scholarly journals Contribution of Sialic Acid to the Voltage Dependence of Sodium Channel Gating

1997 ◽  
Vol 109 (3) ◽  
pp. 327-343 ◽  
Author(s):  
Eric Bennett ◽  
Mary S. Urcan ◽  
Sally S. Tinkle ◽  
Adam G. Koszowski ◽  
Simon R. Levinson
Keyword(s):  
Steady State ◽  
Sialic Acid ◽  
Voltage Dependence ◽  
Channel Gating ◽  
Chinese Hamster ◽  
Glycosylation Sites ◽  
Voltage Dependent ◽  
Negative Surface ◽  
Electrostatic Mechanism ◽  
External Calcium

A potential role for sialic acid in the voltage-dependent gating of rat skeletal muscle sodium channels (rSkM1) was investigated using Chinese hamster ovary (CHO) cells stably transfected with rSkM1. Changes in the voltage dependence of channel gating were observed after enzymatic (neuraminidase) removal of sialic acid from cells expressing rSkM1 and through the expression of rSkM1 in a sialylation-deficient cell line (lec2). The steady-state half-activation voltages (Va) of channels under each condition of reduced sialylation were ∼10 mV more depolarized than control channels. The voltage dependence of the time constants of channel activation and inactivation were also shifted in the same direction and by a similar magnitude. In addition, recombinant deletion of likely glycosylation sites from the rSkM1 sequence resulted in mutant channels that gated at voltages up to 10 mV more positive than wild-type channels. Thus three independent means of reducing channel sialylation show very similar effects on the voltage dependence of channel gating. Finally, steady-state activation voltages for channels subjected to reduced sialylation conditions were much less sensitive to the effects of external calcium than those measured under control conditions, indicating that sialic acid directly contributes to the negative surface potential. These results are consistent with an electrostatic mechanism by which external, negatively charged sialic acid residues on rSkM1 alter the electric field sensed by channel gating elements.

Differential contribution of sialic acid to the function of repolarizing K+ currents in ventricular myocytes

2001 ◽  
Vol 281 (2) ◽  
pp. C464-C474 ◽  
Author(s):  
Carmen A. Ufret-Vincenty ◽  
Deborah J. Baro ◽  
L. F. Santana
Keyword(s):  
Sialic Acid ◽  
Voltage Dependence ◽  
Ventricular Myocytes ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Early Afterdepolarizations ◽  
Kv4 Channels ◽  
Ovary Cell Line ◽  
Differential Contribution ◽  
K Currents

We investigated the contribution of sialic acid residues to the K+ currents involved in the repolarization of mouse ventricular myocytes. Ventricular K+ currents had a rapidly inactivating component followed by slowly decaying and sustained components. This current was produced by the summation of three distinct currents: I to, which contributed to the transient component; I ss, which contributed to the sustained component; and I K,slow, which contributed to both components. Incubation of ventricular myocytes with the sialidase neuraminidase reduced the amplitude of I to without altering I K,slow and I ss. We found that the reduction in I to amplitude resulted from a depolarizing shift in the voltage of activation and a reduction in the conductance of I to. Expression of Kv4.3 channels, a major contributor to I to in the ventricle, in a sialylation-deficient Chinese hamster ovary cell line (lec2) mimicked the effects of neuraminidase on the ventricular I to. Furthermore, we showed that sialylated glycolipids have little effect on the voltage dependence of I to. Finally, consistent with its actions on I to, neuraminidase produced an increase in the duration of the action potential of ventricular myocytes and the frequency of early afterdepolarizations. We conclude that sialylation of the proteins forming Kv4 channels is important in determining the voltage dependence and conductance of I to and that incomplete glycosylation of these channels could lead to arrhythmias.

scholarly journals Uncharged S4 Residues and Cooperativity in Voltage-dependent Potassium Channel Activation

1998 ◽  
Vol 111 (3) ◽  
pp. 421-439 ◽  
Author(s):  
Catherine J. Smith-Maxwell ◽  
Jennifer L. Ledwell ◽  
Richard W. Aldrich
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Potassium Channel ◽  
Voltage Dependence ◽  
Channel Gating ◽  
Ionic Currents ◽  
Channel Activation ◽  
Functional Changes ◽  
Activation Pathway ◽  
Voltage Dependent

Substitution of the S4 of Shaw into Shaker alters cooperativity in channel activation by slowing a cooperative transition late in the activation pathway. To determine the amino acids responsible for the functional changes in Shaw S4, we created several mutants by substituting amino acids from Shaw S4 into Shaker. The S4 amino acid sequences of Shaker and Shaw S4 differ at 11 positions. Simultaneous substitution of just three noncharged residues from Shaw S4 into Shaker (V369I, I372L, S376T; ILT) reproduces the kinetic and voltage-dependent properties of Shaw S4 channel activation. These substitutions cause very small changes in the structural and chemical properties of the amino acid side chains. In contrast, substituting the positively charged basic residues in the S4 of Shaker with neutral or negative residues from the S4 of Shaw S4 does not reproduce the shallow voltage dependence or other properties of Shaw S4 opening. Macroscopic ionic currents for ILT could be fit by modifying a single set of transitions in a model for Shaker channel gating (Zagotta, W.N., T. Hoshi, and R.W. Aldrich. 1994. J. Gen. Physiol. 103:321–362). Changing the rate and voltage dependence of a final cooperative step in activation successfully reproduces the kinetic, steady state, and voltage-dependent properties of ILT ionic currents. Consistent with the model, ILT gating currents activate at negative voltages where the channel does not open and, at more positive voltages, they precede the ionic currents, confirming the existence of voltage-dependent transitions between closed states in the activation pathway. Of the three substitutions in ILT, the I372L substitution is primarily responsible for the changes in cooperativity and voltage dependence. These results suggest that noncharged residues in the S4 play a crucial role in Shaker potassium channel gating and that small steric changes in these residues can lead to large changes in cooperativity within the channel protein.

scholarly journals Voltage-dependent inactivation of slow calcium channels in intact twitch muscle fibers of the frog.

1989 ◽  
Vol 94 (5) ◽  
pp. 937-951 ◽  
Author(s):  
G Cota ◽  
E Stefani
Keyword(s):  
Steady State ◽  
Rate Constant ◽  
Voltage Dependence ◽  
Muscle Fibers ◽  
Dependent Manner ◽  
Inactivation Curve ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Intact Muscle ◽  
Ca2 Channels

Inactivation of slow Ca2+ channels was studied in intact twitch skeletal muscle fibers of the frog by using the three-microelectrode voltage-clamp technique. Hypertonic sucrose solutions were used to abolish contraction. The rate constant of decay of the slow Ca2+ current (ICa) remained practically unchanged when the recording solution containing 10 mM Ca2+ was replaced by a Ca2+-buffered solution (126 mM Ca-maleate). The rate constant of decay of ICa monotonically increased with depolarization although the corresponding time integral of ICa followed a bell-shaped function. The replacement of Ca2+ by Ba2+ did not result in a slowing of the rate of decay of the inward current nor did it reduce the degree of steady-state inactivation. The voltage dependence of the steady-state inactivation curve was steeper in the presence of Ba2+. In two-pulse experiments with large conditioning depolarizations ICa inactivation remained unchanged although Ca2+ influx during the prepulse greatly decreased. Dantrolene (12 microM) increased mechanical threshold at all pulse durations tested, the effect being more prominent for short pulses. Dantrolene did not significantly modify ICa decay and the voltage dependence of inactivation. These results indicate that in intact muscle fibers Ca2+ channels inactivate in a voltage-dependent manner through a mechanism that does not require Ca2+ entry into the cell.

scholarly journals Mechanism of the Voltage Sensitivity of IRK1 Inward-rectifier K+ Channel Block by the Polyamine Spermine

2005 ◽  
Vol 125 (4) ◽  
pp. 413-426 ◽  
Author(s):  
Hyeon-Gyu Shin ◽  
Zhe Lu
Keyword(s):  
Steady State ◽  
Voltage Dependence ◽  
Ion Selectivity ◽  
Inward Rectifier ◽  
Selectivity Filter ◽  
K Channel ◽  
Voltage Sensitivity ◽  
Channel Block ◽  
Head Groups ◽  
Voltage Dependent

IRK1 (Kir2.1) inward-rectifier K+ channels exhibit exceedingly steep rectification, which reflects strong voltage dependence of channel block by intracellular cations such as the polyamine spermine. On the basis of studies of IRK1 block by various amine blockers, it was proposed that the observed voltage dependence (valence ∼5) of IRK1 block by spermine results primarily from K+ ions, not spermine itself, traversing the transmembrane electrical field that drops mostly across the narrow ion selectivity filter, as spermine and K+ ions displace one another during channel block and unblock. If indeed spermine itself only rarely penetrates deep into the ion selectivity filter, then a long blocker with head groups much wider than the selectivity filter should exhibit comparably strong voltage dependence. We confirm here that channel block by two molecules of comparable length, decane-bis-trimethylammonium (bis-QAC10) and spermine, exhibit practically identical overall voltage dependence even though the head groups of the former are much wider (∼6 Å) than the ion selectivity filter (∼3 Å). For both blockers, the overall equilibrium dissociation constant differs from the ratio of apparent rate constants of channel unblock and block. Also, although steady-state IRK1 block by both cations is strongly voltage dependent, their apparent channel-blocking rate constant exhibits minimal voltage dependence, which suggests that the pore becomes blocked as soon as the blocker encounters the innermost K+ ion. These findings strongly suggest the existence of at least two (potentially identifiable) sequentially related blocked states with increasing numbers of K+ ions displaced. Consequently, the steady-state voltage dependence of IRK1 block by spermine or bis-QAC10 should increase with membrane depolarization, a prediction indeed observed. Further kinetic analysis identifies two blocked states, and shows that most of the observed steady-state voltage dependence is associated with the transition between blocked states, consistent with the view that the mutual displacement of blocker and K+ ions must occur mainly as the blocker travels along the long inner pore.

Using lithium to probe sequential cation interactions with GAT1

2012 ◽  
Vol 302 (11) ◽  
pp. C1661-C1675 ◽  
Author(s):  
Anne-Kristine Meinild ◽  
Ian C. Forster
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Voltage Dependence ◽  
Cation Binding ◽  
Experimental Conditions ◽  
Net Charge ◽  
Cation Binding Site ◽  
Voltage Dependent ◽  
Experimental Findings ◽  
Kinetics Of

Li+ interacts with the Na+/Cl−-dependent GABA transporter, GAT1, under two conditions: in the absence of Na+ it induces a voltage-dependent leak current; in the presence of Na+ and GABA, Li+ stimulates GABA-induced steady-state currents. The amino acids directly involved in the interaction with the Na+ and Li+ ions at the so-called “ Na2” binding site have been identified, but how Li+ affects the kinetics of GABA cotransport has not been fully explored. We expressed GAT1 in Xenopus oocytes and applied the two-electrode voltage clamp and 22Na uptake assays to determine coupling ratios and steady-state and presteady-state kinetics under experimental conditions in which extracellular Na+ was partially substituted by Li+. Three novel findings are: 1) Li+ reduced the coupling ratio between Na+ and net charge translocated during GABA cotransport; 2) Li+ increased the apparent Na+ affinity without changing its voltage dependence; 3) Li+ altered the voltage dependence of presteady-state relaxations in the absence of GABA. We propose an ordered binding scheme for cotransport in which either a Na+ or Li+ ion can bind at the putative first cation binding site ( Na2). This is followed by the cooperative binding of the second Na+ ion at the second cation binding site ( Na1) and then binding of GABA. With Li+ bound to Na2, the second Na+ ion binds more readily GAT1, and despite a lower apparent GABA affinity, the translocation rate of the fully loaded carrier is not reduced. Numerical simulations using a nonrapid equilibrium model fully recapitulated our experimental findings.

Aminopyridine inhibition and voltage dependence of K+ currents in smooth muscle cells from cerebral arteries

1994 ◽  
Vol 267 (6) ◽  
pp. C1589-C1597 ◽  
Author(s):  
B. E. Robertson ◽  
M. T. Nelson
Keyword(s):  
Smooth Muscle ◽  
Steady State ◽  
Smooth Muscle Cells ◽  
Voltage Dependence ◽  
Muscle Cells ◽  
Membrane Depolarization ◽  
K Channels ◽  
Voltage Dependent ◽  
Basilar Arteries ◽  
K Currents

Voltage-dependent K+ currents were characterized using the patch-clamp technique in smooth muscle cells isolated from rabbit cerebral (basilar) arteries. This study focused on the voltage dependence and the pharmacology of these K+ currents, since this information will be useful for the investigation of the role of the voltage-dependent K+ channels in arterial function. Currents through Ca(2+)-activated K+ (KCa) channels were minimized by buffering intracellular Ca2+ to low levels and by blockers (tetraethylammonium and iberiotoxin) of these channels. Membrane depolarization increased K+ currents, independent of changes in the driving force for K+ movement. With 140 mM internal and external K+, activation of K+ currents by membrane depolarization was half maximal at about -10 mV and increased as much as e-fold per 11 mV. Inactivation also depended on voltage, with a midpoint at -44 mV. 3,4-Diaminopyridine (3,4-DAP),4-aminopyridine(4-AP),3-amino-pyridine(3-AP), and 2-aminopyridine (2-AP) inhibited voltage-dependent K+ currents. At 0 mV, 3,4-DAP, 4-AP, 3-AP, and 2-AP (5 mM) inhibited the K+ currents by 84, 66, 36, and 8%, respectively. Phencyclidine (100 microM) inhibited the current by 53% at 0 mV. Steady-state whole cell currents through these channels were measured at physiological membrane potentials. At -40 mV, 4-AP (5 mM) reduced the steady-state outward current by 2.5 pA. These results are consistent with the idea that voltage-dependent K+ channels are involved in the regulation of the membrane potential of arterial smooth muscle.

scholarly journals Effects of external Rb+ on inward rectifier K+ channels of bovine pulmonary artery endothelial cells.

1994 ◽  
Vol 103 (4) ◽  
pp. 519-548 ◽  
Author(s):  
M R Silver ◽  
M S Shapiro ◽  
T E DeCoursey
Keyword(s):  
Endothelial Cells ◽  
Steady State ◽  
Pulmonary Artery ◽  
Voltage Dependence ◽  
Reversal Potential ◽  
Inward Rectifier ◽  
Partial Substitution ◽  
Pipette Solution ◽  
K Channels ◽  
Voltage Dependent

Inward rectifier (IR) K+ channels of bovine pulmonary artery endothelial cells were studied using the whole-cell, cell-attached, and outside-out patch-clamp configurations. The effects of Rb+ on the voltage dependence and kinetics of IR gating were explored, with [Rb+]o + [K+]o = 160 mM. Partial substitution of Rb+ for K+ resulted in voltage-dependent reduction of inward currents, consistent with Rb+ being a weakly permeant blocker of the IR. In cells studied with a K(+)-free pipette solution, external Rb+ reduced inward IR currents to a similar extent at large negative potentials but block at more positive potentials was enhanced. In outside-out patches, the single-channel i-V relationship was approximately linear in symmetrical K+, but rectified strongly outwardly in high [Rb+]o due to a reduced conductance for inward current. The permeability of Rb+ based on reversal potential, Vrev, was 0.45 that of K+, whereas the Rb+ conductance was much lower, 0.034 that of K+, measured at Vrev-80 mV. The steady state voltage-dependence of IR gating was determined in Rb(+)-containing solutions by applying variable prepulses, followed by a test pulse to a potential at which outward current deactivation was observed. As [Rb+]o was increased, the half-activation potential, V1/2, changed less than Vrev. In high [K+]o solutions V1/2 was Vrev-6 mV, while in high [Rb+]o V1/2 was Vrev + 7 mV. This behavior contrasts with the classical parallel shift of V1/2 with Vrev in K+ solutions. Steady state IR gating was less steeply voltage-dependent in high [Rb+]o than in K+ solutions, with Boltzmann slope factors of 6.4 and 4.4 mV, respectively. Rb+ decreased (slowed) both activation and deactivation rate constants defined at V1/2, and decreased the steepness of the voltage dependence of the activation rate constant by 42%. Deactivation of IR channels in outside-out patches was also slowed by Rb+. In summary, Rb+ can replace K+ in setting the voltage-dependence of IR gating, but in doing so alters the kinetics.

scholarly journals Characterization of the inward-rectifying potassium current in cat ventricular myocytes.

1988 ◽  
Vol 91 (4) ◽  
pp. 593-615 ◽  
Author(s):  
R D Harvey ◽  
R E Ten Eick
Keyword(s):  
Steady State ◽  
Time Course ◽  
Voltage Dependence ◽  
Ventricular Myocytes ◽  
Reversal Potential ◽  
Inward Current ◽  
Steady State Current ◽  
Voltage Dependent ◽  
K Current ◽  
K Concentration

Whole-cell membrane currents were measured in isolated cat ventricular myocytes using a suction-electrode voltage-clamp technique. An inward-rectifying current was identified that exhibited a time-dependent activation. The peak current appeared to have a linear voltage dependence at membrane potentials negative to the reversal potential. Inward current was sensitive to K channel blockers. In addition, varying the extracellular K+ concentration caused changes in the reversal potential and slope conductance expected for a K+ current. The voltage dependence of the chord conductance exhibited a sigmoidal relationship, increasing at more negative membrane potentials. Increasing the extracellular K+ concentration increased the maximal level of conductance and caused a shift in the relationship that was directly proportional to the change in reversal potential. Activation of the current followed a monoexponential time course, and the time constant of activation exhibited a monoexponential dependence on membrane potential. Increasing the extracellular K+ concentration caused a shift of this relationship that was directly proportional to the change in reversal potential. Inactivation of inward current became evident at more negative potentials, resulting in a negative slope region of the steady state current-voltage relationship between -140 and -180 mV. Steady state inactivation exhibited a sigmoidal voltage dependence, and recovery from inactivation followed a monoexponential time course. Removing extracellular Na+ caused a decrease in the slope of the steady state current-voltage relationship at potentials negative to -140 mV, as well as a decrease of the conductance of inward current. It was concluded that this current was IK1, the inward-rectifying K+ current found in multicellular cardiac preparations. The K+ and voltage sensitivity of IK1 activation resembled that found for the inward-rectifying K+ currents in frog skeletal muscle and various egg cell preparations. Inactivation of IK1 in isolated ventricular myocytes was viewed as being the result of two processes: the first involves a voltage-dependent change in conductance; the second involves depletion of K+ from extracellular spaces. The voltage-dependent component of inactivation was associated with the presence of extracellular Na+.

Transient Voltage-Dependent Potassium Currents Are Reduced in NTS Neurons Isolated From Renal Wrap Hypertensive Rats

2005 ◽  
Vol 94 (6) ◽  
pp. 3849-3859 ◽  
Author(s):  
Sergei Belugin ◽  
Steve Mifflin
Keyword(s):  
Steady State ◽  
Voltage Dependence ◽  
Fluorescent Tracer ◽  
Activation Kinetics ◽  
Whole Cell Patch Clamp ◽  
Outward Currents ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Afferent Inputs ◽  
Made In

Whole cell patch-clamp measurements were made in neurons enzymatically dispersed from the nucleus of the solitary tract (NTS) to determine if alterations occur in voltage-dependent potassium channels from rats made hypertensive (HT) by unilateral nephrectomy/renal wrap for 4 wk. Some rats had the fluorescent tracer DiA applied to the aortic nerve before the experiment to identify NTS neurons receiving monosynaptic baroreceptor afferent inputs. Mean arterial pressure (MAP) was greater in 4-wk HT (165 ± 5 mmHg, n = 26, P < 0.001) rats compared with normotensive (NT) rats (109 ± 3 mmHg measured in 10 of 69 rats). Transient outward currents (TOCs) were observed in 67–82% of NTS neurons from NT and HT rats. At activation voltages from −10 to +10 mV, TOCs were significantly less in HT neurons compared with those observed in NT neurons ( P < 0.001). There were no differences in the voltage-dependent activation kinetics, the voltage dependence of steady-state inactivation, and the rise and decay time constants of the TOCs comparing neurons isolated from NT and HT rats. The 4-aminopyridine–sensitive component of the TOC was significantly less in neurons from HT compared with NT rats ( P < 0.001), whereas steady-state outward currents, whether or not sensitive to 4-aminopyridine or tetraethylammonium, were not different. Delayed excitation, studied under current clamp, was observed in 60–80% of NTS neurons from NT and HT rats and was not different comparing neurons from NT and HT rats. However, examination of the subset of NTS neurons exhibiting somatic DiA fluorescence revealed that DiA-labeled neurons from HT rats had a significantly shorter duration delayed excitation ( n = 8 cells, P = 0.022) than DiA-labeled neurons from NT rats ( n = 7 cells). Neurons with delayed excitation from HT rats had a significantly broader first action potential (AP) and a slower maximal downstroke velocity of repolarization compared with NT neurons with delayed excitation ( P = 0.016 and P = 0.014, respectively). The number of APs in the first 200 ms of a sustained depolarization was greater in HT than NT neurons ( P = 0.012). These results suggest that HT of 4-wk duration reduces TOCs in NTS neurons, and this contributes to reduced delayed excitation and increased AP responses to depolarizing inputs. Such changes could alter baroreflex function in hypertension.

Sign in / Sign up

Export Citation Format

Share Document