Ying Dou

Components of gating charge movement and S4 voltage-sensor exposure during activation of 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.

Normal targeting of a tagged Kv1.5 channel acutely transfected into fresh adult cardiac myocytes by a biolistic method

The transfection of cardiac myocytes is difficult, and so most of the data regarding the regulation of trafficking and targeting of cardiac ion channels have been obtained using heterologous expression systems. Here we apply the fast biolistic transfection procedure to adult cardiomyocytes to show that biolistically introduced exogenous voltage-gated potassium channel, Kv1.5, is functional and, like endogenous Kv1.5, localizes to the intercalated disc, where it is expressed at the surface of that structure. Transfection efficiency averages 28.2 +/- 5.7% of surviving myocytes at 24 h postbombardment. Ventricular myocytes transfected with a tagged Kv1.5 exhibit an increased sustained current component that is approximately 40% sensitive to 100 microM 4-aminopyridine and which is absent in myocytes transfected with a fluorescent protein-encoding construct alone. Kv1.5 deletion mutations known to reduce the surface expression of the channel in heterologous cells similarly reduce the surface expression in transfected ventricular myocytes, although targeting to the intercalated disc per se is generally unaffected by both NH(2)- and COOH-terminal deletion mutants. Expressed current levels in wild-type Kv1.5, Kv1.5DeltaSH3(1), Kv1.5DeltaN209, and Kv1.5DeltaN135 mutants were well correlated with apparent surface expression of the channel at the intercalated disc. Our results conclusively demonstrate functionality of channels present at the intercalated disc in native myocytes and identify determinants of trafficking and surface targeting in intact cells. Clearly, biolistic transfection of adult cardiac myocytes will be a valuable method to study the regulation of surface expression of channels in their native environment.

The neutral, hydrophobic isoleucine at position I521 in the extracellular S4 domain of hERG contributes to channel gating equilibrium

The human ether-a-go-go related (hERG) potassium channel has unusual functional characteristics in that the rates of channel activation and deactivation are much slower than inactivation, which is attributed to specific structural elements within the NH2 terminus and the S1-S4 voltage-sensing domains (VSD). Although the charged residues in the VSD have been extensively modified and mutated as a result, the role and importance of specific hydrophobic residues in the S4 has been much less explored in studies of hERG gating. We found that charged, but not neutral or hydrophobic, amino acid substitution of isoleucine 521 at the outer end of the S4 transmembrane domain resulted in channels activating at much more negative voltages associated with a marked hyperpolarization of the conductance-voltage (G-V) relationship. The contributions of different physicochemical properties to this effect were probed by chemical modification of channels substituted with cysteine at position I521. When positively charged reagents including tetramethyl-rhodamine-5-maleimide (TMRM), 1-(2-maleimidylethyl)-4-[5-(4-methoxyphenyl)oxazol-2-yl] pyridinium methane-sulfonate (PyMPO), [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET), and 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) were bound to the cysteine, I521C channels activated at more negative membrane potentials. To examine the contributions to hERG gating of other residues at the outer end of S4 (520-528), we performed a cysteine scan combined with MTSET modification. Only L520C, along with I521C, shows a substantial hyperpolarizing shift of the G-V relationship upon MTSET modification. The data indicate that the neutral, hydrophobic residue I521 at the extracellular end of S4 is critical for stabilizing the closed conformation of the hERG channel relative to the open state and by comparison with Shaker supports the alignment of hERG I521 with Shaker L361.

Trafficking of an endogenous potassium channel in adult ventricular myocytes

The roles of several small GTPases in the expression of an endogenous potassium current, I(to,f), in adult rat ventricular myocytes have been investigated. The results indicate that forward trafficking of newly synthesized Kv4.2, which underlies I(to,f) in these cells, requires both Rab1 and Sar1 function. Expression of a Rab1 dominant negative (DN) reduced I(to,f) current density by roughly one-half relative to control, mCherry-transfected myocytes. Similarly, expression of a Sar1DN nearly halved I(to,f) current density. Rab11 is not essential to trafficking of Kv4.2, as expression of a Rab11DN had no effect on I(to,f) over the time frames investigated here. In a process dependent on intact endoplasmic reticulum (ER)-to-Golgi transport, however, overexpression of wild-type Rab11 resulted in a doubling of I(to,f) density; block of ER-to-Golgi traffic by Brefeldin A completely abrogated the effect. Also implicated in the trafficking of Kv4.2 are Rab5 and Rab4. Rab5DN expression increased endogenous I(to,f) by two- to threefold, nonadditively with inhibition of dynamin-dependent endocytosis. And, in a phenomenon similar to that previously reported for myoblast-expressed Kv1.5, Rab4DN expression roughly doubled endogenous peak transient currents. Colocalization experiments confirmed the involvement of Rab4 in postinternalization trafficking of Kv4.2. There was little role evident for the lysosome in the degradation of internalized Kv4.2, as overexpression of neither wild-type nor DN isoforms of Rab7 had any effect on I(to,f). Instead, degradation may depend largely on the proteasome; the proteasome inhibitor MG132 significantly increased I(to,f) density.

Biolistic Transfection of Freshly Isolated Adult Ventricular Myocytes

Transfection of mammalian cells has long been an extremely powerful approach for the study of the effects of specific gene expression on cell function. Until recently, however, this approach has been unavailable for the study of gene function in adult cardiac myocytes. Here, an adaptation of the biolistic method to the transfection of adult cardiac myocytes is described. DNA is precipitated onto gold particles in the absence of PVP and the particles are biolistically delivered to freshly isolated adult rat cardiomyocytes via a Bio-Rad Helios System gene gun. The myocytes are cultured in the absence of bovine serum albumin and expression of the introduced genes, in phenotypically intact myocytes, is robust within 12-24 h.