The human ether-à-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel, which is important for cardiac repolarization. Reduction of hERG current due to genetic mutations or drug interferences causes long QT syndrome, leading to cardiac arrhythmias and sudden death. To date, there is no effective therapeutic method to restore or enhance hERG channel function. Using cell biology and electrophysiological methods, we found that the muscarinic receptor agonist carbachol increased the expression and function of hERG, but not ether-à-go-go or K v 1.5 channels stably expressed in human embryonic kidney cells. The carbachol-mediated increase in hERG expression was abolished by the selective M3 antagonist 4-DAMP (1,1-dimethyl-4-diphenylacetoxypiperidinium iodide) but not by the M2 antagonistTreatment of cells with carbachol reduced the hERG-ubiquitin interaction and slowed the rate of hERG degradation. We previously showed that the E3 ubiquitin ligase Nedd4-2 mediates degradation of hERG channels. Here, we found that disrupting the Nedd4-2 binding domain in hERG completely eliminated the effect of carbachol on hERG channels. Carbachol treatment enhanced the phosphorylation level, but not the total level, of Nedd4-2. Blockade of the protein kinase C (PKC) pathway abolished the carbachol-induced enhancement of hERG channels. Our data suggest that muscarinic activation increases hERG channel expression by phosphorylating Nedd4-2 via the PKC pathway.

Kv1.5 channels mediate the ultra-rapidly activating delayed rectifier potassium current (IKur), which is important for atrial repolarization. It has been shown that cell-surface Kv1.5 channels are sensitive to cleavage by the extracellular serine protease, proteinase K (PK). Here, we investigated the effects of extracellular proteolytic digestion on the function of Kv1.5 channels stably expressed in HEK 293 cells. Our data demonstrate that PK treatment cleaved mature membrane-bound (75kDa) Kv1.5 channels at a single locus in the S1-S2 linker, producing 42-kDa N-terminal fragments and 33-kDa C-terminal fragments. Interestingly, such PK treatment did not affect the Kv1.5 current (IKv1.5) recorded using the whole-cell patch clamp technique. Analysis of cell-surface proteins isolated using biotinylation indicated that the PK-generated N- and C-terminal fragments were both present in the plasma membrane. Co-immunoprecipitation (co-IP) experiments indicated that the N- and C-terminal fragments are no longer associated after cleavage. Furthermore, following PK digestion, the N- and C-fragments degraded at different rates. PK is frequently used as a tool to analyze cell-surface localization of membrane proteins, and cleavage of cell-surface channels has been shown to abolish channel function (e.g. hERG). Our data, for the first time, demonstrate that cleavage of cell-surface channels assessed by Western blot analysis does not necessarily correlate with an elimination of the channel activities.

Atrial fibrillation (AF) is the most common form of cardiac arrhythmia. Kv1.5 channel mediated ultra‐rapidly activating delayed rectifier potassium current (IKur) is important for atrial repolarization. Since cardiac IKur is expressed solely in the atria, therapies that target Kv1.5 represent promising strategies for treating AF. Pathologies linked to AF, such as ischemia and hypoxia, have been reported to activate various proteases. It has been shown that cell‐surface Kv1.5 channels are sensitive to cleavage by extracellular proteases, such as proteinase K (PK). In this study, we investigated the consequences of proteolytic digestion on Kv1.5 channels. Our data demonstrate that extracellularly applied PK cleaves mature (75 kDa) Kv1.5 channels at a single locus in the S1‐S2 linker, yielding two digestion products of 42 and 33 kDa, corresponding to the N‐ and C‐terminal fragments, respectively. Surprisingly, whole‐cell patch clamp analysis showed that IKv1.5 was unaltered by the PK cut. Plasma membrane protein isolation via biotinylation indicated that both the N‐ and C‐terminal fragments following PK digestion retain cell‐surface expression over several hours. Co‐immunoprecipitation studies after PK cleavage indicate that the two fragments of the channel do not associate. The time‐dependent decline in the Kv1.5 fragment lacking the N‐terminus and the S1 domain paralleled the amplitude of recorded Kv1.5 currents. Our results suggest that Kv1.5 remains functional without the NH2‐S1 portion of the channel. The resistance of IKv1.5 to proteolytic cleavage may represent an inherent protective mechanism for Kv1.5 function. These findings extend our understanding of the Kv1.5 channel structure‐function relationship.Supported by CIHR & the Heart and Stroke Foundation

The human ether‐a‐go‐go–related gene (hERG) encodes the pore‐forming subunit of the rapidly activating delayed rectifier potassium channel (IKr), which is important for cardiac repolarization. Dysfunction of hERG causes long QT syndrome, arrhythmias and sudden death, which occur in patients with cardiac ischemia. Cardiac ischemia is also associated with activation of various proteases. We characterized the effects of proteases on hERG/IKr with the aim to develop novel strategies to prevent protease‐mediated damage. Using whole‐cell patch clamp and Western blot analysis we demonstrated that the hERG/IKr channel is selectively cleaved by the serine proteases, proteinase K and protease XXIV. Using molecular biology including making chimerical channels between protease K‐sensitive hERG and insensitive hEAG, we identified that the S5‐pore linker of hERG is the target domain for proteinase K. We discovered that the scorpion toxin BeKm‐1, which binds to the S5‐pore linker of hERG, can effectively protect hERG from proteinase K‐mediated damage. The reduction in mature ERG expression was observed in a rabbit cardiac ischemia model and in hERG‐HEK cells cultured in hypoxic conditions. BeKm‐1 effectively prevented the hypoxia‐induced reduction of the mature hERG expression. In conclusion, hERG is uniquely susceptible to proteases, which may contribute to arrhythmias under conditions such as cardiac ischemia. Protecting hERG channels from proteolytic damage using specific peptides may represent a novel strategy to prevent and treat arrhythmias associated with ischemic heart disease.(Supported by the Canadian Institutes of Health Research & the Heart and Stroke Foundation of Ontario)