HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 17/15/2263 02-1057fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by KUPERSHMIDT, S. Articles by BALSER, J. R. Search for Related Content PubMed PubMed Citation Articles by KUPERSHMIDT, S. Articles by BALSER, J. R.

The I_Kr drug response is modulated by KCR1 in transfected cardiac and noncardiac cell lines¹

SABINA KUPERSHMIDT^*,,2,3, IRIS C.-H. YANG^*,2, KENSHI HAYASHI^*, JIAN WEI, SIPRACHANH CHANTHAPHAYCHITH^*, CHRISTINA I. PETERSEN^*, DAVID C. JOHNS, ALFRED L. GEORGE, JR.^,, DAN M. RODEN^, and JEFFREY R. BALSER^*,

Departments of
^* Anesthesiology,
Pharmacology, and
Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA; and
Department of Neurological Surgery, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

³Correspondence: Department of Anesthesiology, Vanderbilt University School of Medicine, 554 Preston Research Bldg., Nashville, TN 37232-6602, USA. E-mail: sabina.kupershmidt{at}vanderbilt.edu

SPECIFIC AIMS

rKCR1 was recently shown to modulate the gating function of the rat neuronal ether-à-go-go (EAG) potassium channel, a relative of the cardiac K⁺ channel I_Kr encoded by the human ether-à-go-go-related gene (HERG). Since drug-induced suppression of I_Kr often provokes abnormal cardiac repolarization and ventricular arrhythmias (torsade de pointes), we investigated the effects of KCR1 on HERG sensitivity to commonly used I_Kr blockers.

PRINCIPAL FINDINGS

1. rKCR1 associates with HERG
We prepared protein extracts from tsA201 cells cotransfected with triple FLAG-tagged KCR1 cDNA (FLAG³-KCR1) and a HERG cDNA, as well as control cells with HERG and the FLAG-epitope expressing vector. Extracts were immunoprecipitated with an anti-FLAG antibody, then assayed in Western blots using an anti-HERG antibody. A band corresponding to HERG (Fig. 1 , IA, lanes 1 and 2) was detected by the anti-HERG antibody after coimmunoprecipitation by the anti-FLAG antibody in cells transfected with HERG and FLAG³-KCR1 (Fig. 1 , IB, lane 1). These findings suggest that HERG and KCR1 associate in a complex and may interact in mammalian cells.

View larger version (34K): [in this window] [in a new window]   Figure 1. I: Coimmunoprecipitation of HERG and KCR1 from mammalian cell extracts. Protein extracts from cells transfected with HERG and FLAG3-KCR1 (lanes 1) HERG plus p3xFLAG-CMV (lanes 2), or nontransfected cells (lanes 3) assayed by Western blot using an anti-HERG antibody. A) 2 µg of input lysate/lane to indicate the position of HERG on the gel. B) 700 µg of total lysate immunoprecipitated with 4 µg of an anti-FLAG antibody before detection by Western blot with the anti-HERG antibody (1:500). C) A mock immunoprecipitation. II: FLAG-epitope labeled KCR1 is detected in the plasma membrane protein fraction. TsA201 cells transfected with a FLAG3-KCR1 (lanes 1), HERG and FLAG3-KCR1 (lanes 2), and HERG plus p3xFLAG-CMV (lanes 3) were treated with biotin (A, B). Whole-cell extracts were prepared, followed by precipitation of the biotin-linked protein fraction using SA-coated agarose beads. C) Extracts from cells treated as in panels A and B but without biotin, thus revealing the level of nonspecific binding of protein to the SA beads. Whole-cell lysates (A), SA-selected (B) and parallel nonbiotinylated samples (C) were analyzed by Western blotting (9% PAGE). A) Top: a horseradish peroxidase-linked anti-FLAG antibody was used (1:500 dilution, Sigma). In the bottom panel, the same blot was stripped and reprobed using anti-HERG (1:500, Sigma) demonstrating that the SA selection is specific for membrane proteins because only the higher molecular mass form of HERG is detected in panel B. No signal was visible for the anti-HERG probed part of panel C.

Using a cell surface biotinylation assay , we directly tested whether KCR1 is localized to the plasma membrane. Cells transfected with FLAG³-KCR1 and HERG (Fig. 1 II, lanes 1), HERG plus a control vector (lanes 2), or mock-transfected cells (lanes 3) were incubated with a membrane-impermeant biotin analog; extracts were prepared, then precipitated with streptavidin (SA)-linked agarose beads, followed by Western blot analysis using an anti-FLAG antibody (Fig. 1 II, top). KCR1 was found in the SA-selected fraction in the presence and absence of HERG, indicating that at least a portion of KCR1 is associated with the plasma membrane (Fig. 1 , IIB, top, lanes 1, 2). In control experiments, only the plasma membrane-associated form of HERG was detected in the SA-selected fraction, indicating that the biotin/SA selection is specific for membrane-associated proteins (Fig. 1 II, bottom).

2. KCR1 has no effect on HERG gating function
Since rKCR1 has marked effects on the gating of rat EAG channels, we tested whether rKCR1 modulates the gating function of HERG. Currents recorded from CHO cells expressing HERG alone or both HERG and rKCR1 were similar, and their current-voltage relationships exhibited characteristics typical of HERG channels. To assess rKCR1 effects on activation gating, we plotted the peak tail current amplitude at -50 mV after each depolarized clamp step. The half-maximal activation voltages were similar for HERG (2.7 mV) and HERG + KCR1 (2.0 mV). The voltage-dependent distribution of channels between the open and inactivated states was also examined. Data from cells expressing HERG alone and HERG + rKCR1 superimposed, indicating that rKCR1 has no effect on the voltage dependence of inactivation.

3. KCR1 reduces drug blockade of HERG in CHO cells and in transfected cardiac myocytes
Dofetilide, a high-affinity blocker of I_Kr and HERG, has been associated with torsades de pointes. As expected, dofetilide block of HERG developed slowly during continuous pulsing. However, the amount of block was significantly reduced by rKCR1 coexpression. After 20 min of exposure to 20 nM dofetilide, only 49 ± 6% of the HERG current remained whereas 74 ± 8% of HERG + rKCR1 current remained. There was no significant time-dependent reduction in either HERG or HERG + rKCR1 currents in drug-free conditions. Exposure of HERG and HERG + rKCR1 to a range of dofetilide concentrations revealed a rightward shift in the dose-response curve. For HERG alone, the IC₅₀ for dofetilide block was 15.2 nM; for HERG + rKCR1 the IC₅₀ was 59.7 nM.

We studied the effect of KCR1 on HERG block by d-sotalol and quinidine, two compounds known to inhibit I_Kr and provoke torsades de pointes. Like dofetilide, block by d-sotalol developed over minutes, and rKCR1 coexpression nearly eliminated the blocking effect. HERG tail current remaining after 20 min of 300 µM d-sotalol exposure was 54 ± 9% of the predrug control for HERG alone, but 95 ± 6% for HERG + rKCR1 (P<0.05 vs. HERG alone). Quinidine (1 µM), by contrast, produced rapid block, reaching an equilibrium level of current inhibition within the first few test pulses. Despite these more rapid blocking characteristics, rKCR1 reduced the extent of quinidine block; by the second pulse, the tail current was 38 ± 3% of the predrug control level for HERG alone, but 48 ± 3% for HERG + rKCR1 (P<0.05 vs. HERG alone).

To assess the effects of exogenously added KCR1 on I_Kr recorded from myocytes, we transfected the cDNA into HL-1 cells and determined the dofetilide sensitivity of I_Kr in transfected and nontransfected control cells. The effect of KCR1 on I_Kr drug block is recapitulated in HL-1 cells (Fig. 2 B). In the presence of 5 and 20 nM dofetilide, the rate of onset of block and the extent of steady-state block were reduced by KCR1, although complete steady-state block could not be achieved within the time limits of cell viability at these low therapeutic concentrations. We estimate the IC₅₀ to be 4.7 nM in the absence of KCR1 vs. 14.8 nM in the presence of KCR1. Whereas the overall dofetilide potency in HL-1 cells was increased, the IC₅₀ value coexpressed with KCR1 more closely resembled the values reported for I_Kr reported in native neonatal mouse myocytes.

View larger version (34K): [in this window] [in a new window]   Figure 2. KCR1 lowers the sensitivity of myocyte IKr to block by dofetilide. Ethidium Bromide stained agarose gel. A) RT-PCR was used to determine KCR1 mRNA expression in RNA isolated from mouse atrium, ventricle and HL-1 cells. M, molecular size standards. B) Currents were recorded from HL-1 cells expressing IKr endogenously. KCR1 was either cotransfected (solid circles) or omitted (open squares). Depolarizing pulses (top, right) were applied every 15 s, and the amplitude of the tail current at -40 mV is plotted relative to the size of the tail current measured before drug exposure. After 3 min of dofetilide, tail currents recorded from untransfected control cells were suppressed more than those recorded from rKCR1 transfected cells.

4. KCR1 antagonizes effects of MiRP1
Prior studies have shown that the Mink-related protein MiRP1 associates with HERG. Hence, we examined whether MiRP1 coexpression would alter the effects of KCR1 on HERG function and drug sensitivity. After 20 min, the currents generated from HERG alone or from HERG + MiRP1 were completely blocked by 100 nM dofetilide. There was far less current blocked when HERG was coexpressed with rKCR1 (38±5% current remained), and expression of HERG with rKCR1 and MiRP1 (HERG+rKCR1+MiRP1) produced an intermediate effect (21±5% current remained). We examined the deactivating HERG current tail in each cell at -120 mV before drug application. MiRP1 coexpression speeded deactivation of HERG. Moreover, whereas KCR1 had no effect on the deactivation kinetics of HERG alone, it antagonized the deactivation gating effects of MiRP1 on HERG. These studies suggest that KCR1 and MiRP1 may interact with HERG in a manner that is functionally competitive: MiRP1 coexpression limits the effect of KCR1 to reduce HERG drug sensitivity; conversely, KCR1 seems to limit the effect of MiRP1 on HERG deactivation gating.

5. KCR1 is expressed in human heart and has effects similar to rKCR1
A human KCR1 cDNA (hKCR1) identified from an EST database exhibited 86% amino acid identity to rat KCR1. We examined mRNA expression of hKCR1 in human tissues using Northern blot analysis. Two mRNA transcripts were detected in multiple tissues, including heart. Each transcript is large enough to encompass the complete human KCR1 coding region and may represent splice variants or possibly independent transcripts from highly similar genes.

6. Human KCR1 has similar pharmacologic effects as the rat isoform
After 20 min of exposure to 20 nM dofetilide, 72 ± 6% of HERG + hKCR1 current remained, while in an additional set of HERG-alone cells only 35 ± 8% of the current remained (P<0.05 vs. HERG+hKCR1). Studies of hKCR1 on HERG gating yielded no effects and suggest that hKCR1, like rKCR1, does not modulate HERG drug block by altering gating function.

7. KCR1 does not act as a drug transporter
hKCR1 shows amino acid conservation to a yeast glucosyltransferase and other sugar transporters. We considered the possibility that KCR1 may limit drug action by transporting the compounds out of the cell (limiting the intracellular drug concentration) rather than by modulating the drug channel interaction. We added dofetilide to the whole-cell patch electrode to "clamp" the intracellular concentration of drug (supplying an infinite reservoir), eliminating the effects of KCR1 on HERG block if KCR1 was behaving as a drug transporter. For HERG alone, dofetilide eliminated nearly all the potassium current (11±1% current remained) while 64 ± 1% of HERG + hKCR1 current remained (P<0.05 vs. HERG alone).

CONCLUSIONS AND SIGNIFICANCE

The physiologic role of KCR1 in humans is unknown. We speculate that the protein may influence the function of K⁺ channels in the CNS and the cardiovascular system. Our study reveals that KCR1 is expressed in the human heart and reduces the sensitivity of HERG K⁺ channels to blockade by commonly used clinical compounds in heterologous cells as well as in myocytes. KCR1 titrates the drug sensitivity of I_Kr to proarrhythmic drug blockade over clinically relevant concentration ranges, where small changes in drug sensitivity could have profound functional effects on cardiac repolarization. We speculate that regulation of hKCR1 in the myocardium may be a target for modifying the proarrhythmic effects of otherwise clinically useful compounds.

View larger version (19K): [in this window] [in a new window]   Figure 3. Schematic hypothesis relating the effects of KCR1 on HERG drug block. The HERG channel is shown as a tetramer shaded in gray, the plasma membrane is shown in yellow, drug molecules in blue, KCR in pink, and MiRP in green. Although the interaction between HERG and KCR1 is shown here to occur at the plasma membrane, it may nevertheless occur in an unidentified intracytoplasmic compartment. A) In the absence of KCR1, drugs have free access to the HERG channel. B) In the presence of KCR1, drugs have reduced access to HERG. This is depicted here as a physical impediment imposed by KCR1, although the true mechanism is unknown. C) KCR1 and MiRP compete for interaction with HERG, which permits limited drug-access to HERG.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1057fje;

² These two authors contributed equally to this work.

This article has been cited by other articles:

				T. Nakajima, K. Hayashi, P. C. Viswanathan, M.-Y. Kim, M. Anghelescu, K. A. Barksdale, W. Shuai, J. R. Balser, and S. Kupershmidt HERG Is Protected from Pharmacological Block by {alpha}-1,2-Glucosyltransferase Function J. Biol. Chem., February 23, 2007; 282(8): 5506 - 5513. [Abstract] [Full Text] [PDF]

				Y.-F. Xiao, E. M. TenBroek, J. J. Wilhelm, P. A. Iaizzo, and D. C. Sigg Electrophysiological characterization of murine HL-5 atrial cardiomyocytes Am J Physiol Cell Physiol, September 1, 2006; 291(3): C407 - C416. [Abstract] [Full Text] [PDF]

				I. J. Rampil, D. H. Moller, and A. H. Bell Isoflurane modulates genomic expression in rat amygdala. Anesth. Analg., May 1, 2006; 102(5): 1431 - 1438. [Abstract] [Full Text] [PDF]

				S. M. White, P. E. Constantin, and W. C. Claycomb Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and function Am J Physiol Heart Circ Physiol, March 1, 2004; 286(3): H823 - H829. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 17/15/2263 02-1057fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by KUPERSHMIDT, S. Articles by BALSER, J. R. Search for Related Content PubMed PubMed Citation Articles by KUPERSHMIDT, S. Articles by BALSER, J. R.