scholarly journals Functional interactions of voltage sensor charges with an S2 hydrophobic plug in hERG channels

2013 ◽  
Vol 142 (3) ◽  
pp. 289-303 ◽  
Author(s):  
Yen May Cheng ◽  
Christina M. Hull ◽  
Christine M. Niven ◽  
Ji Qi ◽  
Charlene R. Allard ◽  
...  
Keyword(s):  
Positive Charge ◽  
Functional Interactions ◽  
Closed State ◽  
Shaker Channels ◽  
Deactivation Kinetics ◽  
Gating Charge ◽  
Herg Channels ◽  
Double Mutant Cycle ◽  
S4 Segment ◽  
Gating Charges

Human ether-à-go-go–related gene (hERG, Kv11.1) potassium channels have unusually slow activation and deactivation kinetics. It has been suggested that, in fast-activating Shaker channels, a highly conserved Phe residue (F290) in the S2 segment forms a putative gating charge transfer center that interacts with S4 gating charges, i.e., R362 (R1) and K374 (K5), and catalyzes their movement across the focused electric field. F290 is conserved in hERG (F463), but the relevant residues in the hERG S4 are reversed, i.e., K525 (K1) and R537 (R5), and there is an extra positive charge adjacent to R537 (i.e., K538). We have examined whether hERG channels possess a transfer center similar to that described in Shaker and if these S4 charge differences contribute to slow gating in hERG channels. Of five hERG F463 hydrophobic substitutions tested, F463W and F463Y shifted the conductance–voltage (G-V) relationship to more depolarized potentials and dramatically slowed channel activation. With the S4 residue reversals (i.e., K525, R537) taken into account, the closed state stabilization by F463W is consistent with a role for F463 that is similar to that described for F290 in Shaker. As predicted from results with Shaker, the hERG K525R mutation destabilized the closed state. However, hERG R537K did not stabilize the open state as predicted. Instead, we found the neighboring K538 residue to be critical for open state stabilization, as K538R dramatically slowed and right-shifted the voltage dependence of activation. Finally, double mutant cycle analysis on the G-V curves of F463W/K525R and F463W/K538R double mutations suggests that F463 forms functional interactions with K525 and K538 in the S4 segment. Collectively, these data suggest a role for F463 in mediating closed–open equilibria, similar to that proposed for F290 in Shaker channels.

scholarly journals Insights on the mechanisms of Ca2+ regulation of connexin26 hemichannels revealed by human pathogenic mutations (D50N/Y)

2013 ◽  
Vol 142 (1) ◽  
pp. 23-35 ◽  
Author(s):  
William Lopez ◽  
Jorge Gonzalez ◽  
Yu Liu ◽  
Andrew L. Harris ◽  
Jorge E. Contreras
Keyword(s):  
Essential Role ◽  
Negative Charge ◽  
Salt Bridge ◽  
Cysteine Residue ◽  
Wild Type ◽  
Closed State ◽  
Large Size ◽  
Deactivation Kinetics ◽  
Connexin Hemichannel

Because of the large size and modest selectivity of the connexin hemichannel aqueous pore, hemichannel opening must be highly regulated to maintain cell viability. At normal resting potentials, this regulation is achieved predominantly by the physiological extracellular Ca2+ concentration, which drastically reduces hemichannel activity. Here, we characterize the Ca2+ regulation of channels formed by wild-type human connexin26 (hCx26) and its human mutations, D50N/Y, that cause aberrant hemichannel opening and result in deafness and skin disorders. We found that in hCx26 wild-type channels, deactivation kinetics are accelerated as a function of Ca2+ concentration, indicating that Ca2+ facilitates transition to, and stabilizes, the closed state of the hemichannels. The D50N/Y mutant hemichannels show lower apparent affinities for Ca2+-induced closing than wild-type channels and have more rapid deactivation kinetics, which are Ca2+ insensitive. These results suggest that D50 plays a role in (a) stabilizing the open state in the absence of Ca2+, and (b) facilitating closing and stabilization of the closed state in the presence of Ca2+. To explore the role of a negatively charged residue at position 50 in regulation by Ca2+, this position was substituted with a cysteine residue, which was then modified with a negatively charged methanethiosulfonate reagent, sodium (2-sulfanoethyl) methanethiosulfonate (MTSES)−. D50C mutant hemichannels display properties similar to those of D50N/Y mutants. Recovery of the negative charge with chemical modification by MTSES− restores the wild-type Ca2+ regulation of the channels. These results confirm the essential role of a negative charge at position 50 for Ca2+ regulation. Additionally, charge-swapping mutagenesis studies suggest involvement of a salt bridge interaction between D50 and K61 in the adjacent connexin subunit in stabilizing the open state in low extracellular Ca2+. Mutant cycle analysis supports a Ca2+-sensitive interaction between these two residues in the open state of the channel. We propose that disruption of this interaction by extracellular Ca2+ destabilizes the open state and facilitates hemichannel closing. Our data provide a mechanistic understanding of how mutations at position 50 that cause human diseases are linked to dysfunction of hemichannel gating by external Ca2+.

scholarly journals Components of gating charge movement and S4 voltage-sensor exposure during activation of hERG channels

2013 ◽  
Vol 141 (4) ◽  
pp. 431-443 ◽  
Author(s):  
Zhuren Wang ◽  
Ying Dou ◽  
Samuel J. Goodchild ◽  
Zeineb Es-Salah-Lamoureux ◽  
David Fedida
Keyword(s):  
Mammalian Cells ◽  
Voltage Sensor ◽  
Delayed Rectifier ◽  
Charge Movement ◽  
Α Subunit ◽  
Time Constants ◽  
Gating Charge ◽  
Activation Gating ◽  
Pore Opening ◽  
Herg Channels

The human ether-á-go-go–related gene (hERG) K+ channel encodes the pore-forming α subunit of the rapid delayed rectifier current, IKr, and has unique activation gating kinetics, in that the α subunit of the channel activates and deactivates very slowly, which focuses the role of IKr current to a critical period during action potential repolarization in the heart. Despite its physiological importance, fundamental mechanistic properties of hERG channel activation gating remain unclear, including how voltage-sensor movement rate limits pore opening. Here, we study this directly by recording voltage-sensor domain currents in mammalian cells for the first time and measuring the rates of voltage-sensor modification by [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET). Gating currents recorded from hERG channels expressed in mammalian tsA201 cells using low resistance pipettes show two charge systems, defined as Q1 and Q2, with V1/2’s of −55.7 (equivalent charge, z = 1.60) and −54.2 mV (z = 1.30), respectively, with the Q2 charge system carrying approximately two thirds of the overall gating charge. The time constants for charge movement at 0 mV were 2.5 and 36.2 ms for Q1 and Q2, decreasing to 4.3 ms for Q2 at +60 mV, an order of magnitude faster than the time constants of ionic current appearance at these potentials. The voltage and time dependence of Q2 movement closely correlated with the rate of MTSET modification of I521C in the outermost region of the S4 segment, which had a V1/2 of −64 mV and time constants of 36 ± 8.5 ms and 11.6 ± 6.3 ms at 0 and +60 mV, respectively. Modeling of Q1 and Q2 charge systems showed that a minimal scheme of three transitions is sufficient to account for the experimental findings. These data point to activation steps further downstream of voltage-sensor movement that provide the major delays to pore opening in hERG channels.

Role of S4 positively charged residues in the regulation of Kv4.3 inactivation and recovery

2007 ◽  
Vol 293 (3) ◽  
pp. C906-C914 ◽  
Author(s):  
Matthew R. Skerritt ◽  
Donald L. Campbell
Keyword(s):  
Positive Charge ◽  
Voltage Sensitivity ◽  
Shaker Channels ◽  
Recovery From Inactivation ◽  
Kv4 Channels ◽  
Charged Residues ◽  
Arginine Residues ◽  
Voltage Sensing Domain ◽  
Positively Charged

The molecular and biophysical mechanisms by which voltage-sensitive K+ (Kv)4 channels inactivate and recover from inactivation are presently unresolved. There is a general consensus, however, that Shaker-like N- and P/C-type mechanisms are likely not involved. Kv4 channels also display prominent inactivation from preactivated closed states [closed-state inactivation (CSI)], a process that appears to be absent in Shaker channels. As in Shaker channels, voltage sensitivity in Kv4 channels is thought to be conferred by positively charged residues localized to the fourth transmembrane segment (S4) of the voltage-sensing domain. To investigate the role of S4 positive charge in Kv4.3 gating transitions, we analyzed the effects of charge elimination at each positively charged arginine (R) residue by mutation to the uncharged residue alanine (A). We first demonstrated that R290A, R293A, R296A, and R302A mutants each alter basic activation characteristics consistent with positive charge removal. We then found strong evidence that recovery from inactivation is coupled to deactivation, showed that the precise location of the arginine residues within S4 plays an important role in the degree of development of CSI and recovery from CSI, and demonstrated that the development of CSI can be sequentially uncoupled from activation by R296A, specifically. Taken together, these results extend our current understanding of Kv4.3 gating transitions.

scholarly journals Gating charge movement precedes ionic current activation in hERG channels

Channels ◽  
2013 ◽  
Vol 8 (1) ◽  
pp. 84-89 ◽  
Author(s):  
Samuel J Goodchild ◽  
David Fedida
Keyword(s):  
Ionic Current ◽  
Charge Movement ◽  
Gating Charge ◽  
Current Activation ◽  
Herg Channels

scholarly journals R1 in the Shaker S4 occupies the gating charge transfer center in the resting state

2011 ◽  
Vol 138 (2) ◽  
pp. 155-163 ◽  
Author(s):  
Meng-chin A. Lin ◽  
Jui-Yi Hsieh ◽  
Allan F. Mock ◽  
Diane M. Papazian
Keyword(s):  
Charge Transfer ◽  
Binding Site ◽  
Resting State ◽  
Slow Component ◽  
Maximum Amplitude ◽  
Voltage Sensor ◽  
Shaker Channels ◽  
Gating Charge ◽  
Voltage Dependent ◽  
Arginine Residues

During voltage-dependent activation in Shaker channels, four arginine residues in the S4 segment (R1–R4) cross the transmembrane electric field. It has been proposed that R1–R4 movement is facilitated by a “gating charge transfer center” comprising a phenylalanine (F290) in S2 plus two acidic residues, one each in S2 and S3. According to this proposal, R1 occupies the charge transfer center in the resting state, defined as the conformation in which S4 is maximally retracted toward the cytoplasm. However, other evidence suggests that R1 is located extracellular to the charge transfer center, near I287 in S2, in the resting state. To investigate the resting position of R1, we mutated I287 to histidine (I287H), paired it with histidine mutations of key voltage sensor residues, and determined the effect of extracellular Zn2+ on channel activity. In I287H+R1H, Zn2+ generated a slow component of activation with a maximum amplitude (Aslow,max) of ∼56%, indicating that only a fraction of voltage sensors can bind Zn2+ at a holding potential of −80 mV. Aslow,max decreased after applying either depolarizing or hyperpolarizing prepulses from −80 mV. The decline of Aslow,max after negative prepulses indicates that R1 moves inward to abolish ion binding, going beyond the point where reorientation of the I287H and R1H side chains would reestablish a binding site. These data support the proposal that R1 occupies the charge transfer center upon hyperpolarization. Consistent with this, pairing I287H with A359H in the S3–S4 loop generated a Zn2+-binding site. At saturating concentrations, Aslow,max reached 100%, indicating that Zn2+ traps the I287H+A359H voltage sensor in an absorbing conformation. Transferring I287H+A359H into a mutant background that stabilizes the resting state significantly enhanced Zn2+ binding at −80 mV. Our results strongly support the conclusion that R1 occupies the gating charge transfer center in the resting conformation.

scholarly journals Voltage-insensitive Gating after Charge-neutralizing Mutations in the S4 Segment of Shaker Channels

1999 ◽  
Vol 113 (1) ◽  
pp. 139-151 ◽  
Author(s):  
Hongxia Bao ◽  
Atiya Hakeem ◽  
Mark Henteleff ◽  
John G. Starkus ◽  
Martin D. Rayner
Keyword(s):  
Single Channel ◽  
Voltage Sensor ◽  
Control Voltage ◽  
Shaker Channels ◽  
Open Time ◽  
Channel Opening ◽  
Electrostatic Coupling ◽  
And Control ◽  
Pore Domain ◽  
S4 Segment

Shaker channel mutants, in which the first (R362), second (R365), and fourth (R371) basic residues in the S4 segment have been neutralized, are found to pass potassium currents with voltage-insensitive kinetics when expressed in Xenopus oocytes. Single channel recordings clarify that these channels continue to open and close from −160 to +80 mV with a constant opening probability (Po). Although Po is low (∼0.15) in these mutants, mean open time is voltage independent and similar to that of control Shaker channels. Additionally, these mutant channels retain characteristic Shaker channel selectivity, sensitivity to block by 4-aminopyridine, and are partially blocked by external Ca2+ ions at very negative potentials. Furthermore, mean open time is approximately doubled, in both mutant channels and control Shaker channels, when Rb+ is substituted for K+ as the permeant ion species. Such strong similarities between mutant channels and control Shaker channels suggests that the pore region has not been substantially altered by the S4 charge neutralizations. We conclude that single channel kinetics in these mutants may indicate how Shaker channels would behave in the absence of voltage sensor input. Thus, mean open times appear primarily determined by voltage-insensitive transitions close to the open state rather than by voltage sensor movement, even in control, voltage-sensitive Shaker channels. By contrast, the low and voltage-insensitive Po seen in these mutant channels suggests that important determinants of normal channel opening derive from electrostatic coupling between S4 charges and the pore domain.

scholarly journals Outwardly Rectifying Anionic Channel from the Plasma Membrane of the Fungus Phycomyces blakesleeanus

2009 ◽  
Vol 8 (9) ◽  
pp. 1439-1448 ◽  
Author(s):  
Miroslav Živić ◽  
Marko Popović ◽  
Nataša Todorović ◽  
Željko Vučinić
Keyword(s):  
Open Channel ◽  
Voltage Dependence ◽  
Present Report ◽  
Channel Activity ◽  
Phycomyces Blakesleeanus ◽  
Patch Clamp Technique ◽  
Electrophysiological Properties ◽  
Deactivation Kinetics ◽  
Gating Charge ◽  
Fast Activation

ABSTRACT In the present report, by using a patch clamp technique, we provide, to our knowledge, the first detailed description of an anionic channel from filamentous fungi. The characterized channel, an outwardly rectifying anionic channel (ORAC), is the most prominent feature of the cell membrane of the fungus Phycomyces blakesleeanus in the absence of energizing substrates. The unitary conductance of the channel is 11.3 ± 0.4 pS. It is characterized by a strong voltage dependence of the open-channel probability (zδ; the gating charge is 2.1 ± 0.1), and the channel is activated by depolarization. The values of the time constants for voltage-induced activation and deactivation of 28 ± 3 ms for τ a and 39 ± 9 ms for τ d show that the ORAC is characterized by fast activation/deactivation kinetics. The ORAC shows strong selectivity for anions over cations and weak selectivity among anions, with a selectivity sequence of I− ≥ NO3 − > Br− > Cl− > SO4 2− = 4.8 > 4.4 > 2.2 > 1 > 0.55, which corresponds to Eisenman series 1. The channel is characterized by two open and two closed states, with dominant long open (τo2 = 35.0 ± 3.9 ms) and long closed (τc2 = 166 ± 28 ms) states occupying 63% ± 8% and 79% ± 3% of total open and closed times, respectively. The ORAC is insensitive to anthracene-9-carboxylic acid (<200 μM), but 2 mM malate reversibly inhibits 59% ± 12% of the channel activity. Based on the electrophysiological properties of the channel, we propose that the ORAC plays a role in anion accumulation and in membrane potential regulation through local membrane depolarization.

Voltage sensing in jellyfish Shaker K+ channels.

1997 ◽  
Vol 200 (22) ◽  
pp. 2919-2926 ◽  
Author(s):  
N G Grigoriev ◽  
J D Spafford ◽  
W J Gallin ◽  
A N Spencer
Keyword(s):  
Segment Length ◽  
Total Charge ◽  
Fast Inactivation ◽  
Activation Curve ◽  
Channel Activation ◽  
Polyorchis Penicillatus ◽  
Shaker Channels ◽  
Critical Residue ◽  
S4 Segment ◽  
Positively Charged

The S4 segment of the jellyfish (Polyorchis penicillatus) Shaker channel jShak1 contains only six positively charged motifs. All other Shaker channels, including the jellyfish Shaker channel jShak2, have seven charges in this segment. Despite their charge differences, both these jellyfish channels produce currents with activation and inactivation curves shifted by approximately +40 mV relative to other Shaker currents. Adding charge without changing segment length by mutating the N-terminal side of jShak1 S4 does not have a pronounced effect on channel activation properties. Adding the positively charged motif RIF on the N-terminal side of K294 (the homologue of K374 in Drosophila Shaker, which is a structurally critical residue) produced a large positive shift in both activation and inactivation without altering the slope of the activation curve of the channel. When IFR was added to the other side of K294, there was a small negative shift in activation and fast inactivation of the channel was prevented. Our results demonstrate that K294 divides the S4 segment into functionally different regions and that the voltage threshold for activation and inactivation of the channel is not determined by the total charge on S4.

scholarly journals Contribution of the S4 Segment to Gating Charge in the Shaker K+ Channel

Neuron ◽  
1996 ◽  
Vol 16 (6) ◽  
pp. 1169-1177 ◽  
Author(s):  
Sanjay Kumar Aggarwal ◽  
Roderick MacKinnon
Keyword(s):  
K Channel ◽  
Gating Charge ◽  
S4 Segment
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