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(Circulation Research. 2004;94:1332.)
© 2004 American Heart Association, Inc.

Cellular Biology

Unique Kir2.x Properties Determine Regional and Species Differences in the Cardiac Inward Rectifier K⁺ Current

Amit S. Dhamoon, Sandeep V. Pandit, Farzad Sarmast, Keely R. Parisian, Prabal Guha, You Li, Suveer Bagwe, Steven M. Taffet, Justus M.B. Anumonwo

From the Department of Pharmacology and Institute for Cardiovascular Research, State University of New York Upstate Medical University, Syracuse.

Correspondence to Justus M.B. Anumonwo, PhD, Department of Pharmacology, State University of New York Upstate Medical University, 766 Irving Ave, Syracuse, NY 13210. E-mail anumonwj{at}upstate.edu

	Abstract

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The inwardly rectifying potassium (Kir) 2.x channels mediate the cardiac inward rectifier potassium current (I_K1). In addition to differences in current density, atrial and ventricular I_K1 have differences in outward current profiles and in extracellular potassium ([K⁺]_o) dependence. The whole-cell patch-clamp technique was used to study these properties in heterologously expressed Kir2.x channels and atrial and ventricular I_K1 in guinea pig and sheep hearts. Kir2.x channels showed distinct rectification profiles: Kir2.1 and Kir2.2 rectified completely at potentials more depolarized than –30 mV (I0 pA). In contrast, rectification was incomplete for Kir2.3 channels. In guinea pig atria, which expressed mainly Kir2.1, I_K1 rectified completely. In sheep atria, which predominantly expressed Kir2.3 channels, I_K1 did not rectify completely. Single-channel analysis of sheep Kir2.3 channels showed a mean unitary conductance of 13.1±0.1 pS in 15 cells, which corresponded with I_K1 in sheep atria (9.9±0.1 pS in 32 cells). Outward Kir2.1 currents were increased in 10 mmol/L [K⁺]_o, whereas Kir2.3 currents did not increase. Correspondingly, guinea pig (but not sheep) atrial I_K1 showed an increase in outward currents in 10 mmol/L [K⁺]_o. Although the ventricles of both species expressed Kir2.1 and Kir2.3, outward I_K1 currents rectified completely and increased in high [K⁺]_o-displaying Kir2.1-like properties. Likewise, outward current properties of heterologously expressed Kir2.1-Kir2.3 complexes in normal and 10 mmol/L [K⁺]_o were similar to Kir2.1 but not Kir2.3. Thus, unique properties of individual Kir2.x isoforms, as well as heteromeric Kir2.x complexes, determine regional and species differences of I_K1 in the heart.

Key Words: Kir2 • extracellular potassium • heteromerization • rectification

	Introduction

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In the heart, the inwardly rectifying potassium (Kir) current (I_K1) stabilizes the resting membrane potential and plays a major role during the final phase of action potential (AP) repolarization.^1–3 The Kir2.x channels mediate cardiac I_K1.³ Previous studies have demonstrated that I_K1 properties are different in atrial and ventricular myocytes.^1,3–6 First, I_K1 current density is higher in the ventricles than in the atria.^6,7 Second, ventricular I_K1 has been described as having a more prominent negative slope conductance at depolarized potentials than atrial I_K1 (ie, atrial I_K1 does not rectify completely).^1,4,5 Also, the outward component of the background potassium current (I_B; consisting mainly of I_K1) is significantly increased in high extracellular potassium ([K⁺]_o) in ventricular but not atrial myocytes.¹ The molecular mechanisms underlying these I_K1 differences are unknown.

The Kir2.x channel expression patterns may determine outward I_K1 properties.^8,9 Outward currents through Kir channels may play an important role in the dynamics of atrial and ventricular fibrillation, as studied in the sheep¹⁰ and guinea pig,¹¹ respectively. However, outward current profiles of the individual Kir2.x isoforms have not been comparatively studied. Moreover, the effect of high [K⁺]_o on outward currents of Kir2.x isoforms has also not been compared. It is possible that the properties of Kir2.x isoforms, existing either as homomers or heteromers, determine regional I_K1 differences in the heart. Although recent studies have shown that Kir2.x subunits heteromerize,^12–14 the rectification and [K⁺]_o dependence of heteromeric Kir2.x channels have not yet been studied.

In this study, we show that individual Kir2.x isoforms have unique outward current profiles as well as differential responses to elevated [K⁺]_o. We also demonstrate differences in Kir2.x expression patterns in the atria and ventricles of the sheep and guinea pig. Our results show that the rectification profile and [K⁺]_o sensitivity of I_K1 in these species are determined by the expression patterns of the underlying Kir2.x isoforms in the atria and the ventricle. We also demonstrate that the rectification and [K⁺]_o sensitivity of the Kir2.1 isoform determine I_K1 properties when heteromeric complexes are formed. Part of this work has been presented in abstract form.^8,15

	Materials and Methods

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Detailed descriptions of the approaches used in the experiments are presented in the expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org. Guinea pig Kir2.1, Kir2.2, and Kir2.3, and sheep Kir2.3 were cloned using the polymerase chain reaction and transiently transfected into human embryonic kidney 293 (HEK293) cells using the Qiagen Effectene protocol. Guinea pig and sheep cardiac myocytes were enzymatically dissociated using the Langendorff-retrograde perfusion method as described previously.¹¹ Inwardly rectifying currents were recorded using the whole-cell and cell-attached patch-clamp techniques. Kir2.x mRNA was measured using the ribonuclease (RNase) protection assay,¹⁶ and Kir2.x protein was measured using the Western blot technique. A mathematical model of the human atrial myocyte AP¹⁷ was implemented in C language on a SUN Ultra-10 workstation platform.

	Results

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Properties of Outward Currents Through Heterologously Expressed Kir2.x Channels
Figure 1A shows representative current-voltage (I-V) relationships of Kir2.1, Kir2.2, and Kir2.3 currents measured in HEK293 cells, as well as I-V relations in an untransfected cell. A voltage-clamp ramp protocol from –100 to 0 mV was used and barium-sensitive currents are shown. The data show marked differences in the rectification profiles of the expressed Kir2.x channels. The trace recorded from an untransfected HEK293 cell was almost indistinguishable from the x axis, showing that endogenous and leak currents contribute virtually no component to the barium-sensitive current. Figure 1B illustrates the rectification profiles of Kir2.x channels normalized to current at –100 mV. Outward current peaked at similar voltages for Kir2.1 (–68.2±1.9 mV; n=6) and Kir2.2 (–74.2±1.7 mV; n=6). However, outward current in Kir2.3 channels peaked at a more positive potential (–54.7±3.1 mV; n=4; P<0.05) than the other 2 isoforms. There were no significant differences in the reversal potential (E_rev) of the different isoforms (Kir2.1 –86±1.1 mV; Kir2.2 –86±1.2 mV; Kir2.3 –85±1.2 mV; ANOVA). To quantify the degree of rectification of the Kir2.x channels, we analyzed relative current (ratio of actual current measured and the current predicted assuming a linear unblocked current)¹⁸ by fitting with the Boltzmann equation. As shown in Figure 1C, a single Boltzmann function was sufficient to fit Kir2.1 and Kir2.2 currents. Kir2.2 currents showed stronger voltage dependence of rectification (z=3.66±0.43; n=6) than Kir2.1 currents (z=2.5±0.07; n=6; P<0.05). In contrast, Kir2.3 currents could only be fit by a double Boltzmann function (z₁=1.31±0.05; z₂=10.9±1.1; n=4).

View larger version (13K): [in this window] [in a new window]   Figure 1. I-V relations of Kir2.x channels expressed in HEK293 cells. A, Comparison of ramp (–100 to 0 mV)-generated, barium-sensitive currents in cells expressing Kir2.x channels. Current in an untransfected cell is superimposed. B, Average data for cells expressing Kir2.1 (n=6), Kir2.2 (n=6), and Kir2.3 (n=4) channels. Data were normalized (norm) to current at –100 mV. C, Average relative (rel) current (ratio of actual current measured and the current predicted assuming a linear unblocked current) as a function of voltage. Data were fit with a single (Kir2.1 and Kir2.2) or a double (Kir2.3) Boltzmann function.

Properties of Outward Cardiac I_K1
Based on the differences in the rectification profiles of the Kir2.x channels shown above, we examined outward atrial and ventricular I_K1 properties in the guinea pig and sheep. We also correlated our data with Kir2.x mRNA and protein expression in the 2 species.

Guinea Pig Atrial and Ventricular I_K1
A comparative analysis of inward rectification properties in guinea pig atrial and ventricular myocytes has been described previously, but in the study,¹ I_K1 was not isolated from the I_B. In Figure 2A, average barium-sensitive I-V relations of guinea pig atrial (n=6) and ventricular I_K1 (n=4) are shown. Peak inward current density (I_p), measured at –100 mV was –4.76±0.53 pA/pF for atrial cells and was significantly greater (–9.18±1.3 pA/pF; P<0.05) in ventricular cells. The outward currents peaked at similar voltages (–67±2.4 mV versus –61±2.6 mV), and the E_rev was not significantly different (–82.9±1.8 mV versus –81.9±1.3 mV) for atrial and ventricular cells, respectively. Note that I_K1 rectified completely for both atrial and ventricular myocytes.

View larger version (16K): [in this window] [in a new window]   Figure 2. Properties of IK1 in guinea pig atrial and ventricular cells. A, Current density-voltage (I-V) relationships of guinea pig atrial (n=6) and ventricular (n=4) IK1. I-V relationships in this as well as in subsequent figures were analyzed as barium-sensitive, ramp (–100 to 0 mV)-generated currents. B, Concentrations of Kir2.1 and Kir2.3 mRNA, normalized to cyclophilin, in the atria and the ventricle of the guinea pig. ND, not detected. *P<0.05. C, Western blot analysis in the guinea pig heart.

We developed an RNase Protection Assay (RPA) to examine Kir2.x expression patterns in the guinea pig heart. Kir2.4 was not studied because there is evidence that these channels are only expressed in neuronal cells of the heart and not in cardiac myocytes.¹⁹ Figure 2B shows the relative concentrations of Kir2.1, Kir2.2, and Kir2.3 mRNA in the atria and the ventricle of the guinea pig, normalized per unit of cyclophilin. The ventricle expressed significant mRNA levels of both Kir2.1 and Kir2.3, whereas only Kir2.1 was present in the atria. Kir2.1 mRNA expression was 5-fold greater in the ventricle compared with the atria. Also, Kir2.2 mRNA was undetectable in both the atria and the ventricle but was detected in the brain. Figure 2C is the Western blot analysis of Kir2.1 (top) and Kir2.3 (bottom) in the guinea pig heart for atrial (left lane) and ventricular (right lane) tissue samples. The data are representative of analysis performed in 3 membrane preparations of guinea pig hearts. The blots show that Kir2.1 and Kir2.3 proteins are expressed in the guinea pig ventricles and that the atria express Kir2.1 but not Kir2.3.

Sheep Atrial and Ventricular I_K1
Given that there are important species-dependent differences in ion channel expression,²⁰ we studied cardiac I_K1 properties in the sheep, a commonly used animal model to study cardiac fibrillation in this and other laboratories. Properties of freshly isolated sheep cardiac myocytes are shown in the online data supplement. Figure 3A illustrates the I-V relationships for atrial (n=7) and ventricular (n=4) I_K1. I_p was –1.7±0.24 pA/pF in atrial cells and was significantly greater (–5.27±2.07 pA/pF; P<0.05) in ventricular cells. Note that at depolarized potentials, ventricular I_K1 rectified completely, whereas atrial I_K1 did not. Furthermore, peak outward I_K1 in the atria was measured at –41±3.5 mV compared with –68±0.32 mV in the ventricle. Values for E_rev in atrial (–82.3±1.5 mV) and ventricular (–87.3±1.4 mV) cells were not significantly different. Figure 3B illustrates atrial and ventricular I_K1 after normalization to I_p. The I-V relationships of sheep atrial and ventricular I_K1 are very similar to heterologously expressed Kir2.3 and Kir2.1 channels, respectively (Figure 1B).

View larger version (36K): [in this window] [in a new window]   Figure 3. Properties of IK1 in sheep atrial and ventricular cells. A, I-V relationships of atrial (n=7) and ventricular (n=4) IK1 B, Atrial and ventricular IK1 normalized (norm) to peak inward current at –100 mV. C, Concentrations of Kir2.1, Kir2.2, and Kir2.3 mRNA in the atria and the ventricle. mRNA amounts have been normalized to cyclophilin concentrations in each lane. ND, not detected. D, Western blot analysis.

These data were correlated with Kir2.x mRNA and protein analysis. Figure 3C shows relative Kir2.x mRNA levels in the sheep atria and ventricles. The sheep ventricle expressed significant amounts of Kir2.1 and Kir2.3 mRNA. Furthermore, similar levels of Kir2.3 mRNA were expressed in the sheep atria and ventricle, whereas atrial Kir2.1 mRNA expression was only 10% of that measured in the ventricle. Kir2.2 mRNA was not detected in the sheep heart but was detected in the brain. Figure 3D is the Western blot analysis of Kir2.1 (top) and Kir2.3 (bottom) in the sheep heart performed using atrial (left lane) or ventricular (right lane) tissue samples. The data are representative of analysis performed in 3 hearts. Our results show that whereas the Kir2.1 and Kir2.3 proteins are expressed in the sheep ventricles, Kir2.3 is the predominant Kir2.x isoform expressed in the sheep atria.

Single-Channel Analyses
Unlike studies in the guinea pig,¹⁹ unitary conductance properties of Kir2.x channels have not been correlated to I_K1 channels expressed in the sheep myocardium. The trace in Figure 4A (top) is a cell-attached recording in a cell transfected with sheep Kir2.3 and the corresponding all-points histogram (top), which shows a single transition level from baseline. The bottom in Figure 4A is an events histogram showing a mean unitary conductance of 13.1±0.1 pS from a total of 232 transitions obtained from 15 patches. Figure 4B (top) is a cell-attached recording from an isolated sheep atrial cell and the corresponding all-points histogram. Note the presence of a distinct peak in the events histogram (Figure 4B, bottom). The mean unitary conductance was 9.9±0.1 pS, obtained from a total of 336 events in 32 patches in cells isolated from 5 sheep hearts. The data in Figure 4C were obtained from cell-attached recordings in sheep left ventricular myocytes. The all-points histogram (top) was obtained from the first 2.5 seconds of the trace shown. In contrast to the data in Figure 4A and 4B, the trace and the histogram in Figure 4C show multilevel transitions (n=270) that represent multiple conductance levels in 23 cell-attached patches from ventricular cells isolated from 5 sheep hearts.

View larger version (24K): [in this window] [in a new window]   Figure 4. Single-channel analysis of sheep Kir2.3 channels and sheep atrial and ventricular IK1, in cell-attached patches. The [K+] in the pipette and the bath solutions was 140 mmol/L, and the transmembrane potential was –120 mV for all of the recordings. In all panels, calibration bars are 1 pA and 500 ms. A, Top, Trace of a representative recording of Kir2.3 channel, and the corresponding all-points histogram. Bottom, Events histogram of averaged unitary conductance data of Kir2.3 channels. B, Top, Trace of unitary IK1 recording from an atrial myocyte and the corresponding all-points histogram. Bottom, Events histogram of averaged unitary conductance data of atrial IK1. C, Top, Representative trace of unitary IK1 events in a ventricular myocyte and the corresponding amplitude histogram. Bottom, Events histogram of averaged unitary conductance data of ventricular IK1.

Outward Current Profiles and [K⁺]_o
The regulation of potassium channels by [K⁺]_o has important physiological implications.³ Therefore, we were interested in determining how the outward current profiles of Kir2.x channels in high [K⁺]_o correlate with changes in I_K1 under similar conditions.

Kir2.x Channels
We characterized the whole-cell [K⁺]_o dependence of outward Kir2.1 and Kir2.3 currents. Figure 5A shows I-V relationships of Kir2.1 channels (n=5) recorded in normal [K⁺]_o (5.4 mmol/L [K⁺]_o) and elevated [K⁺]_o (10 mmol/L [K⁺]_o). Currents were normalized to peak inward current recorded in normal [K⁺]_o. Increasing [K⁺]_o from 5.4 to 10 mmol/L resulted in a Nernstian shift in the E_rev from –82.9±1.6 mV to –69.4±1.1 mV (P<0.05), a 3.2-fold increase in I_p and a 2-fold increase in the peak outward current through Kir2.1 channels. To compare the magnitude of outward current changes, we used the integration voltage, or the area under the curve (AUC) to describe outward currents from E_rev to 0 mV. Elevation of [K⁺]_o resulted in an increase in AUC from 6.15 to 13.9 U of normalized current·mV. Figure 5B shows I-V relationships of Kir2.3 channels recorded in normal and high [K⁺]_o (n=4). Similar to Kir2.1 channels, high [K⁺]_o resulted in a right shift of E_rev from –79±2.8 mV to –61.7±5.4 mV (P<0.05) as well as a 2.8-fold increase in inward currents. Importantly, however, there was no increase in the magnitude of outward currents for Kir2.3 channels in elevated [K⁺]_o.

View larger version (12K): [in this window] [in a new window]   Figure 5. [K+]o dependence of Kir2.1 and Kir2.3 channels expressed in HEK293 cells. A, I-V relationships of Kir2.1 channels (n=5) recorded in 5.4 mmol/L [K+]o and in 10 mmol/L [K+]o. Data were normalized (norm) to peak inward current recorded in 5.4 mmol/L [K+]o. B, I-V relationships of sheep Kir2.3 channels (n=4) recorded in 5.4 mmol/L [K+]o and in 10 mmol/L [K+]o. Data were normalized to peak inward current recorded in 5.4 mmol/L [K+]o.

Sheep Cardiac Myocytes
Figure 6A shows the I-V relationships of sheep ventricular I_K1 in normal [K⁺]_o (data from figure 4A) and from another group of cells in 10 mmol/L [K⁺]_o. Elevated [K⁺]_o resulted in a right shift of E_rev from –87.3±1.45 mV (n=4) to –69.3±1.31 mV (n=3; P<0.05). Additionally, there was an increase in I_p from –5.27±2.07 pA/pF to –35.7±1.47 pA/pF (P<0.05), as well as an increase in AUC from 67.8±11.9 pA·mV/pF to 160.6±26.4 pA·mV/pF (P<0.05), similar to the heterologously expressed Kir2.1 isoform. Figure 6B shows the corresponding analysis in sheep atrial I_K1. Elevated [K⁺]_o resulted in a right shift of E_rev from –82.8±1.47 mV (n=7) to –68.1±1.04 mV (n=4) and an increase in I_p from –1.7±0.24 pA/pF to –4.9±0.8 pA/pF. Importantly, similar to heterologously expressed Kir2.3 channels in high [K⁺]_o, there was no increase in peak outward current for sheep atrial I_K1.

View larger version (26K): [in this window] [in a new window]   Figure 6. [K+]o dependence of atrial and ventricular IK1 in the sheep and the guinea pig. A, I-V relationships of sheep ventricular IK1 recorded in 5.4 mmol/L [K+]o (n=4) and in 10 mmol/L [K+]o (n=3). B, I-V relationships of sheep atrial IK1 recorded in 5.4 mmol/L [K+]o (n=7) and in 10 mmol/L [K+]o (n=4). C, I-V relationships of guinea pig (GP) ventricular IK1 recorded in 5.4 mmol/L [K+]o (n=4) and in 10 mmol/L [K+]o (n=4). D, I-V relationships of GP atrial IK1 recorded in 5.4 mmol/L [K+]o (n=6) and in 10 mmol/L [K+]o (n=5).

Guinea Pig Cardiac Myocytes
Figure 6C illustrates the I-V relationships of ventricular I_K1 in normal [K⁺]_o (data from Figure 2A) and in elevated (10 mmol/L) [K⁺]_o conditions. Elevation of [K⁺]_o resulted in a right shift of E_rev from –81.9±1.28 mV (n=4) to –68.7±0.26 mV (n=4; P<0.05), an increase in I_p from –9.18±1.26 pA/pF to –18.2±1.37 pA/pF (P<0.05), and an increase in AUC from 108.4±19.7 pA·mV/pF to 233.7±21.6 pA·mV/pF (P<0.05). Figure 6D is the I-V relationship of atrial I_K1 in normal [K⁺]_o (data from Figure 2A) and in high [K⁺]_o. I_p in atrial cells increased from –4.76±0.53 pA/pF (n=6) to –18.9±2.2 pA/pF (n=5; P<0.05), and E_rev shifted from –82.9±1.79 mV to –69.4±1.57 mV (P<0.05) as [K⁺]_o was elevated. The AUC was significantly greater for guinea pig atrial I_K1 (34.7±10.7 pA·mV/pF versus 93.8±21.5 pA·mV/pF; P<0.05) as [K⁺]_o was increased.

Rectification and [K⁺]_o Sensitivity of Heteromeric Kir2.x Channels
Heteromeric Kir2.x channels were studied by examining the properties of coexpressed Kir2.1 and Kir2.3 and concatenated Kir2.1-Kir2.3 subunits.¹² Figure 7A shows the rectification profile and [K⁺]_o dependence of coexpressed Kir2.1-Kir2.3 channels. Barium-sensitive I-V relationships (data not illustrated) showed virtually complete rectification from –30 to 0 mV. Relative currents from coexpressed Kir2.1-Kir2.3 subunits were fit with the Boltzmann equation and z=2.34±0.12 (n=3), similar to Kir2.1 (2.5±0.07; n=6). Figure 7A also compares I-V relationships of coexpressed Kir2.1 and Kir2.3 subunits (n=3) in 5.4 mmol/L [K⁺]_o and 10 mmol/L [K⁺]_o. Increasing [K⁺]_o resulted in a shift in E_rev from –83.6±0.9 mV to –70.6±1.1 mV, a 3-fold increase in I_p and a 42% increase in peak outward current. An elevation of [K⁺]_o resulted in an increase in AUC from 11.2 to 14.1 U of normalized current·mV.

View larger version (14K): [in this window] [in a new window]   Figure 7. [K+]o dependence of Kir2.1-Kir2.3 heteromeric channels expressed in HEK293 cells. A, I-V relationships of coexpressed Kir2.1 and Kir2.3 subunits in the pIRES vector (n=3) recorded in 5.4 mmol/L [K+]o and in 10 mmol/L [K+]o. Data were normalized to peak inward current recorded in 5.4 mmol/L [K+]o. B, Barium-sensitive current density-voltage relationships of Kir2.1-Kir2.3 concatemers recorded in 5.4 mmol/L [K+]o (n=4) and in 10 mmol/L [K+]o (n=3).

Figure 7B shows current density-voltage relationships of barium-sensitive currents through Kir2.1-Kir2.3 concatemers in 5.4 mmol/L [K⁺]_o (n=4) and another group of cells in 10 mmol/L [K⁺]_o (n=3). The covalently linked Kir2.1-Kir2.3 constructs displayed a prominent negative slope conductance and passed virtually no outward current at depolarized potentials. Relative currents from Kir2.1-Kir2.3 concatamers were fit with the Boltzmann equation and z=3.34±0.37. Elevating [K⁺]_o resulted in a shift in E_rev from –85.2±1.3 mV to –74.2±0.3 mV, a 4-fold increase in I_p and a 2.6-fold increase in peak outward current. Elevating [K⁺]_o resulted in an increase in the AUC from 76.7±12.2 pA·mV/pF to 207±37 pA·mV/pF (P<0.05). These data suggest that the [K⁺]_o dependence of outward currents through heteromeric Kir2.1-Kir2.3 channels is determined by Kir2.1 and not Kir2.3 subunits.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
A major finding of this study is that outward currents in individual Kir2.x channel isoforms display distinct whole-cell rectification profiles and are modulated differently by elevated [K⁺]_o. Our results demonstrate significant differences in regional Kir2.x expression patterns in the sheep and guinea pig. Species- and tissue-dependent differences in these I_K1 properties are determined primarily by the specific Kir2.x isoforms expressed in the tissue. These data provide novel insight into the properties of I_K1 channels, which shape the cardiac AP and play a role in various pathophysiological states.^3,11,21

Outward Currents of Kir2.x Channels
Although the biophysical properties of Kir2.x channels^19,22–24 have been extensively examined, differences in outward currents within this subfamily have not been studied comparatively. Our data show that Kir2.1 and Kir2.2 channels displayed a prominent negative slope conductance and rectified completely at 40 mV positive to the E_rev. In contrast, Kir2.3 channels did not rectify completely at these depolarized potentials. The relatively weaker rectification of the Kir2.3 isoform has been observed previously but not discussed.^14,24 Interestingly, heteromeric Kir2.1-Kir2.3 channels rectified completely at depolarized potentials, suggesting that Kir2.1 rectification properties are dominant in a Kir2.1-Kir2.3 heteromeric complex. Recent evidence suggests that Kir2.x channels have differences in spermine sensitivity as well as in unblocking kinetics,²⁵ which may contribute to their different rectification profiles.

I_K1 Properties in Atrial Cells
Similar to previous studies, our data show that atrial I_K1 has lower current density than ventricular I_K1.^1,4–6 Consistent with this, our RPA results in the guinea pig and sheep show lower overall expression of Kir2.x mRNA in the atria compared with the ventricle.

Our data demonstrate striking differences in the atrial I_K1 rectification profiles in these species. Our results show that Kir2.3 is the predominant Kir2.x isoform expressed in the sheep atria. Correspondingly, the rectification profile, [K⁺]_o dependence, and single-channel conductance of sheep atrial I_K1 are reminiscent of Kir2.3 channel properties. The smaller value of the single-channel conductance in native cells (9.9±0.1 pS) compared with Kir2.3 (13.1±0.1 pS) may be attributable to interactions with scaffolding proteins as described previously.^19,26 In guinea pig atrial cells, our expression studies as well as rectification and [K⁺]_o properties suggest that Kir2.1 plays a major role in determining atrial I_K1. In a previous investigation,¹ I_K1 was not isolated from the background conductance, and complete rectification of guinea pig atrial I_K1 was not evident.

We explored the functional significance of the different rectification profiles in atrial I_K1 by using a previously published mathematical model of the human atrial AP.¹⁷ The parameters in the equation for I_K1 were modified to obtain fits to the normalized I-V plots for Kir2.1 and Kir2.3, as shown in Figure 8A. Figure 8B depicts APs and the underlying I_K1 currents obtained by fits to Kir2.1 and Kir2.3 data. The AP based on Kir2.3-like I_K1 characteristics displays a shorter AP duration compared with the corresponding Kir2.1 AP. Our results show that I_K1 with Kir2.1-like properties is important only during the terminal phase of repolarization. In contrast, I_K1 with Kir2.3-like characteristics contributes to a repolarizing current during the plateau phase of the AP, in addition to the terminal phase of repolarization. This suggests that differences in the I_K1 rectification profile may be important for determining the relative role of I_K1 in cardiac repolarization (see also Nichols et al²⁷).

View larger version (11K): [in this window] [in a new window]   Figure 8. Simulation of atrial AP (see online text for model details). A, The equation for IK1 was modified to obtain fits to the normalized I-V relationships of Kir2.1 and Kir2.3 shown in Figure 1B. B, Top, Steady-state APs obtained by running the model for 13 seconds at 1 Hz. Bottom, Underlying Kir2.x currents using Kir2.1 and Kir2.3 I-V relationships.

I_K1 Properties in Ventricular Cells
In both sheep and guinea pig ventricles, I_K1 has a prominent negative slope conductance, which is consistent with relatively high levels of Kir2.1 subunits expressed in these tissues. The ventricles also express significant levels of Kir2.3 mRNA and protein. Although regulated by many factors,^24,28,29 the role of Kir2.3 subunits in this tissue is not clear because the rectification and [K⁺]_o properties of this isoform are not apparent in the ventricles. Our data suggest that the properties of Kir2.3 subunits are not evident in the ventricles because in a heteromeric complex, Kir2.1 properties are dominant. Although in our study sheep and guinea pig ventricular I_K1 displayed similar rectification characteristics, previous work has shown that there are species-specific differences in the rectification properties of ventricular I_K1 in other species.³⁰ Our single-channel data of the sheep ventricles show that there are wide distributions of conductances, which may correspond to homomeric Kir2.1 and Kir2.3 channels as well as heteromeric channels. Clearly, further work is required to understand the regulation of heteromeric Kir2.x channels and their role in native I_K1.

The results of our RPA do not show any measurable expression of Kir2.2 mRNA in the guinea pig atria or ventricles. In contrast, Liu et al suggested that Kir2.2 is the major guinea pig ventricular isoform underlying I_K1.¹⁹ This conclusion was primarily based on correlation of single-channel conductances and barium sensitivities of heterologously expressed Kir2.x channels with native guinea pig myocytes. Similarities in barium sensitivities of Kir2.2 and native I_K1 can be interpreted in other ways. For instance, Schram et al¹⁴ have shown that the barium sensitivity of coexpressed Kir2.1 and Kir2.3 channels is very similar to native I_K1 and to homomeric Kir2.2 channels but is very different from that of homomeric Kir2.1 and Kir2.3 channels. Commercially available Kir2.2 antibodies designed against a rat epitope are available through Alomone labs (Jerusalem, Israel). These antibodies did not detect Kir2.2 protein in sheep or guinea pig tissue or transfected cells, perhaps because the corresponding guinea pig Kir2.2 epitope only has 12 of 19 residues identical to the rat epitope. The homology of sheep Kir2.2 to the rat epitope is unknown because this gene has not yet been cloned. Nevertheless, our mRNA results clearly show that Kir2.2 is expressed in the brain but not in the heart of sheep or guinea pigs using species-specific antisense probes.

[K⁺]_o Dependence of Outward Currents in Kir2.x Channels and I_K1
[K⁺]_o is elevated during pathophysiological states such as in ischemia, tachycardia, and fibrillation.³¹ K⁺ ions can accumulate in either intercellular clefts or t-tubules of cardiac myocytes, which is particularly relevant because Kir2.x subunits are expressed in t-tubules^6,32 and the intercalated disks⁶ of cardiac myocytes. Yet, differences in the effect of high [K⁺]_o on outward currents in Kir2.x isoforms have not been described comparatively. We have shown that increasing [K⁺]_o resulted in a Nernstian shift of E_rev and an increase in inward currents for Kir2.1 and Kir2.3 channels. However, whereas Kir2.1 channels showed an increase in outward currents in high [K⁺]_o, Kir2.3 channels did not. Accordingly, sheep atria, which predominantly express Kir2.3 channels, did not exhibit an increase in the outward component of I_K1 in high [K⁺]_o. In contrast, guinea pig atrial I_K1, mediated mainly by Kir2.1, showed an increase in outward current in high [K⁺]_o. Curiously, guinea pig atrial I_K1 showed a more prominent secondary hump at depolarized potentials in high [K⁺]_o. Also, guinea pig and sheep ventricles, which express both Kir2.1 and Kir2.3 subunits, showed an increase in outward currents in high [K⁺]_o, attributable to the dominant role of Kir2.1 in a heteromeric complex.

Potential Limitations
Although our data show that differential expression of Kir2.x isoforms play an important role in determining the rectification properties of native I_K1, other factors, such as differences in levels of polyamines in different tissues and species, may also be important in modulating rectification properties. The single-channel properties (ie, unitary conductance and open probability) of heteromeric Kir2.x channels are unknown, therefore, it is difficult to correlate unitary conductance values of heterologously expressed homomeric Kir2.x isoforms with I_K1 unitary events in the ventricle, which are presumably determined by heteromeric Kir2.x complexes.

In conclusion, our studies show that heterologously expressed Kir2.x channels display important differences in their whole-cell outward current profiles, as well as the [K⁺]_o dependence of their outward currents. The results also show that Kir2.1 rectification properties and [K⁺]_o sensitivity are dominant in a heteromeric Kir2.x complex. Tissue and species-specific expression of these isoforms determine the biophysical and regulatory properties of I_K1 in the heart.

	Acknowledgments

 
This work was supported by grants PO1 HL39707 and HL60843 from the National Heart, Lung, and Blood Institute and a predoctoral fellowship from the American Heart Association (to A.D.). We would like to thank Drs José Jalife and Mario Delmar for critically reading this manuscript, Jiang Jiang for his expert technical assistance, and Dr Eduardo Solessio for his insight. Also, we would like to thank Regina Preisig-Muller for the Kir2.1-Kir2.3 concatemers.

	Footnotes

 
Original received May 17, 2002; first resubmission received March 31, 2003; second resubmission received January 15, 2004; revised resubmission received April 1, 2004; accepted April 7, 2004.

	References

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