1 and E2 conformations. A model is presented for phosphorylation of the ATPase by phosphate as a function of pH, Mg2+, K+ and Ca2+.     

Ca2+ dependence of the Ca2+-selective TRPV6 channel

Microfluorimetry and patch-clamp experiments were performed on TRPV6-expressing HEK cells to determine whether this Ca(2+)-sensing Ca(2+) channel is constitutively active. Intact cells loaded with fura-2 had an elevated intracellular free Ca(2+) concentration ([Ca(2+)](i)), which decreased to the same level such as in non-transfected cells if external Ca(2+) was chelated by EGTA. Whole cell recordings from non-transfected HEK cells and cells expressing human TRPV6 revealed the presence of a basal inward current in both types of cells when the internal solution contained 0.1 mm EGTA and 100 nm [Ca(2+)](i) or if the cytosolic Ca(2+) buffering remained undisturbed in perforated patch-clamp experiments. If recombinantly expressed TRPV6 forms open channels, one would expect Ca(2+)-induced current inhibition, because TRPV6 is negatively regulated by internal Ca(2+). However, dialyzing solutions with high [Ca(2+)] such as 1 microm into TRPV6-expressing cells did not block the basal inward current, which was not different from the recordings from non-transfected cells. In contrast, dialyzing 0.5 mm EGTA into TRPV6-expressing cells readily activated Ca(2+) inward currents, which were undetectable in non-transfected cells. Interestingly, monovalent cations permeated the TRPV6 channels under conditions where no Ca(2+) permeation was detectable, indicating that divalent cations block TRPV6 channels from the extracellular side. Like human TRPV6, the truncated human TRPV6(Delta695-725), which lacks the C-terminal domain required for Ca(2+)-calmodulin binding, does not form constitutive active channels, whereas the human TRPV6(D542A), carrying a point mutation in the presumed pore region, does not function as a channel. In summary, no constitutive open TRPV6 channels were detected in patch-clamp experiments from transfected HEK cells. However, channel activity is highly regulated by intracellular and extracellular divalent cations.     

A kinetic model for Ca2+ efflux mediated by the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum.

We present a model for Ca2+ efflux from vesicles of sarcoplasmic reticulum (SR). It is proposed that efflux is mediated by the Ca2+ + Mg2+-activated ATPase that is responsible for Ca2+ uptake in this system. In the normal ATPase cycle of the ATPase, phosphorylation of the ATPase is followed by a conformational change in which the Ca2+-binding sites change from being outward-facing and of high affinity to being inward-facing and of low affinity. To mediate Ca2+ efflux, it is proposed that the ATPase can adopt a conformation in which the Ca2+-binding sites are of low affinity but still outward-facing. It is shown that experimental data on the rates of Ca2+ efflux can be simulated in terms of this model, with Ca2+-binding-site affinities previously proposed to explain ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227]. Effects of Mg2+ and adenine nucleotides on efflux rates are explained. It is suggested that Ca2+ efflux from SR mediated by the ATPase could be important in excitation-contraction coupling in skeletal muscle.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle: effects of Sr2+ and Cd2+ on Ca2+ uptake and formation of the phosphorylated intermediate of the Ca2+,Mg2+-ATPase

The effects of various divalent cations on the Ca2+ uptake by microsomes from bovine aortic smooth muscle were studied. High concentrations (1 mM) of Co2+, Zn2+, Mn2+, Fe2+, and Ni2+ inhibited neither the Ca2+ uptake by the microsomes nor the formation of the phosphorylated intermediate (E approximately P) of the Ca2+,Mg2+-ATPase of the microsomes. The cadmium ion, however, inhibited both the Ca2+ uptake and the E approximately P formation by the microsomes. Dixon plot analysis indicated Cd2+ inhibited (Ki = 135 microM) the Ca2+ dependent E approximately P formation in a non-competitive manner. The inhibitory effect of Cd2+ was lessened by cysteine or dithiothreitol. The strontium ion inhibited the Ca2+ uptake competitively, while the E approximately P formation increased on the addition of Sr2+ at low Ca2+ concentrations. At a low Ca2+ concentration (1 microM), Sr2+ was taken up by the aortic microsomes in the presence of 1 mM ATP. It is thus suggested that Sr2+ replaces Ca2+ at the Ca2+ binding site on the ATPase.     

Uptake of Ca2+ mediated by the (Ca2+ + Mg2+)-ATPase in reconstituted vesicles

The (Ca2+ + Mg2+)-ATPase was purified from skeletal muscle sarcoplasmic reticulum and reconstituted into sealed phospholipid vesicles by solution in cholate and deoxycholate followed by detergent removal on a column of Sephadex G-50. The level of Ca2+ accumulated by these vesicles, either in the presence or absence of phosphate within the vesicles, increased with increasing content of phosphatidylethanolamine in the phospholipid mixture used for the reconstitution. The levels of Ca2+ accumulated in the absence of phosphate were very low for vesicles reconstituted with egg yolk phosphatidylcholine alone at pH 7.4, but increased markedly with decreasing pH to 6.0. Uptake was also relatively low for vesicles reconstituted with dimyristoleoyl- or dinervonylphosphatidylcholine, and addition of cholesterol had little effect. The level of Ca2+ accumulated increased with increasing external K+ concentration, and was also increased by the ionophores FCCP and valinomycin. Vesicle sizes changed little with changing phosphatidylethanolamine content, and the sidedness of insertion of the ATPase was close to random at all phosphatidylethanolamine contents. It is suggested that the effect of phosphatidylethanolamine on the level of Ca2+ accumulation follows from an effect on the rate of Ca2+ efflux mediated by the ATPase.     

The effect of residual Ca2+ on the stochastic gating of Ca2+-regulated Ca2+ channel models

Single-channel models of intracellular Ca(2+) channels such as the inositol 1,4,5-trisphosphate receptor and ryanodine receptor often assume that Ca(2+)-dependent transitions are mediated by a constant background [Ca(2+)] as opposed to a dynamic [Ca(2+)] representing the formation and collapse of a localized Ca(2+) domain. This assumption neglects the fact that Ca(2+) released by open intracellular Ca(2+) channels may influence subsequent gating through the processes of Ca(2+)-activation or -inactivation. We study the effect of such "residual Ca(2+)" from previous channel opening on the stochastic gating of minimal and realistic single-channel models coupled to a restricted cytoplasmic compartment. Using Monte Carlo simulation as well as analytical and numerical solution of a system of advection-reaction equations for the probability density of the domain [Ca(2+)] conditioned on the state of the channel, we determine how the steady-state open probability (p(open)) of single-channel models of Ca(2+)-regulated Ca(2+) channels depends on the time constant for Ca(2+) domain formation and collapse. As expected, p(open) for a minimal model including Ca(2+) activation increases as the domain time constant becomes large compared to the open and closed dwell times of the channel, that is, on average the channel is activated by residual Ca(2+) from previous openings. Interestingly, p(open) for a channel model that is inactivated by Ca(2+) also increases as a function of the domain time constant when the maximum domain [Ca(2+)] is fixed, because slow formation of the Ca(2+) domain attenuates Ca(2+)-mediated inactivation. Conversely, when the source amplitude of the channel is fixed, increasing the domain time constant leads to elevated domain [Ca(2+)] and decreased open probability. Consistent with these observations, a realistic De Young-Keizer-like IP(3)R model responds to residual Ca(2+) with a steady-state open probability that is a monotonic function of the domain time constant, though minimal models that include both Ca(2+)-activation and -inactivation show more complex behavior. We show how the probability density approach described here can be generalized for arbitrarily complex channel models and for any value of the domain time constant. In addition, we present a comparatively simple numerical procedure for estimating p(open) for models of Ca(2+)-regulated Ca(2+) channels in the limit of a very fast or very slow Ca(2+) domain. When the ordinary differential equation for the [Ca(2+)] in a restricted cytoplasmic compartment is replaced by a partial differential equation for the buffered diffusion of intracellular Ca(2+) in a homogeneous isotropic cytosol, we find the dependence of p(open) on the buffer time constant is qualitatively similar to the above-mentioned results.     

Ca2+-binding of S-100 protein in the presence of K+ and Mg2+

Ca2+-binding of S-100 protein was studied using a Ca2+ electrode at pH 6.80. In the presence of 0.1 M KCl and 10 mM MgCl2 (ionic strength 0.13), Ca2+-binding to S-100 protein occurred in three steps with positive cooperativity. The numbers of bound Ca2+ ions in the three steps were 2, 2, and 4. The Ca2+-binding constants were 6.9 x 10(3) M-1, 2.9 x 10(3) M-1, and 3.7 x 10(2) M-1, respectively. The Ca2+-binding constants of the first and second steps obtained in the presence of 33.3 mM MgCl2 or 0.1 M KCl (ionic strength 0.10) were 1.4 times larger than those described above. This suggests that Mg2+ does not inhibit Ca2+-binding of S-100 protein. The increase of KCl concentration from 0.1 to 0.2 M caused a decrease of the Ca2+-binding constants to ca. 50%.     

Mg2+ homeostasis: the Mg2+nificent TRPM chanzymes

TRPM6 and TRPM7 are distinct from all other ion channels in that they are composed of linked channel and protein kinase domains. Recent studies demonstrate that these 'chanzymes' are essential for Mg(2+) homeostasis, which is critical for human health and cell viability.     

Dependence of Na+-Ca2+ exchange and Ca2+-Ca2+ exchange on monovalent cations

Two mechanisms of passive Ca2+ transport, Na+-Ca2+ exchange and Ca2+-Ca2+ exchange, were studied using highly-purified dog heart sarcolemmal vesicles. About 80% of the Ca2+ accumulated by Na+-Ca2+ exchange or Ca2+-Ca2+ exchange could be released as free Ca2+, while up to 20% was probably bound. Na+-Ca2+ exchange was simultaneous, coupled countertransport of Na+ and Ca2+. The movement of anions during Na+-Ca2+ exchange did not limit the initial rate of Na+-Ca2+ exchange. Na+-Ca2+ exchange was electrogenic, with a reversal potential of about -105 mV. The apparent flux ratio of Na+-Ca2+ exchange was 4 Na+:1 Ca2+. Coupled cation countertransport by the Na+-Ca2+ exchange mechanism required a monovalent cation gradient with the following sequence of ion activation: Na+ much greater than Li+ greater than Cs+ greater than K+ greater than Rb+. In contrast to Na+-Ca2+ exchange, Ca2+-Ca2+ exchange did not require a monovalent cation gradient, but required the presence of Ca2+ plus a monovalent cation on both sides of the vesicle membrane. The sequence of ion activation of Ca2+-Ca2+ exchange was: K+ much greater than Rb+ greater than Na+ greater than Li+ greater than Cs+. Na+ inhibited Ca2+-Ca2+ exchange when Ca2+-Ca2+ exchange was supported by another monovalent cation. Both Na+-Ca2+ exchange and Ca2+-Ca2+ exchange were inhibited, but with different sensitivities, by external MgCl2, quinidine, or verapamil.     

Site-directed disulfide mapping of residues contributing to the Ca2+ and K+ binding pocket of the NCKX2 Na+/Ca2+-K+ exchanger

The Na+/Ca2+-K+ exchanger (NCKX) gene products are polytopic membrane proteins that utilize the existing cellular Na+ and K+ gradients to extrude cytoplasmic Ca2+. NCKX proteins are made up of two clusters of hydrophobic segments, both thought to consist of five putative membrane-spanning alpha-helices, and separated by a large cytoplasmic loop. The two most conserved regions within the NCKX sequence are known as the alpha1 and alpha2 repeats, and are found within the first and second set of transmembrane domains, respectively. The alpha repeats have previously been shown to contain residues critical for transport function. Here we used site-directed disulfide mapping to report that the alpha repeats are found in close proximity in three-dimensional space, bringing together key functional NCKX residues, e.g., the two critical acidic residues, Glu188 and Asp548. Glu188Cys in the alpha1 repeat could form a disulfide cross-link with Asp548Cys in the alpha2 repeat. Surprisingly, cysteine substitutions of Ser185 in the alpha1 repeat could form disulfide cross-links with cysteine substitutions of three residues in the alpha2 repeat (Ser545, Asp548, and Ser552), thought to cover close to two full turns of an alpha helix, implying an area of increased flexibility. Using the same method, Asp575, a residue critical for the K+ dependence of NCKX, was shown to be in the proximity of Ser185 and Glu188, consistent with its role in enabling K+ to bind to a single Ca2+ and K+ binding pocket.