A glutamine to glutamate mutation at position 170 (Q170E) in the rabbit Na+/glucose cotransporter, rSGLT1, enhances binding affinity for Na+

Using cysteine mutagenesis and chemical modification by methanethiosulfonate derivatives, it was demonstrated that the external putative loop, joining transmembrane segments (TM's) IV-V of rabbit Na+/glucose cotransporter, rSGLT1, forms part of a Na+ binding and voltage sensing domain. Within this region, exposure to cationic (2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) inhibited F163C, A166C, and L173C, but anionic sodium (2-sulfonatoethyl)methanethiosulfonate (MTSES) had no effect. Unexpectedly, MTSEA had no effect on Q170C; however, MTSES profoundly altered Q170C charge transfer and turnover, leaving Na+ and sugar binding affinity unchanged, but mutation of glutamine to anionic glutamate (Q170E) shifted V(0.5) to positive potentials, suggesting enhanced Na+ affinity. To clarify the role of glutamine 170 in Na+ interaction, we embarked on a more detailed investigation of Q170E using the two-microelectrode voltage clamping in Xenopus oocytes. Compared to wild-type (wt) rSGLT1, Q170E exhibits (i) a 2-fold decrease in methyl alpha-D-glucopyranoside affinity (-150 to -90 mV), (ii) a 5-fold increase in Na+ affinity (-150 to -100 mV) with less voltage dependency, (iii) reduced Na+ leak, and (iv) two transient current decay constants (tau(fast), tau(slow)) compared to three (tau(fast), tau(medium), tau(slow)) for wt, and computer simulation of Q170E pre-steady-state currents with a four-state kinetic model yields parameters similar to wt SGLT1, except for a reduced Na+ debinding rate constant compared to wt. Taken together, the data strengthen the conclusion that residue 170 lies in the Na+ pathway and provide the first evidence that it participates in determining Na+ binding.     

Conformational dynamics of hSGLT1 during Na+/glucose cotransport

This study examines the conformations of the Na(+)/glucose cotransporter (SGLT1) during sugar transport using charge and fluorescence measurements on the human SGLT1 mutant G507C expressed in Xenopus oocytes. The mutant exhibited similar steady-state and presteady-state kinetics as wild-type SGLT1, and labeling of Cys507 by tetramethylrhodamine-6-maleimide had no effect on kinetics. Our strategy was to record changes in charge and fluorescence in response to rapid jumps in membrane potential in the presence and absence of sugar or the competitive inhibitor phlorizin. In Na(+) buffer, step jumps in membrane voltage elicited presteady-state currents (charge movements) that decay to the steady state with time constants tau(med) (3-20 ms, medium) and tau(slow) (15-70 ms, slow). Concurrently, SGLT1 rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages (DeltaF). The charge vs. voltage (Q-V) and fluorescence vs. voltage (DeltaF-V) relations (for medium and slow components) obeyed Boltzmann relations with similar parameters: zdelta (apparent valence of voltage sensor) approximately 1; and V(0.5) (midpoint voltage) between -15 and -40 mV. Sugar induced an inward current (Na(+)/glucose cotransport), and reduced maximal charge (Q(max)) and fluorescence (DeltaF(max)) with half-maximal concentrations (K(0.5)) of 1 mM. Increasing [alphaMDG](o) also shifted the V(0.5) for Q and DeltaF to more positive values, with K(0.5)'s approximately 1 mM. The major difference between Q and DeltaF was that at saturating [alphaMDG](o), the presteady-state current (and Q(max)) was totally abolished, whereas DeltaF(max) was only reduced 50%. Phlorizin reduced both Q(max) and DeltaF(max) (K(i) approximately 0.4 microM), with no changes in V(0.5)'s or relaxation time constants. Simulations using an eight-state kinetic model indicate that external sugar increases the occupancy probability of inward-facing conformations at the expense of outward-facing conformations. The simulations predict, and we have observed experimentally, that presteady-state currents are blocked by saturating sugar, but not the changes in fluorescence. Thus we have isolated an electroneutral conformational change that has not been previously described. This rate-limiting step at maximal inward Na(+)/sugar cotransport (saturating voltage and external Na(+) and sugar concentrations) is the slow release of Na(+) from the internal surface of SGLT1. The high affinity blocker phlorizin locks the cotransporter in an inactive conformation.     

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.     

Rapid charge translocation by the cardiac Na(+)-Ca2+ exchanger after a Ca2+ concentration jump.

The kinetics of Na(+)-Ca2+ exchange current after a cytoplasmic Ca2+ concentration jump (achieved by photolysis of DM-nitrophen) was measured in excised giant membrane patches from guinea pig or rat heart. Increasing the cytoplasmic Ca2+ concentration from 0.5 microM in the presence of 100 mM extracellular Na+ elicits an inward current that rises with a time constant tau 1 < 50 microseconds and decays to a plateau with a time constant tau 2 = 0.65 +/- 0.18 ms (n = 101) at 21 degrees C. These current signals are suppressed by Ni2+ and dichlorobenzamil. No stationary current, but a transient inward current that rises with tau 1 < 50 microseconds and decays with tau 2 = 0.28 +/- 0.06 ms (n = 53, T = 21 degrees C) is observed if the Ca2+ concentration jump is performed under conditions that promote Ca(2+)-Ca2+ exchange (i.e., no extracellular Na+, 5 mM extracellular Ca2+). The transient and stationary inward current is not observed in the absence of extracellular Ca2+ and Na+. The application of alpha-chymotrypsin reveals the influence of the cytoplasmic regulatory Ca2+ binding site on Ca(2+)-Ca2+ and forward Na(+)-Ca2+ exchange and shows that this site regulates both the transient and stationary current. The temperature dependence of the stationary current exhibits an activation energy of 70 kj/mol for temperatures between 21 degrees C and 38 degrees C, and 138 kj/mol between 10 degrees C and 21 degrees C. For the decay time constant an activation energy of 70 kj/mol is observed in the Na(+)-Ca2+ and the Ca(2+)-Ca2+ exchange mode between 13 degrees C and 35 degrees C. The data indicate that partial reactions of the Na(+)-Ca2+ exchanger associated with Ca2+ binding and translocation are very fast at 35 degrees C, with relaxation time constants of about 6700 s-1 in the forward Na(+)-Ca2+ exchange and about 12,500 s-1 in the Ca(2+)-Ca2+ exchange mode and that net negative charge is moved during Ca2+ translocation. According to model calculations, the turnover number, however, has to be at least 2-4 times smaller than the decay rate of the transient current, and Na+ inward translocation appears to be slower than Ca2+ outward movement.     

Evidence for intraprotein charge transfer during the transport activity of the melibiose permease from Escherichia coli.

Electrogenic activity associated with the activity of the melibiose permease (MelB) of Escherichia coli was investigated by using proteoliposomes containing purified MelB adsorbed onto a solid-supported membrane. Transient currents were selectively recorded by applying concentration jumps of Na+ ions (or Li+) and/or of different sugar substrates of MelB (melibiose, thio-methyl galactoside, raffinose) using a fast-flow solution exchange system. Characteristically, the transient current response was fast, including a single decay exponential component (tau approximately 15 ms) on applying a Na+ (or Li+) concentration jump in the absence of sugar. On imposing a Na+ (or Li+) jump on proteoliposomes preincubated with the sugar, a sugar jump in a preparation preincubated with the cation, or a simultaneous jump of the cation and sugar substrates, the electrical transients were biphasic and comprised both the fast and an additional slow (tau approximately 350 ms) decay components. Finally, selective inactivation of the cosubstrate translocation step by acylation of MelB cysteins with N-ethyl maleimide suppressed the slow response components and had no effect on the fast transient one. We suggest that the fast transient response reflects charge transfer within MelB during cosubstrate binding while the slow component is associated with charge transfer across the proteoliposome membrane. From the time course of the transient currents, we estimate a rate constant for Na+ binding in the absence and presence of melibiose of k > 50 s(-1) and one for melibiose binding in the absence of Na+ of k approximately 10 s(-1).     

Transition states of the high-affinity rabbit Na(+)/glucose cotransporter SGLT1 as determined from measurement and analysis of voltage-dependent charge movements

The charge-membrane voltage (Q-V) distribution of wild-type rabbit Na⁺/glucose transporter (rSGLT1) expressed in Xenopus oocytes was investigated in the absence of glucose, using the two-electrode voltage-clamp technique. Although this distribution is generally believed to be well represented by a two-state Boltzmann equation, we recently provided evidence for the existence of at least four states (Krofchick D and Silverman M. Biophys J 84: 3690–3702, 2003), confirming an earlier finding for human SGLT1 (Chen XZ, Coady MJ, and Lapointe JY. Biophys J 71: 2544–2552, 1996). We now extend our study of rSGLT1 pre-steady-state currents, employing high-resolution measurement and analysis of the Q-V distribution. A ramp, instead of a step, voltage change was used to prevent saturation of the apparatus in the first 1 ms. Transient currents were integrated out to 150 ms, instead of the standard 50–100 ms. Measurements were taken every 10 mV instead of the standard 20 mV. The Q-V distribution was fit with a two-, three-, and four-state Boltzmann equation and was described best by the three-state equation. The three-state fit produced two valences of 0.45 and 1.1 at two V_0.5 values of –48 and –7.7, respectively. Our findings are critically compared with other published studies and the differences are discussed. An implication of the three-state fit is that the turnover rate of rSGLT1 is 34 s^–1, i.e., 54% greater than previously reported (22 s^–1). Our new findings support the concept that the sugar-free model of SGLT1 is more complex than generally accepted, most likely involving a minimum of four transition states. SGLT1; Boltzmann distribution; Xenopus oocyte; sodium/glucose cotransport; two-electrode voltage clamp     

Pre-steady-state currents in neutral amino acid transporters induced by photolysis of a new caged alanine derivative

Na+-Dependent transmembrane transport of small neutral amino acids, such as glutamine and alanine, is mediated, among others, by the neutral amino acid transporters of the solute carrier 1 [SLC1, alanine serine cysteine transporter 1 (ASCT1), and ASCT2] and SLC38 families [sodium-coupled neutral amino acid transporter 1 (SNAT1), SNAT2, and SNAT4]. Many mechanistic aspects of amino acid transport by these systems are not well-understood. Here, we describe a new photolabile alanine derivative based on protection of alanine with the 4-methoxy-7-nitroindolinyl (MNI) caging group, which we use for pre-steady-state kinetic analysis of alanine transport by ASCT2, SNAT1, and SNAT2. MNI-alanine has favorable photochemical properties and is stable in aqueous solution. It is also inert with respect to the transport systems studied. Photolytic release of free alanine results in the generation of significant transient current components in HEK293 cells expressing the ASCT2, SNAT1, and SNAT2 proteins. In ASCT2, these currents show biphasic decay with time constants, tau, in the 1-30 ms time range. They are fully inhibited in the absence of extracellular Na+, demonstrating that Na+ binding to the transporter is necessary for induction of the alanine-mediated current. For SNAT1, these transient currents differ in their time course (tau = 1.6 ms) from previously described pre-steady-state currents generated by applying steps in the membrane potential (tau approximately 4-5 ms), indicating that they are associated with a fast, previously undetected, electrogenic partial reaction in the SNAT1 transport cycle. The implications of these results for the mechanisms of transmembrane transport of alanine are discussed. The new caged alanine derivative will provide a useful tool for future, more detailed studies of neutral amino acid transport.     

How Drugs Interact with Transporters: SGLT1 as a Model

Drugs are transported by cotransporters with widely different turnover rates. We have examined the underlying mechanism using, as a model system, glucose and indican (indoxyl-beta-D: -glucopyranoside) transport by human Na(+)/glucose cotransporter (hSGLT1). Indican is transported by hSGLT1 at 10% of the rate for glucose but with a fivefold higher apparent affinity. We expressed wild-type hSGLT1 and mutant G507C in Xenopus oocytes and used electrical and optical methods to measure the kinetics of glucose (using nonmetabolized glucose analogue alpha-methyl-D: -glucopyranoside, alphaMDG) and indican transport, alone and together. Indican behaved as a competitive inhibitor of alphaMDG transport. To examine protein conformations, we recorded SGLT1 capacitive currents (charge movements) and fluorescence changes in response to step jumps in membrane voltage, in the presence and absence of indican and/or alphaMDG. In the absence of sugar, voltage jumps elicited capacitive SGLT currents that decayed to steady state with time constants (tau) of 3-20 ms. These transient currents were abolished in saturating alphaMDG but only slightly reduced (10%) in saturating indican. SGLT1 G507C rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages. Maximal fluorescence increased approximately 150% in saturating indican but decreased approximately 50% in saturating alphaMDG. Modeling indicated that the rate-limiting step for indican transport is sugar translocation, whereas for alphaMDG it is dissociation of Na(+) from the internal binding sites. The inhibitory effects of indican on alphaMDG transport are due to its higher affinity and a 100-fold lower translocation rate. Our results indicate that competition between substrates and drugs should be taken into consideration when targeting transporters as drug delivery systems.     

Uncoupling of gating charge movement and closure of the ion pore during recovery from inactivation in the Kv1.5 channel

Both wild-type (WT) and nonconducting W472F mutant (NCM) Kv1.5 channels are able to conduct Na(+) in their inactivated states when K(+) is absent. Replacement of K(+) with Na(+) or NMG(+) allows rapid and complete inactivation in both WT and W472F mutant channels upon depolarization, and on return to negative potentials, transition of inactivated channels to closed-inactivated states is the first step in the recovery of the channels from inactivation. The time constant for immobilized gating charge recovery at -100 mV was 11.1 +/- 0.4 ms (n = 10) and increased to 19.0 +/- 1.6 ms (n = 3) when NMG(+)(o) was replaced by Na(+)(o). However, the decay of the Na(+) tail currents through inactivated channels at -100 mV had a time constant of 129 +/- 26 ms (n = 18), much slower than the time required for gating charge recovery. Further experiments revealed that the voltage-dependence of gating charge recovery and of the decay of Na(+) tail currents did not match over a 60 mV range of repolarization potentials. A faster recovery of gating charge than pore closure was also observed in WT Kv1.5 channels. These results provide evidence that the recovery of the gating elements is uncoupled from that of the pore in Na(+)-conducting inactivated channels. The dissociation of the gating charge movements and the pore closure could also be observed in the presence of symmetrical Na(+) but not symmetrical Cs(+). This difference probably stems from the difference in the respective abilities of the two ions to limit inactivation to the P-type state or prevent it altogether.     

Expression of Na+-D-glucose cotransporter SGLT2 in rodents is kidney-specific and exhibits sex and species differences

With a novel antibody against the rat Na(+)-D-glucose cotransporter SGLT2 (rSGLT2-Ab), which does not cross-react with rSGLT1 or rSGLT3, the ～75-kDa rSGLT2 protein was localized to the brush-border membrane (BBM) of the renal proximal tubule S1 and S2 segments (S1 > S2) with female-dominant expression in adult rats, whereas rSglt2 mRNA expression was similar in both sexes. Castration of adult males increased the abundance of rSGLT2 protein; this increase was further enhanced by estradiol and prevented by testosterone treatment. In the renal BBM vesicles, the rSGLT1-independent uptake of [(14)C]-α-methyl-D-glucopyranoside was similar in females and males, suggesting functional contribution of another Na(+)-D-glucose cotransporter to glucose reabsorption. Since immunoreactivity of rSGLT2-Ab could not be detected with certainty in rat extrarenal organs, the SGLT2 protein was immunocharacterized with the same antibody in wild-type (WT) mice, with SGLT2-deficient (Sglt2 knockout) mice as negative control. In WT mice, renal localization of mSGLT2 protein was similar to that in rats, whereas in extrarenal organs neither mSGLT2 protein nor mSglt2 mRNA expression was detected. At variance to the findings in rats, the abundance of mSGLT2 protein in the mouse kidneys was male dominant, whereas the expression of mSglt2 mRNA was female dominant. Our results indicate that in rodents the expression of SGLT2 is kidney-specific and point to distinct sex and species differences in SGLT2 protein expression that cannot be explained by differences in mRNA.     

Voltage clamp study of fast excitatory synaptic currents in bullfrog sympathetic ganglion cells

Excitatory postsynaptic currents (EPSCs) have been studied in voltage- clamped bullfrog sympathetic ganglion B cells. The EPSC was small, rose to a peak within 1-3 ms, and then decayed exponentially over most of its time-course. For 36 cells at --50 mV (21-23 degrees C), peak EPSC size was --6.5 +/- 3.5 nA (mean +/- SD), and the mean decay time constant tau was 5.3 +/- 0.9 ms. tau showed a small negative voltage dependence, which appeared independent of temperature, over the range -- 90 to --30 mV; the coefficient of voltage dependence was --0.0039 +/- 0.0014 mV-1 (n = 29). The peak current-voltage relationship was linear between --120 and --30 mV but often deviated from linearity at more positive potentials. The reversal potential determined by interpolation was approximately --5 mV. EPSC decay tau had a Q10 = 3. The commonly used cholinesterase inhibitors, neostigmine and physostigmine, exhibited complex actions at the ganglia. Neostigmine (1 X 10(-5)M) produced a time-dependent slowing of EPSC decay without consistent change in EPSC size. In addition, the decay phase often deviated from a single exponential function, although it retained its negative voltage dependence. With 1 x 10(-6) M physostigmine, EPSC decay was slowed by the decay phase remained exponential. At higher concentrations of physostigmine, EPSC decay was markedly prolonged and was composed of at least two decay components. High concentrations of atropine (10(-5) to 10(-4) M) produced complex alterations in EPSC decay, creating two or more exponential components; one decay component was faster and the other was slower than that observed in untreated cells. These results suggest that the time-course of ganglionic EPSC decay is primarily determined by the kinetics of the receptor-channel complex rather than hydrolysis or diffusion of transmitter away from the postsynaptic receptors.     

Transport model of the human Na+-coupled L-ascorbic acid (vitamin C) transporter SVCT1

Vitamin C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant and as a cofactor in many enzymatic reactions. Intestinal absorption and renal reabsorption of the vitamin is mediated by the epithelial apical L-ascorbic acid cotransporter SVCT1 (SLC23A1). We explored the molecular mechanisms of SVCT1-mediated L-ascorbic acid transport using radiotracer and voltage-clamp techniques in RNA-injected Xenopus oocytes. L-ascorbic acid transport was saturable (K(0.5) approximately 70 microM), temperature dependent (Q(10) approximately 5), and energized by the Na(+) electrochemical potential gradient. We obtained a Na(+)-L-ascorbic acid coupling ratio of 2:1 from simultaneous measurement of currents and fluxes. L-ascorbic acid and Na(+) saturation kinetics as a function of cosubstrate concentrations revealed a simultaneous transport mechanism in which binding is ordered Na(+), L-ascorbic acid, Na(+). In the absence of L-ascorbic acid, SVCT1 mediated pre-steady-state currents that decayed with time constants 3-15 ms. Transients were described by single Boltzmann distributions. At 100 mM Na(+), maximal charge translocation (Q(max)) was approximately 25 nC, around a midpoint (V(0.5)) at -9 mV, and with apparent valence approximately -1. Q(max) was conserved upon progressive removal of Na(+), whereas V(0.5) shifted to more hyperpolarized potentials. Model simulation predicted that the pre-steady-state current predominantly results from an ion-well effect on binding of the first Na(+) partway within the membrane electric field. We present a transport model for SVCT1 that will provide a framework for investigating the impact of specific mutations and polymorphisms in SLC23A1 and help us better understand the contribution of SVCT1 to vitamin C metabolism in health and disease.     

The glutamate transporter subtypes EAAT4 and EAATs 1-3 transport glutamate with dramatically different kinetics and voltage dependence but share a common uptake mechanism

Here, we report the application of glutamate concentration jumps and voltage jumps to determine the kinetics of rapid reaction steps of excitatory amino acid transporter subtype 4 (EAAT4) with a 100-micros time resolution. EAAT4 was expressed in HEK293 cells, and the electrogenic transport and anion currents were measured using the patch-clamp method. At steady state, EAAT4 was activated by glutamate and Na+ with high affinities of 0.6 microM and 8.4 mM, respectively, and showed kinetics consistent with sequential binding of Na(+)-glutamate-Na+. The steady-state cycle time of EAAT4 was estimated to be >300 ms (at -90 mV). Applying step changes to the transmembrane potential, V(m), of EAAT4-expressing cells resulted in the generation of transient anion currents (decaying with a tau of approximately 15 ms), indicating inhibition of steady-state EAAT4 activity at negative voltages (<-40 mV) and activation at positive V(m) (>0 mV). A similar inhibitory effect at V(m) < 0 mV was seen when the electrogenic glutamate transport current was monitored, resulting in a bell-shaped I-V(m) curve. Jumping the glutamate concentration to 100 muM generated biphasic, saturable transient transport and anion currents (K(m) approximately 5 microM) that decayed within 100 ms, indicating the existence of two separate electrogenic reaction steps. The fast electrogenic reaction was assigned to Na+ binding to EAAT4, whereas the second reaction is most likely associated with glutamate translocation. Together, these results suggest that glutamate uptake of EAAT4 is based on the same molecular mechanism as transport by the subtypes EAATs 1-3, but that its kinetics and voltage dependence are dramatically different from the other subtypes. EAAT4 kinetics appear to be optimized for high affinity binding of glutamate, but not rapid turnover. Therefore, we propose that EAAT4 is a high-affinity/low-capacity transport system, supplementing low-affinity/high-capacity synaptic glutamate uptake by the other subtypes.     

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.     

Dynamic changes of cardiac conduction during rapid pacing

Slow conduction and unidirectional conduction block (UCB) are key mechanisms of reentry. Following abrupt changes in heart rate, dynamic changes of conduction velocity (CV) and structurally determined UCB may critically influence arrhythmogenesis. Using patterned cultures of neonatal rat ventricular myocytes grown on microelectrode arrays, we investigated the dynamics of CV in linear strands and the behavior of UCB in tissue expansions following an abrupt decrease in pacing cycle length (CL). Ionic mechanisms underlying rate-dependent conduction changes were investigated using the Pandit-Clark-Giles-Demir model. In linear strands, CV gradually decreased upon a reduction of CL from 500 ms to 230-300 ms. In contrast, at very short CLs (110-220 ms), CV first decreased before increasing again. The simulations suggested that the initial conduction slowing resulted from gradually increasing action potential duration (APD), decreasing diastolic intervals, and increasing postrepolarization refractoriness, which impaired Na(+) current (I(Na)) recovery. Only at very short CLs did APD subsequently shorten again due to increasing Na(+)/K(+) pump current secondary to intracellular Na(+) accumulation, which caused recovery of CV. Across tissue expansions, the degree of UCB gradually increased at CLs of 250-390 ms, whereas at CLs of 180-240 ms, it first increased and subsequently decreased. In the simulations, reduction of inward currents caused by increasing intracellular Na(+) and Ca(2+) concentrations contributed to UCB progression, which was reversed by increasing Na(+)/K(+) pump activity. In conclusion, CV and UCB follow intricate dynamics upon an abrupt decrease in CL that are determined by the interplay among I(Na) recovery, postrepolarization refractoriness, APD changes, ion accumulation, and Na(+)/K(+) pump function.     

Time-dependent outward current in guinea pig ventricular myocytes. Gating kinetics of the delayed rectifier

Several conflicting models have been used to characterize the gating behavior of the cardiac delayed rectifier. In this study, whole-cell delayed rectifier currents were measured in voltage-clamped guinea pig ventricular myocytes, and a minimal model which reproduced the observed kinetic behavior was identified. First, whole-cell potassium currents between -10 and +70 mV were recorded using external solutions designed to eliminate Na and Ca currents and two components of time-dependent outward current were found. One component was a La3(+)-sensitive current which inactivated and resembled the transient outward current described in other cell types; single-channel observations confirmed the presence of a transient outward current in these guinea pig ventricular cells (gamma = 9.9 pS, [K]o = 4.5 mM). Analysis of envelopes of tail amplitudes demonstrated that this component was absent in solutions containing 30-100 microM La3+. The remaining time-dependent current, IK, activated with a sigmoidal time course that was well-characterized by three time constants. Nonlinear least-squares fits of a four-state Markovian chain model (closed - closed - closed - open) to IK activation were therefore compared to other models previously used to characterize IK gating: n2 and n4 Hodgkin-Huxley models and a Markovian chain model with only two closed states. In each case the four-state model was significantly better (P less than 0.05). The failure of the Hodgkin-Huxley models to adequately describe the macroscopic current indicates that identical and independent gating particles should not be assumed for this K channel. The voltage-dependent terms describing the rate constants for the four-state model were then derived using a global fitting approach for IK data obtained over a wide range of potentials (-80 to +70 mV). The fit was significantly improved by including a term representing the membrane dipole forces (P less than 0.01). The resulting rate constants predicted long single-channel openings (greater than 1 s) at voltages greater than 0 mV. In cell-attached patches, single delayed rectifier channels which had a mean chord conductance of 5.4 pS at +60 mV ([K]o = 4.5 mM) were recorded for brief periods. These channels exhibited behavior predicted by the four-state model: long openings and latency distributions with delayed peaks. These results suggest that the cardiac delayed rectifier undergoes at least two major transitions between closed states before opening upon depolarization.     

Perturbation analysis of the voltage-sensitive conformational changes of the Na+/glucose cotransporter

Conformational changes of the human Na(+)/glucose cotransporter (hSGLT1) were studied using voltage-jump methods. The cotransporter was expressed in Xenopus laevis oocytes, and SGLT1 charge movements were measured in the micro- to millisecond time scale using the cut-open oocyte preparation and in the millisecond to second time scale using the two-electrode voltage clamp method. Simultaneous charge and fluorescence changes were studied using tetramethylrhodamine-6-maleimide-labeled hSGLT1 Q457C. In 100 mM external [Na(+)], depolarizing voltage steps evoked a charge movement that rose initially to a peak (with time constant tau = 0.17 ms) before decaying to steady state with two time constants (tau = 2-30 and 25-150 ms). The time to peak (0.9 ms) decreased with [Na(+)], and was not observed in 0 mM [Na(+)]. In absence of Na(+), charge movement decayed monotonically to steady state with three time constants (0.2, 2, and 150 ms). Charge movement was accompanied by fluorescence changes with similar time courses, indicating that global conformational changes monitored by charge movement are reflected by local environmental changes at or near Q457C. Our results indicate that the major voltage-dependent step of the Na(+)/glucose transport cycle is the return of the empty carrier from inward to outward facing conformations. Finally, we observed subtle differences between time constants for charge movement and for optical changes, suggesting that optical recordings can be used to monitor local conformational changes that underlie the global conformational changes of cotransporters.     

Simulation of the voltage dependence of the Na, K pump applied to cardiac cells

We use simulation to study the dependence of the Na, K pump on membrane potential. Two consecutive mechanisms for the Na, K pump, based on a reduced Post-Albers scheme, are examined: one with six steps called GV3 and one with seven steps called MGV3. In GV3, a single voltage-dependent step combines both Na+ translocation and Na+ release into the extracellular medium. In MGV3, these two processes are allocated to two separate consecutive steps, but only the Na+ translocation step is voltage-dependent. Using the optimization software SCoPfit, numerical values of rate coefficients, symmetry factor (beta), and pump site density were found by fitting the models to published experimental data so that both GV3 and MGV3 could quantitatively reproduce steady-state current-voltage relationships for both forward and backward running of the pump, as well as [Na+]in and [K+]out activation curves. Using the rate coefficient values found by SCoPfit, we simulated a voltage-clamp experiment with both models running under their Na(+)-Na+ exchange mode, and we computed the transient currents generated following voltage steps in both depolarizing and hyperpolarizing directions from a basic potential of -40 mV. The voltage dependence of the rate constant (1/tau) of decay of the transient currents could qualitatively be reproduced when beta = 0.884 for GV3, and 0.932 for MGV3. The quantitative discrepancy between published experimental data and the theoretical curve generated by GV3 at potentials more negative than -20 mV was considerably reduced by using model MGV3. This finding alone suggests that a more detailed mechanism containing a single voltage-dependent step may reproduce all major steady-state and transient characteristics of the Na, K pump without the need of a second voltage sensitive step. However, the quantitative discrepancy between published experimental data and the theoretical curve generated by MGV3 at potentials more negative than -60 mV may be fully removed if either beta itself is voltage-dependent, or if a second voltage-dependent step is included in the model.     

Properties of an early outward current in single cells of the mouse ventricle

Single ventricular myocytes of adult mice were prepared by enzymatic dissociation for voltage clamp experiments with the one suction pipette dialysis method. After blocking the Na current by 10(-4) mol/l TTX early outward currents (IEO) with incomplete inactivation could be elicited by clamping from -50 mV to test potentials (VT) positive to -30 mV. Interfering Ca currents were very small (less than 0.6 nA at VT = 0 mV). The approximation of IEO by the q4r-model showed a pronounced decrease in the time constant of activation (tau q) to more positive potentials. At 50 ms test pulses the time course of the incomplete inactivation could be described by two exponentials and a constant. The time constant of the fast exponential (tau r1) showed a slight decline towards more positive test potentials (8.1 +/- 1.0 ms at -10 mV; 5.8 +/- 1.2 ms at +50 mV, mean +/- SD, n = 5) whereas the time constant of the slow exponential (tau r2) was voltage independent (41.1 +/- 7.9 ms, mean +/- SD, n = 5). The contributions of the fast exponential and the pedestal increased towards positive test potentials. The Q10 value for the time constants of activation and fast inactivation was 2.36 +/- 0.19 and 2.51 +/- 0.09 (mean +/- SD, n = 3), respectively. After an initial delay the recovery of IEO at a recovery potential of -50 mV could be fitted monoexponentially with a time constant of 16.3 +/- 2.9 ms (mean +/- SD, n = 3). The time course of the onset of inactivation determined with the double pulse protocol was slower than the decay at the same potential, and could be described as sum of a fast (tau = 18.4 +/- 6.0 ms) and a slow (tau = 62.1 +/- 19.9ms, mean +/- SD, n = 3) exponential. IEO could be blocked completely by 1 mmol/l 4-aminopyridine at potentials up to +20 mV. Stronger depolarizations had an unblocking effect.