Purpose
Tissue engineered heart valves (TEHV) are being investigated to address the limitations of currently available valve prostheses. In order to advance a wide variety of TEHV approaches, the ...goal of this study was to develop a cardiac valve bioreactor system capable of conditioning living valves with a range of hydrodynamic conditions as well as capable of assessing hydrodynamic performance to ISO 5840 standards.
Methods
A bioreactor system was designed based on the Windkessel approach. Novel features including a purpose-built valve chamber and pressure feedback control were incorporated to maintain asepsis while achieving a range of hydrodynamic conditions. The system was validated by testing hydrodynamic conditions with a bioprosthesis and by operating with cell culture medium for 4 weeks and living cells for 2 weeks.
Results
The bioreactor system was able to produce a range of pressure and flow conditions from static to resting adult left ventricular outflow tract to pathological including hypertension. The system operated aseptically for 4 weeks and cell viability was maintained for 2 weeks. The system was also able to record the pressure and flow data needed to calculate effective orifice area and regurgitant fraction.
Conclusions
We have developed a single bioreactor system that allows for step-wise conditioning protocols to be developed for each unique TEHV design as well as allows for hydrodynamic performance assessment.
The location of the tetraethylammonium (TEA) binding site in the outer vestibule of K
+ channels, and the mechanism by which external TEA slows C-type inactivation, have been considered ...well-understood. The prevailing model has been that TEA is coordinated by four amino acid side chains at the position equivalent to
Shaker T449, and that TEA prevents a constriction that underlies inactivation via a foot-in-the-door mechanism at this same position. However, a growing body of evidence has suggested that this picture may not be entirely correct. In this study, we reexamined these two issues, using both the Kv2.1 and
Shaker potassium channels. In contrast to results previously obtained with
Shaker, substitution of the tyrosine at Kv2.1 position 380 (equivalent to
Shaker 449) with a threonine or cysteine had a relatively minor effect on TEA potency. In both Kv2.1 and
Shaker, modification of cysteines at position 380/449 by 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET) proceeded at identical rates in the absence and presence of TEA. Additional experiments in
Shaker demonstrated that TEA bound well to C-type inactivated channels, but did not interfere with MTSET modification of C449 in inactivated channels. Together, these findings rule out the possibility that TEA binding involves an intimate interaction with the four side chains at the position equivalent to
Shaker 449. Moreover, these results argue against the model whereby TEA slows inactivation via a foot-in-the-door mechanism at position 449, and also argue against the hypothesis that the position 449 side chains move toward the center of the conduction pathway during inactivation. Occupancy by TEA completely prevented MTSET modification of a cysteine in the outer-vestibule turret (Kv2.1 position 356/
Shaker position 425), which has been shown to interfere with both TEA binding and the interaction of K
+ with an external binding site. Together, these data suggest that TEA is stabilized in a more external position in the outer vestibule, and does not bind via direct coordination with any specific outer-vestibule residues.
Current magnitude in Kv2.1 potassium channels is modulated by external K+. In contrast to behavior expected from the change in electrochemical driving force, outward current through Kv2.1 channels ...becomes larger when extracellular K+ is increased within the physiological range. The mechanism that underlies this unusual property involves the opening of Kv2.1 channels into one of two different outer vestibule conformations, which are defined by their sensitivity to TEA. Channels that open into a TEA-sensitive conformation generate larger macroscopic currents, whereas channels that open into a TEA-insensitive conformation generate smaller macroscopic currents. At higher K+, more channels open into the TEA-sensitive conformation. In this manuscript, we examined the mechanism by which the conformational change produced a change in current magnitude. We started by testing the simplest hypothesis: that each pharmacologically defined channel conformation produces a different single channel conductance, one smaller and one larger, and that the K+-dependent change in current magnitude reflects the K+-dependent change in the percentage of channels that open into each of the two conformations. Using single channel and macroscopic recordings, as well as hidden Markov modeling, we were able to quantitatively account for K+-dependent regulation of macroscopic current with this model. Combined with previously published work, these results support a model whereby an outer vestibule lysine interferes with K+ flux through the channel, and that the K+-dependent change in orientation of this lysine alters single channel conductance by changing the level of this interference. Moreover, these results provide an experimental example of single channel conductance being modulated at the outer end of the conduction pathway by a mechanism that involves channel activation into open states with different outer vestibule conformations.
The Kv2.1 potassium channel contains a lysine in the outer vestibule (position 356) that markedly reduces open channel sensitivity to changes in external K(+). To investigate the mechanism underlying ...this effect, we examined the influence of this outer vestibule lysine on three measures of K(+) and Na(+) permeation. Permeability ratio measurements, measurements of the lowest K(+) required for interaction with the selectivity filter, and measurements of macroscopic K(+) and Na(+) conductance, were all consistent with the same conclusion: that the outer vestibule lysine in Kv2.1 interferes with the ability of K(+) to enter or exit the extracellular side of the selectivity filter. In contrast to its influence on K(+) permeation properties, Lys 356 appeared to be without effect on Na(+) permeation. This suggests that Lys 356 limited K(+) flux by interfering with a selective K(+) binding site. Combined with permeation studies, results from additional mutagenesis near the external entrance to the selectivity filter indicated that this site was located external to, and independent from, the selectivity filter. Protonation of a naturally occurring histidine in the same outer vestibule location in the Kv1.5 potassium channel produced similar effects on K(+) permeation properties. Together, these results indicate that a selective, functional K(+) binding site (e.g., local energy minimum) exists in the outer vestibule of voltage-gated K(+) channels. We suggest that this site is the location of K(+) hydration/dehydration postulated to exist based on the structural studies of KcsA. Finally, neutralization of position 356 enhanced outward K(+) current magnitude, but did not influence the ability of internal K(+) to enter the pore. These data indicate that in Kv2.1, exit of K(+) from the selectivity filter, rather than entry of internal K(+) into the channel, limits outward current magnitude. We discuss the implications of these findings in relation to the structural basis of channel conductance in different K(+) channels.
We previously demonstrated that the outer vestibule of activated Kv2.1 potassium channels can be in one of two conformations, and that K(+) occupancy of a specific selectivity filter site determines ...which conformation the outer vestibule is in. These different outer vestibule conformations result in different sensitivities to internal and external TEA, different inactivation rates, and different macroscopic conductances. The K(+)-dependent switch in outer vestibule conformation is also associated with a change in rate of channel activation. In this paper, we examined the mechanism by which changes in K(+) modulate the rate of channel activation. Elevation of symmetrical K(+) or Rb(+) from 0 to 3 mM doubled the rate of on-gating charge movement (Q(on)), measured at 0 mV. Cs(+) produced an identical effect, but required 40-fold higher concentrations. All three permeant ions occupied the selectivity filter over the 0.03-3 mM range, so simple occupancy of the selectivity filter was not sufficient to produce the change in Q(on). However, for each of these permeant ions, the speeding of Q(on) occurred with the same concentration dependence as the switch between outer vestibule conformations. Neutralization of an amino acid (K356) in the outer vestibule, which abolishes the modulation of channel pharmacology and ionic currents by the K(+)-dependent reorientation of the outer vestibule, also abolished the K(+)-dependence of Q(on). Together, the data indicate that the K(+)-dependent reorientation in the outer vestibule was responsible for the change in Q(on). Moreover, similar K(+)-dependence and effects of mutagenesis indicate that the K(+)-dependent change in rate of Q(on) can account for the modulation of ionic current activation rate. Simple kinetic analysis suggested that K(+) reduced an energy barrier for voltage sensor movement. These results provide strong evidence for a direct functional interaction, which is modulated by permeant ions acting at the selectivity filter, between the outer vestibule of the Kv2.1 potassium channel and the voltage sensor.
The Kv2.1 potassium channel contains a lysine in the outer vestibule (position 356) that markedly reduces open channel sensitivity to changes in external K+. To investigate the mechanism underlying ...this effect, we examined the influence of this outer vestibule lysine on three measures of K+ and Na+ permeation. Permeability ratio measurements, measurements of the lowest K+ required for interaction with the selectivity filter, and measurements of macroscopic K+ and Na+ conductance, were all consistent with the same conclusion: that the outer vestibule lysine in Kv2.1 interferes with the ability of K+ to enter or exit the extracellular side of the selectivity filter. In contrast to its influence on K+ permeation properties, Lys 356 appeared to be without effect on Na+ permeation. This suggests that Lys 356 limited K+ flux by interfering with a selective K+ binding site. Combined with permeation studies, results from additional mutagenesis near the external entrance to the selectivity filter indicated that this site was located external to, and independent from, the selectivity filter. Protonation of a naturally occurring histidine in the same outer vestibule location in the Kv1.5 potassium channel produced similar effects on K+ permeation properties. Together, these results indicate that a selective, functional K+ binding site (e.g., local energy minimum) exists in the outer vestibule of voltage-gated K+ channels. We suggest that this site is the location of K+ hydration/dehydration postulated to exist based on the structural studies of KcsA. Finally, neutralization of position 356 enhanced outward K+ current magnitude, but did not influence the ability of internal K+ to enter the pore. These data indicate that in Kv2.1, exit of K+ from the selectivity filter, rather than entry of internal K+ into the channel, limits outward current magnitude. We discuss the implications of these findings in relation to the structural basis of channel conductance in different K+ channels. PUBLICATION ABSTRACT
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