The current topological model of the Na ؉ -Ca 2؉ exchanger consists of 11 transmembrane segments with extracellular loops a, c, e, g, i, and k and cytoplasmic loops b, d, f, h, and j. Cytoplasmic loop f, which plays a role in regulating the exchanger, is large and separates the first five from the last six transmembrane segments. We have tested this topological model by mutating residues near putative transmembrane segments to cysteine and then examining the effects of intracellular and extracellular applications of sulfhydryl-modifying reagents on exchanger activity. To aid in our topological studies, we also constructed a cysteineless Na ؉ -Ca 2؉ exchanger. This mutant is fully functional in Na ؉ gradientdependent 45 Ca 2؉ uptake measurements and displays wild-type regulatory properties. It is concluded that the 15 endogenous cysteine residues are not essential for either activity or regulation of the exchanger. Our data support the current model by placing loops c and e at the extracellular surface and loops d, j, and l at the intracellular surface. However, the data also support placing Ser-788 of loop h at the extracellular surface and Gly-837 of loop i at the intracellular surface. To account for these data, we propose a revision of the model that places transmembrane segment 6 in cytoplasmic loop f. Additionally, we propose that putative transmembrane segment 9 does not span the membrane, but may form a "P-loop"-like structure.

Large conductance calcium-and voltagesensitive K ؉ (MaxiK) channels share properties of voltageand ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca 2؉ favor pore opening. We demonstrate the presence of voltage sensor rearrangements with voltage (gating currents) whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca 2؉ concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4-5 elementary charges.

This paper is the third in a series of reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation–contraction coupling and arrhythmias: Na+ channel and Na+ transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on cardiac Na+/Ca2+ exchange (NCX) and Na+/K+-ATPase (NKA). While the relevance of Ca2+ homeostasis in cardiac function has been extensively investigated, the role of Na+ regulation in shaping heart function is often overlooked. Small changes in the cytoplasmic Na+ content have multiple effects on the heart by influencing intracellular Ca2+ and pH levels thereby modulating heart contractility. Therefore it is essential for heart cells to maintain Na+ homeostasis. Among the proteins that accomplish this task are the Na+/Ca2+ exchanger (NCX) and the Na+/K+ pump (NKA). By transporting three Na+ ions into the cytoplasm in exchange for one Ca2+ moved out, NCX is one of the main Na+ influx mechanisms in cardiomyocytes. Acting in the opposite direction, NKA moves Na+ ions from the cytoplasm to the extracellular space against their gradient by utilizing the energy released from ATP hydrolysis. A fine balance between these two processes controls the net amount of intracellular Na+ and aberrations in either of these two systems can have a large impact on cardiac contractility. Due to the relevant role of these two proteins in Na+ homeostasis, the emphasis of this review is on recent developments regarding the cardiac Na+/Ca2+ exchanger (NCX1) and Na+/K+ pump and the controversies that still persist in the field.

The Na ؉ -Ca 2؉ exchanger plays a central role in cardiac contractility by maintaining Ca 2؉ homeostasis. Two Ca 2؉ -binding domains, CBD1 and CBD2, located in a large intracellular loop, regulate activity of the exchanger. Ca 2؉ binding to these regulatory domains activates the transport of Ca 2؉ across the plasma membrane. Previously, we solved the structure of CBD1, revealing four Ca 2؉ ions arranged in a tight planar cluster. Here, we present structures of CBD2 in the Ca 2؉ -bound (1.7-Å resolution) and -free (1.4-Å resolution) conformations. Like CBD1, CBD2 has a classical Ig fold but coordinates only two Ca 2؉ ions in primary and secondary Ca 2؉ sites. In the absence of Ca 2؉ , Lys 585 stabilizes the structure by coordinating two acidic residues (Asp 552 and Glu 648 ), one from each of the Ca 2؉ -binding sites, and prevents a substantial protein unfolding. We have mutated all of the acidic residues that coordinate the Ca 2؉ ions and have examined the effects of these mutations on regulation of exchange activity. Three mutations (E516L, D578V, and E648L) at the primary Ca 2؉ site completely remove Ca 2؉ regulation, placing the exchanger into a constitutively active state. These are the first data defining the role of CBD2 as a regulatory domain in the Na ؉ -Ca 2؉ exchanger.calcium binding ͉ calcium regulation

Large conductance calcium-activated K+ (KCa) channels are rapidly activated by niflumic acid dose-dependently and reversibly. External niflumic acid was about 5 times more potent than internal niflumic acid, and its action was characterized by an increase in the channel affinity for [Ca2+], a parallel left shift of the voltage-activation curve, and a decrease of the channel long-closed states. Niflumic acid applied from the external side did not interfere with channel block by charybdotoxin, suggesting that its site of action is not at or near the charybdotoxin receptor. Accordingly, partial tetraethylammonium blockade did not interfere with channel activation by niflumic acid. Flufenamic acid and mefenamic acid also stimulated KCa channel activity and, as niflumic acid, they were more potent from the external than from the internal side. Fenamates applied from the external side displayed the following potency sequence: flufenamic acid approximately niflumic acid >> mefenamic acid. These results indicate that KCa channels possess at least one fenamatereceptor whose occupancy leads to channel opening.

In the present report we studied the interaction between the skeletal muscle ryanodine receptor and the ubiquitous S100A1 Ca2+ binding protein. S100A1 did not affect equilibrium [3H]ryanodine binding to purified rabbit skeletal muscle terminal cisternae at 100 microM free [Ca2+]. At nanomolar free [Ca2+], however, S100A1 activated by 40 +/- 6.7% (mean +/- SE, n = 5) the [3H]ryanodine binding activity; the half-maximal concentration for stimulation of [3H]ryanodine binding was approximately 70 nM, a value well below the estimated S100A1 concentration in skeletal muscle fibers. Scatchard analysis of [3H]ryanodine binding performed in the presence of 100 microM EGTA indicates that S100A1 increases the apparent affinity of the receptor for ryanodine (Kd = 191 vs 383 nM in the presence and in the absence of 100 nM S100A1, respectively). The effect of S100A1 was also tested on the single-channel gating properties of the purified ryanodine receptor after reconstitution into a lipid planar bilayer. Currents carried by purified ryanodine receptor channels were modulated by both cis Ca2+ and ruthenium red. In the presence of nanomolar [Ca2+], S100A1 activated the channel by increasing (6.0 +/- 2.8)-fold (mean +/- SE, n = 3) the normalized open probability. The interaction between S100A1 and the purified RYR was verified using the optical biosensor BIAcore: we show that the two proteins interact directly both at millimolar and at nanomolar calcium concentrations. We next mapped the regions of the skeletal muscle RYR involved in the interaction with S100A1 by performing ligand overlays on a panel RYR of fusion proteins in the presence of 100 nM S100A1. Our results indicate that the skeletal muscle RYR contains three potential S100A1 binding domains. Binding of S100A1 to the RYR fusion proteins occurred at both nanomolar and millimolar free [Ca2+]. S100A1 binding domain 1 binds the ligand in the presence of 1 mM free [Ca2+] or 1 mM EGTA. Maximal binding to S100A1#2 was achieved in the presence of 1 mM free [Ca2+]. The S100A1#3 domain, which overlaps with calcium-dependent calmodulin binding domain 3 (CaM 3), exhibits weak and strong S100A1 binding activity in the presence of either millimolar or nanomolar Ca2+, respectively. The interaction between S100A1 and the purified RYR complex was also investigated by affinity chromatography: in the presence of nanomolar Ca2+, we observed binding of native RYR complex to S100A1-conjugated Sepharose. This interaction could be inhibited by the presence of RYR polypeptides encompassing S100A1 binding sites S100A1#1, S100A1#2, and S100A1#3.

Large conductance voltage and Ca 2؉ -dependent K ؉ channels (BKCa) are activated by both membrane depolarization and intracellular Ca 2؉ . Recent studies on bacterial channels have proposed that a Ca 2؉ -induced conformational change within specialized regulators of K ؉ conductance (RCK) domains is responsible for channel gating. Each pore-forming ␣ subunit of the homotetrameric BKCa channel is expected to contain two intracellular RCK domains. The first RCK domain in BKCa channels (RCK1) has been shown to contain residues critical for Ca 2؉ sensitivity, possibly participating in the formation of a Ca 2؉ -binding site. The location and structure of the second RCK domain in the BKCa channel (RCK2) is still being examined, and the presence of a high-affinity Ca 2؉ -binding site within this region is not yet established. Here, we present a structure-based alignment of the C terminus of BK Ca and prokaryotic RCK domains that reveal the location of a second RCK domain in human BK Ca channels (hSloRCK2). hSloRCK2 includes a high-affinity Ca 2؉ -binding site (Ca bowl) and contains similar secondary structural elements as the bacterial RCK domains. Using CD spectroscopy, we provide evidence that hSloRCK2 undergoes a Ca 2؉ -induced change in conformation, associated with an ␣-to-␤ structural transition. We also show that the Ca bowl is an essential element for the Ca 2؉ -induced rearrangement of hSloRCK2. We speculate that the molecular rearrangements of RCK2 likely underlie the Ca 2؉ -dependent gating mechanism of BKCa channels. A structural model of the heterodimeric complex of hSloRCK1 and hSloRCK2 domains is discussed.BK channel ͉ circular dichroism ͉ MaxiK ͉ RCK ͉ structural modeling B K Ca channels are formed by the assembly of four identical pore-forming ␣ subunits. They can couple the membrane potential to the intracellular Ca 2ϩ level (1-4), playing critical roles in cell excitability, for example, by controlling smooth muscle tone and neurotransmitter release (1, 5-7). Each BK Ca ␣ subunit possesses a transmembrane voltage sensor (8-10) and two distinct high-affinity Ca 2ϩ sensors (11-15) located within the large intracellular carboxyl terminus. A well studied Ca 2ϩ -binding site corresponds to a C-terminal region that includes five consecutive negatively charged aspartates (D894-D898), christened the ''Ca bowl'' by the Salkoff laboratory (16,17). The Ca bowl binds Ca 2ϩ with high affinity (18-21) and strongly contributes to the channel's Ca 2ϩ sensitivity (18)(19)(20) [supporting information (SI) Fig. 6]. A second high-affinity Ca 2ϩ -sensing region that is impaired by neutralization of two aspartates (D362/D367) (11, 15) or methionine 513 (22) has been identified Ϸ400 aa upstream the Ca bowl.Most likely, these two high-affinity Ca 2ϩ -binding sites form parts of a complex functional domain that converts the free energy of Ca 2ϩ binding into mechanical work to open the channel. Indeed, specialized intracellular motifs regulating the conductance of K ϩ channels (RCK domains) have been recently described in prok...

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