Transient Charge Movements Research Articles

External Ion Access in the Na/K Pump: Kinetics of Na+, K+, and Quaternary Amine Interaction

Na/K pumps build essential ion gradients across the plasmalemma of animal cells by coupling the extrusion of three Na+, with the import of two K+ and the hydrolysis of one ATP molecule. The mechanisms of selectivity and competition between Na+, K+, and inhibitory amines remain unclear. We measured the effects of external tetrapropylammonium (TPA+) and ethylenediamine (EDA2+) on three different Na/K pump transport modes in voltage-clamped Xenopus oocytes: 1) outward pump current (IP), 2) passive inward H+ current at negative voltages without Na+ or K+ (IH), and 3) transient charge movement reporting the voltage-dependent extracellular binding/release of Na+ (QNa). Both amines competed with K+ to inhibit IP. TPA+ inhibited IH without competing with H+, whereas EDA2+ did not alter IH at pH 7.6. TPA+ competed with Na+ in QNa measurements, reducing Na+-apparent affinity, evidenced by a ∼−75 mV shift in the charge-voltage curve (at 20 mM TPA+) without reduction of the total charge moved (Qtot). In contrast, EDA2+ and K+ did not compete with Na+ to inhibit QNa; both reduced Qtot without decreasing Na+-apparent affinity. EDA2+ (15 mM) right-shifted the charge-voltage curve by ∼+50 mV. Simultaneous occlusion of EDA2+ and Na+ by an E2P conformation unable to reach E1P was demonstrated by voltage-clamp fluorometry. Trypsinolysis experiments showed that EDA2+-bound pumps are much more proteolysis-resistant than Na+-, K+-, or TPA+-bound pumps, therefore uncovering unique EDA2+-bound conformations. K+ effects on QNa and IH were also evaluated in pumps inhibited with beryllium fluoride, a phosphate mimic. K+ reduced Qtot without shifting the charge-voltage curve, indicating noncompetitive effects, and partially inhibited IH to the same extent as TPA+ in non-beryllium-fluorinated pumps. These results demonstrate that K+ interacts with beryllium-fluorinated pumps inducing conformational changes that alter QNa and IH, suggesting that there are two external access pathways for proton transport by IH.

Inhibition of Na,K ATPase partial reactions by organic amines

The Na,K‐ATPase (NKA) transports 3 Na ions out of the cell and 2 K ions into the cell for every one ATP molecule hydrolyzed. Two of the three ion‐binding sites within the protein reciprocally bind two K or two Na ions (shared sites), while the other site exclusively binds Na). Three modes of transport can be studied with two electrode voltage clamp (TEVC) in Xenopus oocytes to evaluate NKA function and its partial reactions involving external ion binding: 1) outward pump current (IP) induced by forward NKA cycling when extracellular K is added, 2) transient charge movement (QNa) induced by voltage‐dependent binding and release of Na, rate limited by the NKA conformational change and 3) uncoupled proton import at negative voltages without external Na or K the pump transports H down their electrochemical gradient (IH). Tetrapropylammonium (TPA) and ethylenediamine (EDA), are known competitive inhibitors of extracellular K binding and their mechanisms of action are not fully understood (particularly for EDA). We used TEVC to evaluate the effect of TPA and EDA on IP, QNa and IH. TPA acted as a competitive inhibitor of K binding by reducing its apparent affinity for IP activation in a voltage‐independent manner IC50 of 8.78 ± 0.12 mM. EDA also acted as a K competitor, but its IC50 changed from 8.96 ± 0.48 mM at −100 mV to 15.92 ± 1.84 mM a 0 mV. When evaluating QNa, the position of the charge voltage curve (Q‐V), parameterized by the curve's center voltage (V1/2) related to the apparent affinity for external Na (more negative V1/2 indicating a reduction of Na affinity and viceversa), was −54.39 ± 1.65 mV in the absence of amines, −120.88 ± 2.15 mV in the presence of 20 mM TPA and −10.49 ± 6.13 mV in the presence of 15 mM EDA. While TPA did not significantly affect the total charge moved (related to the number of pumps performing the Na binding reactions) EDA reduced it by 53.6 ± 0.3%. This indicates that TPA acts as a prototypical competitive inhibitor of external Na reducing Na apparent affinity, but EDA does not. Finally, TPA inhibited IH (~70% maximal inhibition) at both pH 7.6 and pH 6.0, while EDA had no effect on IH at pH 7.6, but increased IH (by 2 fold) at pH 6.0 at −180 mV. Our results are consistent with the larger TPA acting as a large extracellular access‐channel blocker therefore competing with Na, K and H without reaching the ion‐binding sites, while EDA reaches the shared sites domain, probably becoming occluded there, therefore making NKAs unavailable to induce QNa. The complex effects of EDA on IH may reflect the complex nature of its occlusion there and are still under evaluation.Support or Funding InformationWork supported by NIH GM 061583 to CG and NSF MCB‐1243842 to PA.

Functional Effect of Human FXYD2, FXYD4, FXYD6, and FXYD7 on Human Alpha 1 Beta 1 Sodium-Potassium ATPase

The Na+,K+-ATPase builds and maintains the Na+ and K+ gradient across the plasmalemma. Functional Na+,K+-ATPase requires association of α- and β-subunits. A third single transmembrane protein of the FXYD family (FXYD1-FXYD7) modulate Na+,K+-ATPase function in a tissue specific manner. Here, we used electrophysiology on oocytes expressing human α1β1 Na+,K+-ATPase to evaluate the functional effects of association with human FXYD2, 4, 6 & 7. Under two-electrode voltage clamp, in the presence of 125 mM Na+o, application of K+o activated outward currents (due to 3Na+/2K+ exchange) with voltage-dependent apparent dissociation constant. For α1β1, K0.5,0 = 1.1 ±0.1 mM and K0.5100= 1.5 ±0.3 mM (at 0, n=6, and −100 mV, n=6, respectively). The K0.5,0 for all FXYDs was 1-1.2 mM; the K0.5100 was ∼1 mM for FXYD2 and FXYD6 and ∼2 mM for FXYD 4 and FXYD7. Thus, these FXYDs induced changes in K+o affinity could be relevant at hyperpolarized potentials. We measured the transient charge movement observed in the absence of K+o, which describes the voltage-dependent equilibrium between Na+-bound and externally open Na+-free states. The steady-state charge-voltage curves were fitted with a Boltzmann distribution. At constant [Na+]o a shift in the distribution's center (V1/2) to positive voltages signals an increase in Na+o affinity while a shift to negative voltages indicates a reduction, with a 25 mV per two fold change in Na+o affinity. The V1/2 were (in mV) −41 ±0.5 (α1β1, n=6), −16 ±2 (FXYD4, n=4), −26 ± 0.3 (FXYD7, n=2), −57 ±2 (FXYD2, n=7), and −60 ±3 (FXYD6, n=7). These results suggest similar increase in Na+o affinity by FXYD4 or FXYD7 and identical reduction in Na+o affinity by FXYD2 and FXYD6. Measurement of K0.5 for Na+K+-ATPase activation by intracellular Na+ in inside-out patches is underway. Supported by R15-NS081570-01A1.

Regulation of ClC-3 Cl-/H+ Transport and “Gating” Transients by Chloride Pathway Residues and External Protons

We investigated ClC-3 ion transport properties by employing a “ClC-5/3” plasmid, consisting of the ClC-5 N-terminal (M1-A46) linked to the core ClC-3 protein. This ClC-3 protein is redirected to the plasma membrane of HEK cells. Functionally it exhibits rapidly activated, outwardly rectifying sustained ion currents (ISS) representing coupled Cl-/H+ transport, and prominent “on/off” transient gating charge (Q) movements, as recently described for a ClC-3 mutant (Guzman et al, 2013, ACS Chem Neurosci). Interpreting Q to represent movements of unprotonated (ie, incompletely cycling) “Gluext”/ E224, ClC-3 exhibits low transport efficiency compared to ClC-5 or ClC-4, which exhibit larger ISS and much smaller Q. Replacement of external Cl- with SCN- increases ClC-3 currents by 3-4 fold, and reduces H+ coupling by ∼90%. Removal of a conserved tyrosine (Y630S, V) positioned at the intersection of the Cl-/H+ pathways (Accardi et al, 2006, JMB) greatly increases currents and decreases Q. For Y630S, H+ coupling is reduced by ∼50%; SCN substitution decreases rather than increases current, and eliminates H+ transport. An M568A mutant moderately impacts transport, increasing currents and decreasing Q by <50%. Reduction of external pH (pH 6 or 5) weakly inhibits ClC-3 ISS, but markedly reduces Q, and shifts the Q(V) relationship toward more positive voltages. These results are consistent with external protons inhibiting cycling by neutralization of Gluext. An endogenous Cl- current activated at pH5 (Iacid) has slow activation kinetics and lacks gating transients, is inhibited by 60-120 microM phloretin, and is decreased by ClC-3 expression. Thus, Iacid is readily distinguished from expressed ClC-3 transport. These data indicate that ClC-3 H+ coupling and transport is influenced by anion interaction, and by external protons, as suggested for the bacterial ClCec-1.

Voltage Dependent Reactions in the Non-Gastric H/K-ATPase

The non-gastric H/K-ATPase (ngHKA) is the closest relative to the Na/K-ATPase (NKA). Its catalytic subunit associates with the NKA β1 subunit to form a functional, ouabain-sensitive HKA. To study ion binding and unbinding reactions in ngHKA, we measured 86Rb uptake and ouabain-sensitive (20 mM) currents under two-electrode voltage clamp in Na+-loaded Xenopus oocytes. Although HKA transports ions in an electroneutral fashion, HKA injected oocytes presented clamp speed-limited ouabain-sensitive charge movement in response to pulses between −180 to +40 mV from −50 mV in 125 mM external N-methyl D-glucamine (NMG). The charge-voltage curve did not saturate at extreme voltages at pH 7.6, but saturated at negative voltages at pH 5.0. The charge moved by a pulse from −50 to −180 mV was 1.29 ± 0.15 nC at pH 7.6 and 0.35 ± 0.08 nC at pH 5.0. This charge was absent in uninjected oocytes (60 ± 23 pC). External K+ abolished charge movement with mildly voltage-dependent K0.5 (∼ 0.15 mM at −180 mV), while the charge moved in the presence of Na+ at pH 7.6 presented nearly identical saturation at negative voltages as NMG at pH 5.0. This demonstrates the existence of mildly voltage-dependent transitions in ngHKAs which, like the charge movement in NKA, are inhibited by the external presence of the imported ion (K+) and modified by high concentrations of the exported ions (Na+ or H+, respectively). To further address the ion binding reactions and their selectivity, we mutated K793A in the catalytic subunit to make the pump electrogenic. As expected, this mutant concomitantly displayed 86Rb uptake and Rb+-induced steady-state outward currents as well as altered transient charge movement. Unexpectedly, K793A largely reduced the pump's affinity for K+o. Supported by NSF-MCB-1243842 & NIH-NS081570-01, to PA; GM 061583 to CG

β-Exodomain Role in Na/K-ATPase α-β Subunit Interactions

We disrupted the three conserved disulfide bonds in Xenopus β3 (C127−C144, C154-C170, C191-C248), one at a time, using Cys to Ala/Ser mutations and coexpression with relatively ouabain-resistant (C113Y) Xenopus α1 in oocytes. We assessed function with two-microelectrode voltage clamp in 1µM ouabain to silence endogenous Na/K pumps. Single C127S/A or C154S, or double C127S+C154S β3 mutants yielded steady Na/K pump currents (saturating [Ko]) and transient Na charge movements (without Ko) like those of C113Y α1 coexpressed with wild-type β3, suggesting that neither assembly, trafficking, nor Na/K pump function require intact first and/or second disulfide bonds.

Membrane Potential-Dependent Inhibition of the Na+,K+-ATPase bypara-Nitrobenzyltriethylammonium Bromide

Membrane potential (V(M))-dependent inhibitors of the Na(+),K(+)-ATPase are a new class of compounds that may have inherent advantages over currently available drugs targeting this enzyme. However, two questions remain unanswered regarding these inhibitors: (1) what is the mechanism of V(M)-dependent Na(+),K(+)-ATPase inhibition, and (2) is their binding affinity high enough to consider them as possible lead compounds? To address these questions, we investigated how a recently synthesized V(M)-dependent Na(+),K(+)-ATPase inhibitor, para-nitrobenzyltriethylamine (pNBTEA), binds to the enzyme by measuring the extracellular pNBTEA concentration and V(M) dependence of ouabain-sensitive transient charge movements in whole-cell patch-clamped rat cardiac ventricular myocytes. By analyzing the kinetics of charge movements and the steady-state distribution of charge, we show that the V(M)-dependent properties of pNBTEA binding differ from those for extracellular Na(+) and K(+) binding, even though inhibitor binding is competitive with extracellular K(+). The data were also fit to specific models for pNBTEA binding to show that pNBTEA binding is a rate-limiting V(M)-dependent reaction that, in light of homology models for the Na(+),K(+)-ATPase, we interpret as a transfer reaction of pNBTEA from a peripheral binding site in the enzyme to a site near the known K(+) coordination sites buried within the transmembrane helices of the enzyme. These models also suggest that binding occurs with an apparent affinity of 7 μM. This apparent binding affinity suggests that high-affinity V(M)-dependent Na(+),K(+)-ATPase inhibitors should be feasible to design and test as specific enzyme inhibitors.

D-Enantiomers Take a Close Look at the Functioning of a Cardiac Cationic L-Amino Acid Transporter

Cationic amino acid transporters are highly selective for L-enantiomers such as L-arginine (L-Arg). Because of this stereoselectivity, little is known about the interaction of these transporters with D-isomers. To study whether these compounds can provide information on the molecular mechanism of transport, inward currents activated by L-Arg with low apparent affinity were measured in whole-cell voltage-clamped cardiomyocytes as a function of extracellular L-Arg and D-Arg concentrations. D-Arg inhibited L-Arg currents in a membrane-potential ( V M)-dependent competitive manner, indicating the presence of D-Arg binding sites in the carrier. Analysis of these steady-state currents showed that L- and D-Arg binding reactions dissipate a similar small fraction of the membrane electric field. Since D-Arg is not transported, these results suggest that enantiomer recognition occurs at conformational transitions that initiate amino acid translocation. The V M dependence of maximal current levels suggests that inward currents arise from the slow outward movement of negative charges in the unliganded transporter. Translocation of the L-Arg-bound complex, on the other hand, appears to be electroneutral. D-Arg-dependent transient charge movements, also detected in these cells, displayed a V M-dependent charge distribution and kinetics that are consistent with amino acid binding in an ion well in a shallow, water-filled extracellular binding pocket.

The Effect of Holding Potential on Charge Translocation by the Na+/K+-ATPase in the Absence of Potassium

The Na(+)/K(+)-ATPase exports 3Na(+) and imports 2K(+) at the expense of the hydrolysis of 1 ATP. In the absence of K(+), it carries on electroneutral, Na(+)-dependent transient charge movement (also known as "electroneutral Na(+)/Na(+) exchange mode") and produces a transient current containing faster and slower components in response to a sudden voltage step. Components with different speeds represent sequential release of Na(+) ions from three binding sites. The effect of holding potential on slow charge movement was studied in the presence of different concentrations of ADP(i), Na (i) (+) and Na (o) (+) with the intention of improving our understanding of Na (i) (+) binding. However, the manipulation of [ADP](i) and [Na(+)](i) did not cause as pronounced changes as predicted in the magnitude of charge movement (Q (tot)), which indicated that our experimental conditions were not able to backwardly drive reaction across the energy barrier to Na (i) (+) release/rebinding steps. On the contrary, lowering [Na(+)](o) caused evident dependence of Q (tot) on holding potential, with characteristics suggesting that pumps were escaping from E2P through the uncoupled Na(+) efflux activity.

The two C-terminal tyrosines stabilize occluded Na/K pump conformations containing Na or K ions

Interactions of the three transported Na ions with the Na/K pump remain incompletely understood. Na/K pump crystal structures show that the extended C terminus of the Na,K–adenosine triphosphatase (ATPase) α subunit directly contacts transmembrane helices. Deletion of the last five residues (KETYY in almost all Na/K pumps) markedly lowered the apparent affinity for Na activation of pump phosphorylation from ATP, a reflection of cytoplasmic Na affinity for forming the occluded E1P(Na3) conformation. ATPase assays further suggested that C-terminal truncations also interfere with low affinity Na interactions, which are attributable to extracellular effects. Because extracellular Na ions traverse part of the membrane’s electric field to reach their binding sites in the Na/K pump, their movements generate currents that can be monitored with high resolution. We report here electrical measurements to examine how Na/K pump interactions with extracellular Na ions are influenced by C-terminal truncations. We deleted the last two (YY) or five (KESYY) residues in Xenopus laevis α1 Na/K pumps made ouabain resistant by either of two kinds of point mutations and measured their currents as 10-mM ouabain–sensitive currents in Xenopus oocytes after silencing endogenous Xenopus Na/K pumps with 1 µM ouabain. We found the low affinity inhibitory influence of extracellular Na on outward Na/K pump current at negative voltages to be impaired in all of the C-terminally truncated pumps. Correspondingly, voltage jump–induced transient charge movements that reflect pump interactions with extracellular Na ions were strongly shifted to more negative potentials; this signals a several-fold reduction of the apparent affinity for extracellular Na in the truncated pumps. Parallel lowering of Na affinity on both sides of the membrane argues that the C-terminal contacts provide important stabilization of the occluded E1P(Na3) conformation, regardless of the route of Na ion entry into the binding pocket. Gating measurements of palytoxin-opened Na/K pump channels additionally imply that the C-terminal contacts also help stabilize pump conformations with occluded K ions.

Altered Transport In Na/K pumps With α‐Subunits Truncated At Its 5 C‐Terminal Residues

Truncation of the α‐subunit's last 5 residues renders Na/K pumps with a reduced affinity for intracellular Na (Nai), without effects on affinity for external K (Ko) (Morth et al, 2007). We used two electrode voltage clamp, and cut‐open oocyte to investigate the effect of the ΔKESYY truncation on electrogenic transport by Xenopus Na/K pumps (Q120RN131D‐α1+β3). Pump currents (IP) were measured 2‐3 days after cRNA injection in Nai‐loaded oocytes. The K0.5 for IP activation by Ko in Na‐free NMDG external solutions was ≈0.2 mM, both in eggs expressing truncated (TP) or normal (NP) pumps; TP expressing eggs showed larger IP (IPmax≈300 nA) than NP expressing eggs (IPmax≈150 nA). Western blots suggest better expression of TP at the plasma membrane. Surprisingly, in Ko‐free solutions, TP but not NP, presented a voltage‐ and Nao‐dependent inward current of ≈ ‐250 nA at ‐160 mV with 135 mM Nao, that is reduced by ≈50% at 67 mM Nao. Consistent with a low Nao affinity in TP, the voltage‐dependent distribution of transient charge movement (which describes the E1‐E2 occupancy in the Na branch of transport) is shifted by ≈‐100 mV compared with NP. The effect of combining ΔKESYY with other mutations that modify external Na affinity will also be presented.

Truncation Of The Na/K Pump's C-terminus Attenuates Voltage-dependent Binding Of External Na+ By Destabilizing Na+ Occlusion

In the recent Na/K pump crystal structure the C-terminus directly contacts the transmembrane region, and deletion of its last five residues (KETYY in pig Na/K pumps) strongly lowered (26-fold) apparent affinity for Na+-dependent phosphorylation, which reflects cytoplasmic-side Na+ binding (Morth et al., 2007). Here we use two-microelectrode voltage-clamp recordings in Xenopus oocytes to investigate the consequence of this C-terminal truncation on the external Na+ dependence of steady-state, and transient, Na/K pump-mediated currents. The corresponding deletion (ΔKESYY in Xenopus α1) was introduced in Xenopus α1β3 pumps made ouabain resistant by either Q120R-N131D (RD) or C113Y (C-Y) mutations. All extracellular solutions then contained 1 μM ouabain to silence the endogenous Xenopus Na/K pumps, and the mutant pump currents were determined by addition of 10 mM ouabain. Inhibition of steady outward Na/K pump current (at 15 mM external K+) by 125 mM extracellular Na+ at negative voltages was weaker in both RD and C-Y pumps than in wild-type Xenopus Na/K pumps, but was almost completely absent (at -120 mV) in RD-ΔKESYY or C-Y-ΔKESYY pumps. Transient, external Na+-dependent, pump currents were similarly measured as 10 mM ouabain-sensitive currents, but in K+-free solution. The voltage dependence of these transient charge movements was shifted to more negative potentials for RD-ΔKESYY and C-Y-ΔKESYY pumps compared to RD or C-Y pumps, similar to the shift caused in wild-type pumps by decreasing the extracellular Na+ concentration. Together, these results suggest that the C-terminal deletion lowers the apparent affinity with which extracellular Na+ ions bind to the Na/K pump to reverse Na+-ion deocclusion. The C-terminus therefore seems important for stabilizing the occluded conformation of the phosphorylated Na/K pump containing 3 bound Na+ ions. [NIH HL36783]

Relative Movement Of The α-Subunit's First And Last External Loops Throughout Na/K-Pump Cycle

Structural models of the Na/K-pump α-subunit show that conserved residues D121 (loop 1-2, pig-α1 numbering) and R972 (loop 9-10) are 5-7 A apart in E2, and more than 12 A away in E1. To check whether these residues reach conformation-dependent atomic proximity, the substitutions D121C and R972C were introduced (individually and concurrently) into the ouabain-insensitive C104Y-Xenopus-α1. Each mutant was coexpressed with Xenopus-β3 in Xenopus oocytes and their function tested in Na+-loaded oocytes under two-electrode voltage clamp. K+o-activated Na/K-pump currents were observed in oocytes injected with D121C or R972C, but not in those injected with D121C-R972C, unless the eggs were exposed to TCEP (10-50 mM, ∼20 min), consistent with the presence of a pump-inhibiting disulfide.To identify the conformation locked by the disulfide, we used palytoxin to transform Na/K-pumps into channels. Palytoxin-induced currents (IPTX) in outside-out patches from oocytes expressing D121C-R972C, bathed in Na+ solutions, were insensitive to MTSET+ application. Patch exposure to DTT restored MTSET+-sensitivity (∼65% IPTX reduction) without affecting IPTX amplitude. Palytoxin stabilizes an E2P-like structure; thus, the lack of DTT effect on IPTX suggest that cross-linking between D121C and R972C occurs in E2P, with the external cation pathway open. Moreover, pump inhibition by spontaneous disulfide formation indicates that conformational mobility between these residues is required for the E2 to E1 transition.The slow component of ouabain-sensitive transient charge movement in 125 mM Nao+ was measured in Na+-loaded oocytes expressing these mutants. The center of the equilibrium distribution of charge (voltage of equal occupancy of E1 and E2) for R972C (V1/2= -34±8 mV) was identical to that of the C104Y-α1 template (V1/2= -35±3 mV). Therefore, it appears that a D121-R972 salt bridge is not necessary for E2P stabilization among Na+-occupied states.

Conformational dynamics of the Na + /K + -ATPase probed by voltage clamp fluorometry

The method of voltage clamp fluorometry combined with site-directed fluorescence labeling was used to detect local protein motions of the fully active Na(+)K(+)-ATPase in real time under physiological conditions. Because helix M5 extends from the cytoplasmic site of ATP hydrolysis into the cation binding region, we chose the extracellular M5-M6 loop of the sheep alpha(1)-subunit for the insertion of cysteine residues to identify reporter positions for conformational rearrangements during the catalytic cycle. After expression of the single cysteine mutants in Xenopus oocytes and covalent attachment of tetramethylrhodamine-6-maleimide, only mutant N790C reported molecular rearrangements of the M5-M6 loop by showing large, ouabain-sensitive fluorescence changes ( approximately 5%) on addition of extracellular K(+). When the enzyme was subjected to voltage jumps under Na(+)Na(+)-exchange conditions, we observed fluorescence changes that directly correlated to transient charge movements originating from the E(1)P-E(2)P transition of the transport cycle. The voltage jump-induced fluorescence changes and transient currents were abolished after replacement of Na(+) by tetraethylammonium or on addition of ouabain, showing that conformational flexibility is impaired under these conditions. Voltage-dependent fluorescence changes could also be observed in the presence of subsaturating K(+) concentrations. This allowed to monitor the time course of voltage-dependent relaxations into a new stationary distribution of states under turnover conditions, showing the acceleration of relaxation kinetics with increasing K(+) concentrations. As a result, the stationary distribution between E(1) and E(2) states and voltage-dependent relaxation times can be determined at any time and membrane potential under Na(+)Na(+) exchange as well as Na(+)K(+) turnover conditions.

Computing transient gating charge movement of voltage-dependent ion channels.

The opening of voltage-gated sodium, potassium, and calcium ion channels has a steep relationship with voltage. In response to changes in the transmembrane voltage, structural movements of an ion channel that precede channel opening generate a capacitative gating current. The net gating charge displacement due to membrane depolarization is an index of the voltage sensitivity of the ion channel activation process. Understanding the molecular basis of voltage-dependent gating of ion channels requires the measurement and computation of the gating charge, Q. We derive a simple and accurate semianalytic approach to computing the voltage dependence of transient gating charge movement (Q-V relationship) of discrete Markov state models of ion channels using matrix methods. This approach allows rapid computation of Q-V curves for finite and infinite length step depolarizations and is consistent with experimentally measured transient gating charge. This computational approach was applied to Shaker potassium channel gating, including the impact of inactivating particles on potassium channel gating currents.

Expression and Localization of Prestin and the Sugar Transporter GLUT-5 during Development of Electromotility in Cochlear Outer Hair Cells

Electromotility, i.e., the ability of cochlear outer hair cells (OHCs) to contract and elongate at acoustic frequencies, is presumed to depend on the voltage-driven conformational changes of "motor" proteins present in the OHC lateral plasma membrane. Recently, two membrane proteins have been proposed as candidates for the OHC motor. A sugar transporter, GLUT-5, was proposed based on its localization in the OHCs and on the observation that sugar transport alters the voltage sensitivity of the OHC motor mechanism. Another candidate, "prestin," was identified from a subtracted OHC cDNA library and shown to impart voltage-driven shape changes to transfected cultured cells. We used antibodies specific for these two proteins to show that they are highly expressed in the lateral membrane of OHCs. We also compared the postnatal expression patterns of these proteins with the development of electromotility in OHCs of the apical turn of the rat organ of Corti. The patch-clamp recording of transient charge movement associated with electromotility indicates that half of the maximal expression of the motor protein occurs at postnatal day 9. Prestin incorporation in the plasma membrane begins from postnatal day 0 and increases progressively in a time course coinciding with that of electromotility. GLUT-5 is not incorporated into the lateral plasma membrane of apical OHCs until postnatal day 15. Our results suggest that, although GLUT-5 may be involved in the control of electromotility, prestin is likely to be a fundamental component of the OHC membrane motor mechanism.

Extracellular pH modulates kinetics of the Na+,K+-ATPase

To investigate effects of pH on the Na+,K+-ATPase, we used the Xenopus oocytes to measure transient charge movements in the absence of extracellular K+, and steady-state currents mediated by the pump as well as ATPase activity. The activity of purified Na+,K+-ATPase strongly depends on pH, which has been attributed to protonation of intracellular sites. The steady-state current reflects pump activity, the transient charge movement voltage-dependent interaction of external Na+ ions with the pump molecule and/or conformational changes during Na+/Na+ exchange. The steady-state current exhibits a characteristic voltage dependence with maximum at about 0 mV at low external K+ (≤2 mM) and with 50 Na+. This dependency is not significantly affected by changes in external pH in the range from pH 9 to pH 6. Only below pH 6, the voltage dependence of pump current becomes less steep, and may be attributed to a pH-dependent inhibition of the forward pump cycle by external Na+. External stimulation of the pump by K+ in the absence of Na+ can be described by a voltage-dependent Km value with an apparent valency zK. At higher external pH the zK value is reduced. The transient current signal in the absence of external K+ can be described by the sum of three exponentials with voltage-dependent time constants of about 50 ms, 700 μs and less than 100 μs during pulses to 0 mV. The charge distribution was calculated by integration of the transient current signals. The slowest component and the associated charge distributions do not significantly depend on external pH changes. The intermediate component of the transients is represented by a voltage-dependent rate constant which shows a minimum at about −120 mV and increases with decreasing pH. Nevertheless, the contribution to the charge movement is not altered by pH changes due to a simultaneous increase of the amplitude of this component. We conclude that reduction of external pH counteracts external K+ and Na+ binding.

Effect of mutation of glycosylation sites on the Na + dependence of steady-state and transient currents generated by the neuronal GABA transporter

The GABA (γ-aminobutyric acid) transporter (GAT1) belongs to a superfamily of secondary active uptake systems for neurotransmitters that depend on the electrochemical gradients for Na + and Cl −. In the GAT1, two Na + ions and one Cl − ion are co-transported with one GABA molecule. Steady-state transport activity and transient charge movements during partial reactions of the transport cycle of the GAT1 of mouse brain expressed in Xenopus oocytes were investigated by two-electrode voltage clamp. Functional expression was demonstrated by Na +-dependent [ 3H]GABA uptake. Effects of mutation of two out of three N-glycosylation sites located in the extracellular loop between transmembrane domains 3 and 4 (Asn 176, Asn 181, Asn 184) were analysed. Simultaneous substitution of two Asn by Asp leaves the transport system intact but leads to a reduction in turnover and complex changes in the interaction of external Na + with the transport protein. If Asn 176 is mutated to Asp and simultaneously Asn 181 to Gly, no transport and no charge movements can be detected. In conclusion, mutations of the glycosylation sites result in altered transport, and the local conformation at Asn 181 is critical for expression of transport activity.

The Na +,K +-ATPase carrying the carboxy-terminal Ca 2+/calmodulin binding domain of the Ca 2+ pump has 2Na +,2K + stoichiometry and lost charge movement in Na +/Na + exchange

An altered ion-transport stoichiometry from 3Na +,2K + to 2Na +,2K + is observed in a chimeric Na +,K +-ATPase, which carries the Ca 2+/calmodulin binding domain (CBD) of the plasma membrane Ca 2+-ATPase at its carboxy-terminus [Zhao et al., FEBS Lett. 408 (1997) 271–275]. The ouabain-resistant mutant of this chimera (ORα1-CBD) was constructed to further investigate the effect of the CBD on ion-transport properties. The ORα1-CBD still shows the 2Na +,2K + stoichiometry. The loss of electrogenicity is accompanied by the disappearance of transient charge movements in the Na +/Na + exchange mode. We conclude that the binding of the third Na + ion, but not of the two others, in 3Na +,2K + transport mode apparently senses the electric field, and that the voltage-dependent Na + binding is likely to be lost in the chimera with CBD.

Electrogenic K+ transport by the Na(+)-K+ pump in rat cardiac ventricular myocytes.

1. The involvement of electrogenic reaction steps in K+ transport by the Na+, K(+)-ATPase was determined in rat cardiac ventricular myocytes using whole-cell patch clamp techniques. 2. Under K(+)-K+ exchange conditions and in the presence of extracellular K+ or Tl+ at concentrations that stimulated submaximal levels of steady-state Na+,K(+)-ATPase activity, ouabain-sensitive transient currents were observed during ('on') and after ('off') step changes in membrane potential (Vm). 3. The quantity of charge moved during the transient currents depended, in a saturable manner, on the magnitude of the voltage step. Maximal ouabain-sensitive 'on' and 'off' charges were calculated to be 9.6 +/- 0.9 and 9.1 +/- 0.4 fC pF-1 (n = 4), respectively, with an effective valeney of 0.48 +/- 0.07 (n = 7). 4. Kinetics of the transient currents were independent of Vm and Tl+o at positive potentials, but became more rapid at increasingly negative Vm values in an ion concentration-dependent fashion. 5. Those data demonstrate that electrogenic steps participate in K+ transport by the Na+,K(+)-ATPase and that the electrogenic step is extracellular ion binding. 6. The temperature- and Vm-dependent properties of transient charge movements were compared under K(+) -K+ and Na(+) -Na+ exchange conditions. The data suggest that extracellular K+ and Na+ binding occur at different sites in the enzyme or to different enzyme conformations. The sum of the effective valencies, 1.14 +/- 0.12, demonstrates that the electrogenicity of extra-cellular ion binding can explain the Vm dependence of ion transport by the Na+,K(+)-ATPase.