Position 170 of Rabbit Na+/glucose cotransporter (rSGLT1) lies in the Na+ pathway; modulation of polarity/charge at this site regulates charge transfer and carrier turnover

Positions 163, 166, and 173, within the putative external loop joining transmembrane segments IV and V of rabbit Na(+)/glucose cotransporter, form part of its Na(+) interaction and voltage-sensing domain. Since a Q170C mutation within this region exhibits anomalous behavior, its function was further investigated. We used Xenopus oocytes coinjected with mouse T-antigen to enhance Q170C expression, and the two-microelectrode voltage-clamp technique. For Q170C, alpha-methyl D-glucopyranoside, phloridzin, and Na(+) affinity values are equivalent to those of wild-type; but turnover is reduced approximately 50%. Decreased [Na(+)] reduces Q170C, but not wild-type, charge transfer. Q170C presteady-state currents exhibit three time constants, tau, identical to wild-type. MTSES decreases maximal alpha-methyl D-glucopyranoside-induced currents by approximately 64% and Na(+) leak by approximately 55%; phloridzin and Na(+) affinity are unchanged. MTSES also reduces charge transfer (dithiothreitol-reversible) and Q170C turnover by approximately 60-70%. MTSEA and MTSET protect against MTSES, but neither affect Q170C function. MTSES has no obvious effect on the tau-values. Q170A behaves the same as Q170C. The mutation Q170E affects voltage sensitivity and reduces turnover, but also appears to influence Na(+) interaction. We conclude that 1), glutamine 170 lies in the Na(+) pathway in rabbit Na(+)/glucose cotransporter and 2), altered polarity and charge at position 170 affect a cotransporter conformational state and transition, which is rate-limiting, but probably not associated with empty carrier reorientation.     

Novel structural determinants of mu-conotoxin (GIIIB) block in rat skeletal muscle (mu1) Na+ channels

mu-Conotoxin (mu-CTX) specifically occludes the pore of voltage-dependent Na(+) channels. In the rat skeletal muscle Na(+) channel (mu1), we examined the contribution of charged residues between the P loops and S6 in all four domains to mu-CTX block. Conversion of the negatively charged domain II (DII) residues Asp-762 and Glu-765 to cysteine increased the IC(50) for mu-CTX block by approximately 100-fold (wild-type = 22.3 +/- 7.0 nm; D762C = 2558 +/- 250 nm; E765C = 2020 +/- 379 nm). Restoration or reversal of charge by external modification of the cysteine-substituted channels with methanethiosulfonate reagents (methanethiosulfonate ethylsulfonate (MTSES) and methanethiosulfonate ethylammonium (MTSEA)) did not affect mu-CTX block (D762C: IC(50, MTSEA+) = 2165.1 +/- 250 nm; IC(50, MTSES-) = 2753.5 +/- 456.9 nm; E765C: IC(50, MTSEA+) = 2200.1 +/- 550.3 nm; IC(50, MTSES-) = 3248.1 +/- 2011.9 nm) compared with their unmodified counterparts. In contrast, the charge-conserving mutations D762E (IC(50) = 21.9 +/- 4.3 nm) and E765D (IC(50) = 22.0 +/- 7.0 nm) preserved wild-type blocking behavior, whereas the charge reversal mutants D762K (IC(50) = 4139.9 +/- 687.9 nm) and E765K (IC(50) = 4202.7 +/- 1088.0 nm) destabilized mu-CTX block even further, suggesting a prominent electrostatic component of the interactions between these DII residues and mu-CTX. Kinetic analysis of mu-CTX block reveals that the changes in toxin sensitivity are largely due to accelerated toxin dissociation (k(off)) rates with little changes in association (k(on)) rates. We conclude that the acidic residues at positions 762 and 765 are key determinants of mu-CTX block, primarily by virtue of their negative charge. The inability of the bulky MTSES or MTSEA side chain to modify mu-CTX sensitivity places steric constraints on the sites of toxin interaction.     

An external determinant in the S5-P linker of the pacemaker (HCN) channel identified by sulfhydryl modification

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels underlie spontaneous rhythmic activities in the heart and brain. Sulfhydryl modification of ion channels is a proven approach for studying their structure-function relationships; here we examined the effects of the hydrophilic sulfhydryl-modifying agents methanethiosulfonate ethylammonium (MTSEA(+)) and methanethiosulfonate ethylsulfonate (MTSES(-)) on wild-type (WT) and engineered HCN1 channels. External application of MTSEA(+) to WT channels irreversibly reduced whole-cell currents (I(MTSEA)/I(Control) = 42 +/- 2%), slowed activation and deactivation kinetics ( approximately 7- and approximately 3-fold at -140 and -20 mV, respectively), and produced hyperpolarizing shifts of steady-state activation (V(12)((MTSEA)) = -125.8 +/- 9.0 mV versus V(12)((Control)) = -76.4 +/- 1.6 mV). Sequence inspection revealed the presence of five endogenous cysteines in the transmembrane domains of HCN1: three are putatively close to the extracellular milieu (Cys(303), Cys(318), and Cys(347) in the S5, S5-P, and P segments, respectively), whereas the remaining two are likely to be cytoplasmic or buried. To identify the molecular constituent(s) responsible for the effects of MTSEA(+), we mutated the three "external" cysteines individually to serine. C303S did not yield measurable currents. Whereas C347S channels remained sensitive to MTSEA(+), C318S was not modified (I(MTSEA)/I(Control) = 101 +/- 2%, V(12)((MTSEA)) = -78.4 +/- 1.1 mV, and V(12)((Control)) = -79.8 +/- 2.3 mV). Likewise, WT (but not C318S) channels were sensitive to MTSES(-). Despite their opposite charges, MTSES(-) produced changes directionally similar to those effected by MTSEA(+) (I(MTSES)/I(Control) = 22 +/- 1.6% and V(12)((MTSES)) = -145.9 +/- 4.9 mV). We conclude that S5-P Cys(318) of HCN1 is externally accessible and that the external pore vestibule and activation gating of HCN channels are allosterically coupled.     

Substituted cysteine accessibility of the third transmembrane domain of the creatine transporter: defining a transport pathway

Twenty-two amino acid residues from transmembrane domain 3 of the creatine transporter were replaced, one at a time, with cysteine. The background for mutagenesis was a C144S mutant retaining approximately 75% of wild-type transport activity but resistant to methanethiosulfonate (MTS) reagents. Each substitution mutant was tested for creatine transport activity and sensitivity to the following MTS reagents: 2-aminoethyl methanethiosulfonate (MTSEA), 2-(trimethylammonium) ethyl methanethiosulfonate (MTSET), and 2-sulfonatoethyl methanethiosulfonate (MTSES). Two mutants (G134C and Y148C) were inactive, but most mutants showed significant levels of creatine transport. Treatment with MTSEA inhibited the activity of the W154C, Y147C, and I140C mutants. Creatine partially protected I140C from inactivation, and this residue, like Cys-144 in the wild-type CreaT, is predicted to be close to a creatine binding site. MTSEA inactivation of Y147C was dependent on Na+ and Cl- suggesting that solvent accessibility was ion-dependent. Helical wheel and helical net projections indicate that the three MTSEA-sensitive mutants (W154C, Y147C, and I140C) and two inactive mutants (V151C and Y148C) are aligned on a face of an alpha-helix, suggesting that they form part of a substrate pathway. The W154C mutant, located near the external face of the membrane, was accessible to the larger MTS reagents, whereas those implicated in creatine binding were only accessible to the smaller MTSEA. Consideration of our data, together with a study on the serotonin transporter (Chen, J. G., Sachpatzidis, A., and Rudnick, G. (1997) J. Biol. Chem. 272, 28321-28327), suggests that involvement of residues from transmembrane domain 3 is a common feature of the substrate pathway of Na+- and Cl- -dependent neurotransmitter transporters.     

Properties of the mutant Ser-460-Cys implicate this site in a functionally important region of the type IIa Na(+)/P(i) cotransporter protein.

The substituted cysteine accessibility approach, combined with chemical modification using membrane-impermeant alkylating reagents, was used to identify functionally important structural elements of the rat type IIa Na(+)/P(i) cotransporter protein. Single point mutants with different amino acids replaced by cysteines were made and the constructs expressed in Xenopus oocytes were tested for function by electrophysiology. Of the 15 mutants with substituted cysteines located at or near predicted membrane-spanning domains and associated linker regions, 6 displayed measurable transport function comparable to wild-type (WT) protein. Transport function of oocytes expressing WT protein was unchanged after exposure to the alkylating reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA, 100 microM), which indicated that native cysteines were inaccessible. However, for one of the mutants (S460C) that showed kinetic properties comparable with the WT, alkylation led to a complete suppression of P(i) transport. Alkylation in 100 mM Na(+) by either cationic ([2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET), MTSEA) or anionic [sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES)] reagents suppressed the P(i) response equally well, whereas exposure to methanethiosulfonate (MTS) reagents in 0 mM Na(+) resulted in protection from the MTS effect at depolarized potentials. This indicated that accessibility to site 460 was dependent on the conformational state of the empty carrier. The slippage current remained after alkylation. Moreover, after alkylation, phosphonoformic acid and saturating P(i) suppressed the slippage current equally, which indicated that P(i) binding could occur without cotransport. Pre-steady state relaxations were partially suppressed and their kinetics were significantly faster after alkylation; nevertheless, the remaining charge movement was Na(+) dependent, consistent with an intact slippage pathway. Based on an alternating access model for type IIa Na(+)/P(i) cotransport, these results suggest that site 460 is located in a region involved in conformational changes of the empty carrier.     

Slow-inactivation induced conformational change in domain 2-segment 6 of cardiac Na+ channel

To examine conformational changes during slow inactivation involving domain 2-segment 6 (D2-S6) of human cardiac Na(+) channel (hNav1.5), we applied the substituted-cysteine accessibility method (SCAM) using methanethiosulfonate ethylammonium (MTSEA). We substituted cysteine (C) for native valine (V) at position 930 of D2-S6 in the MTSEA-resistant hNav1.5 mutant C373Y to produce the double mutant C373Y-V930C. Whole-cell Na(+) currents were recorded using patch-clamp techniques in transiently transfected HEK cells. In C373Y-V930C, we find that MTSEA (1.5 mM) applied in the closed state (-160 mV) has no significant effect on whole-cell Na(+) current, while MTSEA applied in the slow-inactivated state (prolonged depolarization at 0 mV) decreases current. We propose that D2-S6 in hNav1.5 undergoes molecular rearrangement during slow inactivation exposing the side chain of residue 930 such that it becomes accessible to modification by MTSEA.     

Evidence for the involvement of Ala 166 in coupling Na(+) to sugar transport through the human Na(+)/glucose cotransporter

We mutated residue 166, located in the putative Na(+) transport pathway between transmembrane segments 4 and 5 of human Na(+)/glucose cotransporter (hSGLT1), from alanine to cysteine (A166C). A166C was expressed in Xenopus laevis oocytes, and electrophysiological methods were used to assay function. The affinity for Na(+) was unchanged compared to that of hSGLT1, whereas the sugar affinity was reduced and sugar specificity was altered. There was a reduction in the turnover rate of the transporter, and in contrast to that of hSGLT1, the turnover rate depended on the sugar molecule. Exposure of A166C to MTSEA and MTSET, but not MTSES, abolished sugar transport. Accessibility of A166C to alkylating reagents was independent of protein conformation, indicating that the residue is always accessible from the extracellular surface. Sugar and phlorizin did not protect the residue from being alkylated, suggesting that residue 166 is not located in the sugar pathway. MTSEA, MTSET, and MTSES all changed the pre-steady-state kinetics of A166C, independent of pH, and sugars altered these kinetics. The inability of MTSEA-labeled A166C to transport sugar was reversed (with no major change in Na(+) and sugar affinity) if the positive charge on MTSEA was neutralized by increasing the external pH to 9.0. These studies suggest that the residue at position 166 is involved in the interaction between the Na(+) and sugar transport pathways.     

Electrophysiological analysis of the mutated Na,K-ATPase cation binding pocket

Na,K-ATPase mediates net electrogenic transport by extruding three Na+ ions and importing two K+ ions across the plasma membrane during each reaction cycle. We mutated putative cation coordinating amino acids in transmembrane hairpin M5-M6 of rat Na,K-ATPase: Asp776 (Gln, Asp, Ala), Glu779 (Asp, Gln, Ala), Asp804 (Glu, Asn, Ala), and Asp808 (Glu, Asn, Ala). Electrogenic cation transport properties of these 12 mutants were analyzed in two-electrode voltage-clamp experiments on Xenopus laevis oocytes by measuring the voltage dependence of K+-stimulated stationary currents and pre-steady-state currents under electrogenic Na+/Na+ exchange conditions. Whereas mutants D804N, D804A, and D808A hardly showed any Na+/K+ pump currents, the other constructs could be classified according to the [K+] and voltage dependence of their stationary currents; mutants N776A and E779Q behaved similarly to the wild-type enzyme. Mutants E779D, E779A, D808E, and D808N had in common a decreased apparent affinity for extracellular K+. Mutants N776Q, N776D, and D804E showed large deviations from the wild-type behavior; the currents generated by mutant N776D showed weaker voltage dependence, and the current-voltage curves of mutants N776Q and D804E exhibited a negative slope. The apparent rate constants determined from transient Na+/Na+ exchange currents are rather voltage-independent and at potentials above -60 mV faster than the wild type. Thus, the characteristic voltage-dependent increase of the rate constants at hyperpolarizing potentials is almost absent in these mutants. Accordingly, dislocating the carboxamide or carboxyl group of Asn776 and Asp804, respectively, decreases the extracellular Na+ affinity.     

Investigating the conformational states of the rabbit Na+/glucose cotransporter

The Na(+) and voltage-dependence of transient rabbit Na(+)/glucose cotransporter (rSGLT1) kinetics was studied with the two-electrode voltage-clamp technique and Xenopus laevis oocytes. Using step changes in membrane potential, in the absence of glucose but with 100 or 10 mM Na(+), transient currents were measured corresponding to binding/debinding of Na(+) and conformational changes of the protein. Previously, only a single time constant has been published for rSGLT1. We, however, observed three decay components; a fast (tau(f), 0.5-1 ms) voltage- and Na(+)-independent decay, and medium (tau(m), 0.5-4 ms) and slow (tau(s), 8-50 ms) voltage- and Na(+)-dependent decays. Transient currents were simulated and fit using a four-state model to obtain kinetic parameters for the system. The four-state model was able to reconstitute an assortment of experimental data.     

Methanethiosulfonate-modification Alters Local Anesthetic Block in rNavl.4 Cysteine-substituted Mutants S1276C and L1280C

We previously showed that lysine substitutions at two residues in segment 6 of domain 3 in voltage-gated Na(+) channel rNav1.4 (S1276K, L1280K) reduced steady-state inactivated local anesthetic block. Here we studied cysteine substitutions at the same residues (S1276C, L1280C). We used whole-cell recordings to determine local anesthetic block (100 microM bupivacaine) before and after cysteine modification with 1.5 mM 2-aminoethyl methanethiosulfonate (MTSEA). Compared with rNav1.4, steady-state resting bupivacaine block at -180 mV was increased in S1276C, while inactivated block at -50 mV was not different in the mutants. After application of MTSEA at -160 mV, rNav1.4 showed enhanced bupivacaine block and a negative shift in V(1/2) of the bupivacaine affinity curve, while L1280C and S1276C showed a decrease in inactivated bupivacaine block after MTSEA. Application of MTSEA at 0 mV produced similar results in rNav1.4 and L1280C, but an opposite effect in S1276C, i.e., enhancement of bupivacaine block, with a large negative shift in V(1/2) of the bupivacaine affinity curve similar to that found in rNav1.4. We conclude that 1) MTSEA modification of 1276C or 1280C decreases inactivated bupivacaine block similar to that found in L1280K and S1276K, 2) residue 1276C is only accessible to MTS-modification in the resting state, and 3) MTSEA may modify a native cysteine in rNav1.4 that produces an allosteric, indirect effect on bupivacaine affinity.     

Fast voltage clamp discloses a new component of presteady-state currents from the Na(+)-glucose cotransporter.

The human Na(+)-glucose cotransporter (hSGLT1) has been shown to generate, in the absence of sugar, presteady-state currents in response to a change in potential, which could be fitted with single exponentials once the voltage had reached a new constant value. By the cut-open oocyte technique (voltage rising-speed approximately 1 mV/microsecond), phlorizin-sensitive transient currents could be detected with a higher time resolution during continuous intracellular perfusion. In the absence of sugar and internal Na+, and with 90 mM external Na+ concentration ([Na+]o), phlorizin-sensitive currents exhibited two relaxation time-constants: tau 1 increased from 2 to 10 ms when Vm decreased from +60 mV to -80 mV and remained at 10 ms for more negative Vm; tau 2 ranged from 0.4 to 0.8 ms in a weakly voltage-dependent manner. According to a previously proposed model, these two time constants could be accounted for by 1) Na+ crossing a fraction of the membrane electrical field to reach its binding site on the carrier and 2) conformational change of the free carrier. To test this hypothesis, the time constants were measured as [Na+]o was progressively reduced to 0 mM. At 30 and 10 mM external Na+, tau 1 reached the same plateau value of 10 ms but at more negative potentials (-120 and -160 mV, respectively). Contrary to the prediction of the model, two time constants continued to be detected in the bilateral absence of Na+ (at pH 8.0). Under these conditions, tau 1 continuously increased through the whole voltage range and did not reach the 10 ms level even when Vm had attained -200 mV while tau 2 remained in the range of 0.4-0.8 ms. These results indicate that 1) conformational change of the free carrier across the membrane must occur in more than one step and 2) Na+ binding/debinding is not responsible for either of the two observed exponential components of transient currents. By use of the simplest kinetic model accounting for the portion of the hSGLT1 transport cycle involving extracellular Na+ binding/debinding and the dual-step conformational change of the free carrier, tau 1 and tau 2 were fitted throughout the voltage range, and a few sets of parameters were found to reproduce the data satisfactorily. This study shows that 1) tau 1 and tau 2 correspond to two steps in the conformational change of the free carrier, 2) Na+ binding/debinding modulates the slow time constant (tau 1) and 3) a voltage-independent slow conformational change of the free carrier accounts for the observed plateau value of 10 ms.     

Inactivation of batrachotoxin-modified Na+ channels in GH3 cells. Characterization and pharmacological modification

Batrachotoxin (BTX)-modified Na+ currents were characterized in GH3 cells with a reversed Na+ gradient under whole-cell voltage clamp conditions. BTX shifts the threshold of Na+ channel activation by approximately 40 mV in the hyperpolarizing direction and nearly eliminates the declining phase of Na+ currents at all voltages, suggesting that Na+ channel inactivation is removed. Paradoxically, the steady-state inactivation (h infinity) of BTX-modified Na+ channels as determined by a two-pulse protocol shows that inactivation is still present and occurs maximally near -70 mV. About 45% of BTX-modified Na+ channels are inactivated at this voltage. The development of inactivation follows a sum of two exponential functions with tau d(fast) = 10 ms and tau d(slow) = 125 ms at -70 mV. Recovery from inactivation can be achieved after hyperpolarizing the membrane to voltages more negative than -120 mV. The time course of recovery is best described by a sum of two exponentials with tau r(fast) = 6.0 ms and tau r(slow) = 240 ms at -170 mV. After reaching a minimum at -70 mV, the h infinity curve of BTX-modified Na+ channels turns upward to reach a constant plateau value of approximately 0.9 at voltages above 0 mV. Evidently, the inactivated, BTX-modified Na+ channels can be forced open at more positive potentials. The reopening kinetics of the inactivated channels follows a single exponential with a time constant of 160 ms at +50 mV. Both chloramine-T (at 0.5 mM) and alpha-scorpion toxin (at 200 nM) diminish the inactivation of BTX-modified Na+ channels. In contrast, benzocaine at 1 mM drastically enhances the inactivation of BTX-modified Na+ channels. The h infinity curve reaches minimum of less than 0.1 at -70 mV, indicating that benzocaine binds preferentially with inactivated, BTX-modified Na+ channels. Together, these results imply that BTX-modified Na+ channels are governed by an inactivation process.     

Cysteine-scanning mutagenesis and thiol modification of the Rickettsia prowazekii ATP/ADP translocase: evidence that TM VIII faces an aqueous channel

The contribution of transmembrane region VIII of the Rickettsia prowazekii ATP/ADP translocase to the structure of the water-filled channel through which ATP is transported was evaluated from the accessibility of three hydrophilic, thiol reactive, methanethiosulfonate reagents to a library of 21 single-cysteine substitution mutants expressed in Escherichia coli. A negatively charged reagent (MTSES) and two positively charged reagents (MTSET and MTSEA) were used. Mutants Q323C and G327C did not tolerate cysteine substitution and were almost completely deficient in ATP transport. The remaining mutants exhibited 25-226% of the cysteine-less parent's transport activity. Five patterns of inhibition of ATP transport by the MTS reagents were observed. (i) ATP transport was not inhibited by any of the three MTS reagents in mutants Q321C, F324C, A332C, and L335C and only marginally in F333C. (ii) Transport activity of mutants F322C, Q326C, and A330C was markedly inhibited by all three reagents. (iii) ATP transport was inhibited by MTSEA in only the largest group of mutants (M334C, I336C, G337C, S338C, N339C, I340C, and I341C). (iv) Transport activity was inhibited by MTSET and MTSEA, whereas high concentrations of MTSES were required to inhibit mutants W328C, V329C, and I331C. However, mutant W328C could be inhibited by MTSES in the presence of sub-K(m) concentrations of the substrate. (v) ATP transport by mutant Y325C was unaffected by MTSEA, but inhibited approximately 50% by MTSET and MTSES. Transport of ATP protected mutants (F322C, W328C, V329C, A330C, and I331C) from MTS inhibition. Mutants in the half of TM VIII that is closest to the cytoplasm were not inhibited well by MTSES or MTSET in either whole cells or inside-out vesicles. The results indicate that TM VIII makes a major contribution to the structure of the aqueous translocation pathway, that the accessibility to impermeant thiol reagents is influenced (blocked or stimulated) by substrate, and that there is great variation in accessibility to MTS reagents along the length of TM VIII.     

Pore residues critical for mu-CTX binding to rat skeletal muscle Na+ channels revealed by cysteine mutagenesis.

We have studied mu-conotoxin (mu-CTX) block of rat skeletal muscle sodium channel (rSkM1) currents in which single amino acids within the pore (P-loop) were substituted with cysteine. Among 17 cysteine mutants expressed in Xenopus oocytes, 7 showed significant alterations in sensitivity to mu-CTX compared to wild-type rSkM1 channel (IC50 = 17.5 +/- 2.8 nM). E758C and D1241C were less sensitive to mu-CTX block (IC50 = 220 +/- 39 nM and 112 +/- 24 nM, respectively), whereas the tryptophan mutants W402C, W1239C, and W1531C showed enhanced mu-CTX sensitivity (IC50 = 1.9 +/- 0.1, 4.9 +/- 0.9, and 5.5 +/- 0.4 nM, respectively). D400C and Y401C also showed statistically significant yet modest (approximately twofold) changes in sensitivity to mu-CTX block compared to WT (p < 0.05). Application of the negatively charged, sulfhydryl-reactive compound methanethiosulfonate-ethylsulfonate (MTSES) enhanced the toxin sensitivity of D1241C (IC50 = 46.3 +/- 12 nM) while having little effect on E758C mutant channels (IC50 = 199.8 +/- 21.8 nM). On the other hand, the positively charged methanethiosulfonate-ethylammonium (MTSEA) completely abolished the mu-CTX sensitivity of E758C (IC50 > 1 microM) and increased the IC50 of D1241C by about threefold. Applications of MTSEA, MTSES, and the neutral MTSBN (benzyl methanethiosulfonate) to the tryptophan-to-cysteine mutants partially or fully restored the wild-type mu-CTX sensitivity, suggesting that the bulkiness of the tryptophan's indole group is a determinant of toxin binding. In support of this suggestion, the blocking IC50 of W1531A (7.5 +/- 1.3 nM) was similar to W1531C, whereas W1531Y showed reduced toxin sensitivity (14.6 +/- 3.5 nM) similar to that of the wild-type channel. Our results demonstrate that charge at positions 758 and 1241 are important for mu-CTX toxin binding and further suggest that the tryptophan residues within the pore in domains I, III, and IV negatively influence toxin-channel interaction.     

The conserved cysteine 7.38 residue is differentially accessible in the binding-site crevices of the mu, delta, and kappa opioid receptors

Binding pockets of the opioid receptors are presumably formed among the transmembrane domains (TMDs) and are accessible from the extracellular medium. In this study, we determined the sensitivity of binding of [(3)H]diprenorphine, an antagonist, to mu, delta, and kappa opioid receptors to charged methanethiosulfonate (MTS) derivatives and identified the cysteine residues within the TMDs that conferred the sensitivity. Incubation of the mu opioid receptor expressed in HEK293 cells with MTS ethylammonium (MTSEA), MTS ethyltrimethylammonium (MTSET), or MTS ethylsulfonate (MTSES) inhibited [(3)H]diprenorphine binding with the potency order of MTSEA > MTSET > MTSES. Pretreatment of mu, delta, and kappa opioid receptors with MTSEA dose-dependently inhibited [(3)H]diprenorphine binding with MTSEA sensitivity in the order of kappa > mu > delta. The effects of MTSEA occurred rapidly, reaching the maximal inhibition in 10 min. (-)-Naloxone, but not (+)-naloxone, prevented the MTSEA effect, demonstrating that the reaction occurs within or in the vicinity of the binding pockets. Each cysteine residue in the TMDs of the three receptors was mutated singly, and the effects of MTSEA treatment were examined. The mutants had similar affinities for [(3)H]diprenorphine, and C7. 38(321)S, C7.38(303)S, and C7.38(315)S mutations rendered mu, delta, and kappa opioid receptors less sensitive to the effect of MTSEA, respectively. These results indicate that the conserved Cys7.38 is differentially accessible in the binding-site crevice of these receptors. The second extracellular loop of the kappa receptor, which contains several acidic residues, appears to play a role, albeit small, in its higher sensitivity to MTSEA, whereas the negative charge of Glu6.58(297) did not. To the best of our knowledge, this is the first report to show that a conserved residue among highly homologous G protein-coupled receptors is differentially accessible in the binding-site crevice. In addition, this represents the first successful generation of MTSEA-insensitive mutants of mu, delta, and kappa opioid receptors, which will allow determination of residues accessible in the binding-site crevices of these receptors by the substituted cysteine accessibility method.     

Charge translocation by the Na+/K+ pump under Na+/Na+ exchange conditions: intracellular Na+ dependence

The effect of intracellular (i) and extracellular (o) Na+ on pre-steady-state transient current associated with Na+/Na+ exchange by the Na+/K+ pump was investigated in the vegetal pole of Xenopus oocytes. Current records in response to 40-ms voltage pulses from -180 to +100 mV in the absence of external Na+ were subtracted from current records obtained under Na+/Na+ exchange conditions. Na+-sensitive transient current and dihydroouabain-sensitive current were equivalent. The quantity of charge moved (Q) and the relaxation rate coefficient (ktot) of the slow component of the Nao+-sensitive transient current were measured for steps to various voltages (V). The data were analyzed using a four-state kinetic model describing the Na+ binding, occlusion, conformational change, and release steps of the transport cycle. The apparent valence of the Q vs. V relationship was near 1.0 for all experimental conditions. When extracellular Na+ was halved, the midpoint voltage of the charge distribution (Vq) shifted -25.3+/-0.4 mV, which can be accounted for by the presence of an extracellular ion-well having a dielectric distance delta=0.69+/-0.01. The effect of changes of Nai+ on Nao+-sensitive transient current was investigated. The midpoint voltage (Vq) of the charge distribution curve was not affected over the Nao+ concentration range 3.13-50 mM. As Nai+ was decreased, the amount of charge measured and its relaxation rate coefficient decreased with an apparent Km of 3.2+/-0.2 mM. The effects of lowering Nai+ on pre-steady-state transient current can be accounted for by decreasing the charge available to participate in the fast extracellular Na+ release steps, by a slowly equilibrating (phosphorylation/occlusion) step intervening between intracellular Na+ binding and extracellular Na+ release.     

Time- and state-dependent effects of methanethiosulfonate ethylammonium (MTSEA) exposure differ between heart and skeletal muscle voltage-gated Na(+) channels

The substituted-cysteine scanning method (SCAM) is used to study conformational changes in proteins. Experiments using SCAM involve site-directed mutagenesis to replace native amino acids with cysteine and subsequent exposure to a methanethiosulfonate (MTS) reagent such as methanethiosulfonate ethylammonium (MTSEA). These reagents react with substituted-cysteines and can provide functional information about relative positions of amino acids within a protein. In the human heart voltage-gated Na(+) channel hNav1.5 there is a native cysteine at position C373 that reacts rapidly with MTS reagents resulting in a large reduction in whole-cell Na(+) current (I(Na)). Therefore, in order to use SCAM in studies in this isoform, this native cysteine is mutated to a non-reactive residue, e.g., tyrosine. This mutant, hNav1.5-C373Y, is resistant to the MTS-mediated decrease in I(Na). Here we show that this resistance is time- and state-dependent. With relatively short exposure times to MTSEA (<4min), there is little effect on I(Na). However, with longer exposures (4-8min), there is a large decrease in I(Na), but this effect is only found when hNav1.5-C373Y is inactivated (fast or slow) - MTSEA has little effect in the closed state. Additionally, this long-term, state-dependent effect is not seen in human skeletal muscle Na(+) channel isoform hNav1.4, which has a native tyrosine at the homologous site C407. We conclude that differences in molecular determinants of inactivation between hNav1.4 and hNav1.5 underlie the difference in response to MTSEA exposure.     

The transport activity of the Na+-Ca2+ exchanger NCX1 expressed in HEK 293 cells is sensitive to covalent modification of intracellular cysteine residues by sulfhydryl reagents

Membrane permeable N-ethylmaleimide (NEM) and (2-aminoethyl)methanethiosulfonatehydrobromide (MTSEA) inhibited the rat brain Na(+)-Ca(2+) exchanger RBE-2 (NCX1.5) expressed in HEK 293 cells in a dose dependent manner. 50% inhibition was obtained at 1 mm MTSEA and 1.65 mm NEM. External application of membrane impermeable [2-(trimethylammonium) ethyl]methanethiosulfonatebromide (MTSET) and sodium(2-sulfonatoethyl)methanethiosulfonate (MTSES) did not inhibit the transport activity in whole cells. Following reconstitution, however, of RBE-2 transfected cell proteins into proteoliposomes, external application of MTSET and MTSES led to a decrease in transport activity to 42.7 (S.D. = 9.1) and 51% (S.D. = 10.14), respectively. Similar results were obtained also when the rat heart isoform RHE-1 (NCX1.1) or the rat brain isoform RBE-1 (NCX1.4) was expressed. NEM and MTSEA inhibited Na(+) gradient-dependent Ca(2+) uptake also in HEK 293 cells expressing RBE-2/C14A/C20S/ C122S/C780S (numbering corresponds to RBE-2), a mutant in which all putative extracellular cysteines were exchanged. To study the accessibility of different cysteines to covalent modification, surface biotinylation of cells expressing the wild type exchanger and its mutants was carried out using 3-(N-maleimidylpropionyl)biocytin. Surface biotinylation revealed immunoreactive protein derived from the wild type Na(+)-Ca(2+) exchanger only if the transfected cells were exposed to the reducing agent Tris(2-carboxyethyl)phosphine. No reduction was needed when the single cysteine mutants of RBE-2, C14A, C20S, and C780S, were expressed. Treatment of the cells expressing these mutants with MTSET before biotinylation, led to a decrease in the amount of exchanger protein that was revealed. No immunoreactive protein was detected when the quadruple mutant RBE-2, C14A/C20S/C122S/C780S, was biotinylated, suggesting that no additional cysteines are accessible directly from the extracellular face of the membrane. Permeabilizing the cells expressing RBE-2/C14A/C20S/ C122S/C780S with streptolysin O resulted in biotinylation of the exchanger protein. Its amount decreased if exposure to NEM preceded streptolysin O treatment. Our results suggest that Na(+)-Ca(2+) exchange activity is inhibited by covalent modification with sulfhydryl reagents of cysteine residues that are accessible from the cytoplasmic face of the membrane.     

Transmembrane IV of the high-affinity sodium-glucose cotransporter participates in sugar binding

Investigation of the structure/function relationships of the sodium-glucose transporter (SGLT1) is crucial to understanding the cotransporter mechanism. In the present study, we used cysteine-scanning mutagenesis and chemical modification by methanethiosulfonate (MTS) derivatives to test whether predicted transmembrane IV participates in sugar binding. Five charged and polar residues (K139, Q142, T156, K157, and D161) and two glucose/galactose malabsorption missense mutations (I147 and S159) were replaced with cysteine. Mutants I147C, T156C, and K157C exhibited sufficient expression to be studied in detail using the two-electrode voltage-clamp method in Xenopus laevis oocytes and COS-7 cells. I147C was similar in function to wild-type and was not studied further. Mutation of lysine-157 to cysteine (K157C) causes loss of phloridzin and alpha-methyl-D-glucopyranoside (alphaMG) binding. These functions are restored by chemical modification with positively charged (2-aminoethyl) methanethiosulfonate hydrobromide (MTSEA). Mutation of threonine-156 to cysteine (T156C) reduces the affinity of alphaMG and phloridzin for T156C by approximately 5-fold and approximately 20-fold, respectively. In addition, phloridzin protects cysteine-156 in T156C from alkylation by MTSEA. Therefore, the presence of a positive charge or a polar residue at 157 and 156, respectively, affects sugar binding and sugar-induced Na(+) currents.     

Binding of benzocaine in batrachotoxin-modified Na+ channels. State- dependent interactions

Hille (1977. Journal of General Physiology. 69:497-515) first proposed a modulated receptor hypothesis (MRH) to explain the action of benzocaine in voltage-gated Na+ channels. Using the MRH as a framework, we examined benzocaine binding in batrachotoxin (BTX)-modified Na+ channels under voltage-clamp conditions using either step or ramp command signals. We found that benzocaine binding is strongly voltage dependent. At -70 mV, the concentration of benzocaine that inhibits 50% of BTX-modified Na+ currents in GH3 cells (IC50) is 0.2 mM, whereas at +50 mV, the IC50 is 1.3 mM. Dose-response curves indicate that only one molecule of benzocaine is required to bind with one BTX-modified Na+ channel at -70 mV, whereas approximately two molecules are needed at +50 mV. Upon treatment with the inactivation modifier chloramine-T, the binding affinity of benzocaine is reduced significantly at -70 mV, probably as a result of the removal of the inactivated state of BTX- modified Na+ channels. The same treatment, however, enhances the binding affinity of cocaine near this voltage. External Na+ ions appear to have little effect on benzocaine binding, although they do affect cocaine binding. We conclude that two mechanisms underlie the action of local anesthetics in BTX-modified Na+ channels. Unlike open-channel blockers such as cocaine and bupivacaine, neutral benzocaine binds preferentially with BTX-modified Na+ channels in a closed state. Furthermore, benzocaine can be modified chemically so that it behaves like an open-channel blocker. This compound also elicits a use- dependent block in unmodified Na+ channels after repetitive depolarizations, whereas benzocaine does not. The implications of these findings for the MRH theory will be discussed.