HERG1a and hERG1b potassium channel subunits directly interact and preferentially form heteromeric channels

Beth A Mcnally,Zeus D Pendon,Matthew C Trudeau

doi:10.1074/jbc.m117.816488

Beth A Mcnally, Zeus D Pendon + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m117.816488

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Abstract

Voltage-activated human ether-á-go-go-related gene (hERG) potassium channels are critical for the repolarization of cardiac action potentials and tune-spike frequency adaptation in neurons. Two isoforms of mammalian ERG1 channel subunits, ERG1a and ERG1b, are the principal subunits that conduct the IKr current in the heart and are also broadly expressed in the nervous system. However, there is little direct evidence that ERG1a and ERG1b form heteromeric channels. Here, using electrophysiology, biochemistry, and fluorescence approaches, we systematically tested for direct interactions between hERG1a and hERG1b subunits. We report 1) that hERG1a dominant-negative subunits suppress hERG1b currents (and vice versa), 2) that disulfide bonds form between single cysteine residues experimentally introduced into an extracellular loop of hERG1a and hERG1b subunits and produce hERG1a-hERG1b dimers, and 3) that hERG1a and hERG1b subunits tagged with fluorescent proteins that are FRET pairs exhibit robust energy transfer at the plasma membrane. Thus, multiple lines of evidence indicated a physical interaction between hERG1a and hERG1b, consistent with them forming heteromeric channels. Moreover, co-expression of variable ratios of hERG1a and hERG1b RNA yielded channels with deactivation kinetics that reached a plateau and were different from those of hERG1b channels, consistent with a preference of hERG1b subunits for hERG1a subunits. Cross-linking studies revealed that an equal input of hERG1a and hERG1b yields more hERG1a-hERG1a or hERG1a-hERG1b dimers than hERG1b-hERG1b dimers, also suggesting that hERG1b preferentially interacts with hERG1a. We conclude that hERG1b preferentially forms heteromeric ion channels with hERG1a at the plasma membrane.

Highlights

ERG Kϩ channels are expressed in the heart [1, 2], central nervous system [3], and in a variety of other tissues, including tumor cells [4, 5]
Using disulfide bond formation between a cysteine residue introduced into the extracellular loop between the S5 and P-loop domains of hERG1a subunits (L589C) and the equivalent site in hERG1b subunits (L249C) we found robust disulfide bonds between hERG1a and hERG1b subunits, indicating that these subunits were in close proximity
Using different mixtures of hERG1a and hERG1b subunit cRNA ratios we found that the kinetics of deactivation became faster with increasing amounts of hERG1b cRNA (Fig. 1, B–D)

Summary

Results

To determine the functional properties of hERG1a subunits in the presence of hERG1b subunits (Fig. 1A), we co-expressed hERG1a with various ratios of hERG1b RNA in Xenopus oocytes and performed two-electrode voltage-clamp recordings. We performed purifications in the presence of ␤-mercaptoethanol, a reducing agent, which abolished the bands at the predicted sizes for dimers, suggesting that the dimers were formed by disulfide bonds between introduced cysteines (Fig. 3C, lanes 5–7) Together, these results suggested that specific disulfide bonds were formed between hERG1a– hERG1a, hERG1b– hERG1b, or hERG1a– hERG1b subunits and that hERG1a and hERG1b subunits were within close enough proximity to form disulfide bonds. ECFP and hERG1b–Citrine (Fig. 5E) indicating that FRET was independent of the donor and acceptor pair This result shows that hERG1a and hERG1b subunits were in close proximity at the plasma membrane. HERG1b–Citrine at the membrane, which is consistent with the small measurable currents from hERG1b (Fig. 1), we detected FRET between hERG1b–eCFP or hERG1b–Citrine subunits (Fig. 5E) These results showed that hERG1a subunits were in close proximity with hERG1b subunits at the plasma membrane suggesting they were in heteromeric channels. This suggests that hERG1a increases the amount of hERG1b at the membrane, which is consistent with results indicating that the hERG1b maturation is enhanced by hERG1a [18, 30]

Discussion

Experimental procedures

F FRET 458