A Direct Demonstration of Closed-State Inactivation of K+ Channels at Low pH

Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltage-clamp fluorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fluorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker α-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fluorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fluorescence demonstrated slow unquenching that also reflects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fluorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fluorescence at low pH reflects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K+ or the mutation T449V) prevented fluorescence loss at low pH, and the fluorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fluorescence amplitude (pKa = 5.9) in channels held continuously at −80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fluorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state

Fast free of acrylamide clearing tissue (FACT) for clearing, immunolabelling and three-dimensional imaging of partridge tissues

Fast free of acrylamide clearing tissue (FACT) is a modified sodium dodecyl sulfate-based clearing protocol for the chemical clearing of lipids that completely preserves fluorescent signals in the cleared tissues. The FACT protocol was optimized to image translucent immunostained brain and non-nervous tissues. For this purpose adult male Chukar partridge (Alectoris chukar) was used as a model. After clearing the tissues, 1 or 2 mm-thickness sections of tissues were immunolabeled. The paraventricular nucleus in the hypothalamus (2-mm section) was cleared with FACT, and then was stained with gonadotropin-inhibitory hormone (GnIH) antibody and Hoechst. Simultaneously, immunohistochemical (IHC) staining of cryosectioned brain (30 μm) was done by GnIH-antibody. The FACT protocol and staining of cell nuclei of nine other tissues were done by a z-stack motorized fluorescent microscope. GnIH-immunoreactive neurons were found by FACT and IHC during the breeding season in male partridges. Deep imaging of the kidney, duodenum, jejunum, lung, pancreas, esophagus, skeletal muscle, trachea, and testis were also done. The FACT protocol can be used for the three-dimensional imaging of various tissues and immunostained evaluation of protein markers

Structural and genetic modulators of voltage-gated potassium channel activation kinetics

Voltage-gated potassium (Kv) channels regulate membrane excitability and are therefore critical determinants of cellular function. However, the detailed mechanisms by which Kv channel activity is modulated are not well understood. This thesis investigates the modulation of activation of Kv1.2 and KCNQ1 channels. These studies reveal that Kv1.2 can activate via two different pathways that produce two distinct gating phenotypes/modes. In the 'slow' gating mode, the activation V 1/2 was shifted by +30 mV and activation kinetics were at least 20-fold slower than those of channels gating through the 'fast' mode. This offers an explanation for the wide variations in the reported activation kinetics of Kv1.2 in the literature. Introduction of a positive charge at or around threonine 252 (T252) in the S2-S3 cytoplasmic linker of Kv1.2 trapped channels in the 'fast' activation mode, suggesting that this region may act as the molecular switch in Kv1.2. Consistent with this, the S2-S3 linker was shown to mediate the gating-modifying effect of a mutation (T46V) in the cytoplasmic T1 domain of Kv1.2. Excision of patches containing Kv1.2 also trapped channels in the 'fast' gating mode, indicating cytoplasmic regulators may also modify the gating mode via the S2-S3 linker. We have ruled out cytoplasmic regulation by PIP₂, polyamines and phoshporylation. Interestingly, one kinase inhibitor, KN-93, a commonly used calcium/calmodulin-dependent protein kinase II inhibitor, was found to be a direct extracellular blocker of many different Kv channels including Kv1.2. Finally, a novel missense mutation at the intracellular end of the S3 helix in a mutant KCNQ1 channel (V205M), detected in an aboriginal community with a high prevalence of long QT syndrome and sudden death, was shown to cause a depolarizing shift in the voltage dependence of activation and a slowing of activation kinetics. This resulted in reduced repolarization reserve during the cardiac action potential and a likely increased susceptibility to the initiation of arrhythmias. The close positioning of this mutation to the S2-S3 linker provides a putative structural working model for the gating switch in Kv1.2 that involves changes in the hydrophobic packing of the S3 helix and its influence on S4 voltage-sensor movement.Medicine, Faculty ofCellular and Physiological Sciences, Department ofGraduat

4-Aminopyridine Prevents the Conformational Changes Associated with P/C-Type Inactivation in Shaker Channels

ABSTRACT The effect of 4-aminopyridine (4-AP) on K v channel activation has been extensively investigated, but its interaction with inactivation is less well understood. Voltage-clamp fluorimetry was used to directly monitor the action of 4-AP on conformational changes associated with slow inactivation of Shaker channels. Tetramethylrhodamine-5-maleimide was used to fluorescently label substituted cysteine residues in the S3-S4 linker (A359C) and pore (S424C). Activation-and inactivation-induced changes in fluorophore microenvironment produced fast and slow phases of fluorescence that were modified by 4-AP. In Shaker A359C, 4-AP block reduced the slow-phase contribution from 61 Ϯ 3 to 28 Ϯ 5%, suggesting that binding inhibits the conformational changes associated with slow inactivation and increased the fast phase that reports channel activation from 39 Ϯ 3 to 72 Ϯ 5%. In addition, 4-AP enhanced both fast and slow phases of fluorescence return upon repolarization ( reduced from 87 Ϯ 15 to 40 Ϯ 1 ms and from 739 Ϯ 83 to 291 Ϯ 21 ms, respectively), suggesting that deactivation and recovery from inactivation were enhanced. In addition, the effect of 4-AP on the slow phase of fluorescence was dramatically reduced in channels with either reduced (T449V) or permanent P-type (W434F) inactivation. Interestingly, the slow phase of fluorescence return of W434F channels was enhanced by 4-AP, suggesting that 4-AP prevents the transition to C-type inactivation in these channels. These data directly demonstrate that 4-AP prevents slow inactivation of K v channels and that 4-AP can bind to P-type-inactivated channels and selectively inhibit the onset of C-type inactivation

Voltage dependence of the dequenching of fluorescence and ionic current activation

(A) Mean values for the time constants of the dequenching component of fluorescence measured from TMRM attached to either M394C or A397C and the time constants of the associated ionic current activation, measured between +10 and +60 mV. (B) Relative amplitude of the dequenching component of fluorescence, normalized to the total fluorescence deflection on depolarization, measured from TMRM attached at either M394C or A397C.<p><b>Copyright information:</b></p><p>Taken from "Voltage Clamp Fluorimetry Reveals a Novel Outer Pore Instability in a Mammalian Voltage-gated Potassium Channel"</p><p></p><p>The Journal of General Physiology 2008;132(2):209-222.</p><p>Published online Jan 2008</p><p>PMCID:PMC2483330.</p><p></p