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R. T. Mathias, I. S. Cohen, J. Gao, and Y. Wang are in the Department of Physiology and Biophysics and Institute of Molecular Cardiology, State University of New York at Stony Brook, Stony Brook, New York 11794-8661.
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Abstract |
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Introduction |
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 Top Introduction Isoforms of the Na+-K+...Physical factors affecting the...Autonomic regulation of the...SummaryReferences |
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The past decade has provided significant advances in our knowledge of the regulation of the Na+-K+ pump but little understanding of its physiological purpose. Figure 1
illustrates the stoichiometry of the pump and some possible targets of regulatory changes in pump activity in the heart. In general, regulatory changes in Na+-K+ pump current (IP) are one part of an integrated, multifaceted control system that adjusts cardiac output to suit the needs of the organism. For example, activation of the sympathetic nervous system causes increases in the levels of circulating epinephrine and release of norepinephrine (NE) in the vicinity of heart cells. This results in activation of 
- and ß-adrenergic receptors that are coupled to changes in Na+, K+, Cl–, and Ca2+ currents that affect the action potential, changes in pacemaker activity that increase heart rate, changes in excitation/contraction coupling that increase cardiac output, and changes in Na+-K+ pump current. As suggested in Fig. 1, regulatory changes in IP may set intracellular Na+ concentration ([Na+]i) at some desired level, which through the Na+/Ca2+ exchanger could participate in regulation of intracellular Ca2+ concentration ([Ca2+]i). Indeed, regulation of IP is not only isoform specific, it is also [Ca2+]i dependent, suggesting a close relationship. Outside of the cell, IP directly affects extracellular K+ concentration ([K+]o), particularly in small restricted volumes such as the t-system lumen or between tightly packed cells. Lastly, the 3Na+-for-2K+ stoichiometry means that the Na+-K+ pumps generate a net outward current, IP, which is a significant component of total net current during the cardiac action potential plateau phase. Hence regulatory changes in IP may directly affect the duration of the plateau phase, or indeed any electrical activity in the heart that is associated with small net current (e.g., pacemaking). With these possible functions in mind, we will review isoform-specific regulation of IP and point out cases in which the regulatory response of each isoform may or may not contribute to a particular function.
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Isoforms of the Na+-K+ pump |
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TopIntroductionIsoforms of the Na+-K+...Physical factors affecting the...Autonomic regulation of the...SummaryReferences |
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- and ß-subunit, which may dimerize with another -ß complex. The -subunit binds Na+ and K+, hydrolyzes ATP, and binds ouabain, which blocks ATPase activity. Thus the functional properties of the Na+-K+ pump are generally thought to reside in the -subunit. However, there are multiple ß-isoforms that may have specific functional roles that are not yet understood. There are at least three -isoforms (14), and in most species the 1-isoform has a much (100-fold) lower affinity for ouabain than the 2- or 3-isoform. The 1-isoform is most widely expressed, whereas the 2- or 3-isoform tends to be associated with excitable cells, though there are many exceptions. In heart, all three isoforms are expressed (14), and guinea pig ventricular myocytes coexpress the 1- and 2-isoforms (9).
The first evidence for multiple isoforms in guinea pig myocytes came from data on IP blockade as a function of dihydroouabain (DHO) concentration (10). The blockade data from Ref. 5 are shown in Fig. 2A. There are clearly two inhibitory sites with roughly a 100-fold difference in affinity. Gao et al. (5) reported that the two dissociation constants were 0.75 µM and 72 µM. This difference in affinity immediately suggested that the low DHO affinity pumps were the 1-isoform and the high DHO affinity pumps were either the 2- or 3-isoform. Gao et al. (9) used RNase protection assays and found mRNA for the 1- and 2-isoforms. Thus, in guinea pig ventricular myocytes, total IP is the sum of current contributed by the 1-isoform (IP1) plus that by the 2-isoform (IP2): IP = IP1 + IP2.
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. A glass pipette is pressed against the membrane of an acutely isolated guinea pig ventricular myocyte. Suction is applied to the pipette, causing it to seal to the membrane, and with further suction, the patch of membrane beneath the tip ruptures. Within ~5 min, the solution in the pipette diffuses into the cell, exchanging with the cell's contents and allowing a reasonable degree of control of intracellular Na+, K+, Ca2+, and ATP, the ions that are known to interact with the Na+-K+ pumps. A command voltage (VC) is applied to the cell, and the holding current (Ih) necessary to maintain VC constant is recorded. DHO, a specific inhibitor of the Na+-K+ pump, is superfused in the bathing solution. The change in IP elicited by application of DHO (
IP) measures the change in Na+-K+-ATPase activity. The vertical dashed lines in Fig. 2A are at 5 µM DHO, which blocks essentially all transport by the 2-isoform with little effect on the 1-isoform, and 1 mM DHO, which blocks essentially all transport by both the 1- and 2-isoforms. Thus 5 µM DHO was used to determine IP2, and 1 mM DHO was used to determine IP = IP1 + IP2, then IP1 = IP – IP2. In some experiments, 5 µM DHO was continuously superfused; then when 1 mM DHO was added, the resulting IP approximately equaled IP1. |
Physical factors affecting the Na+-K+ pump |
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TopIntroductionIsoforms of the Na+-K+...Physical factors affecting the...Autonomic regulation of the...SummaryReferences |
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summarizes the effects of the physical environment on the 1- and 2-isoforms in guinea pig ventricular myocytes. The graphs show the normalized activation curves, whereas the numbers below represent half-maximal activation for each isoform (5). Both isoforms are half-maximally activated by [Na+]i of 9 mM, which is very close to normal physiological [Na+]i. Hence each isoform is poised to respond to any change in [Na+]i and provide negative feedback. In contrast, at normal physiological [K+]o of ~4 mM, the [K+]o dependence of IP2 is near saturation, whereas that of IP1 is half-maximally activated. Thus the 1-isoform provides negative feedback regulation of [K+]o, but the 2-isoform does not. Small changes in extracellular H+ concentration (pHo) occur with normal changes in cardiac activity, but the major stimulus for changes in pHo is ischemia. IP1 is specifically inhibited by a drop in pHo, whereas IP2 is completely insensitive to pHo over the range of 6–8. The purpose of this isoform-specific response is difficult to understand. With extracellular accumulation of H+, the H+ flux into cells will increase, and it should be advantageous to increase the transport of H+ out of the cell. Instead, a drop in pHo reduces IP1 and thus increases [Na+]i, which in turn reduces H+ extrusion by Na+/H+ exchange. Evidently, there is a more complex, indirect effect that may be part of an overall cellular protective response to ischemia.
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shows that both isoforms depend on membrane voltage (Vm). Gadsby and Nakao (1) first measured the voltage dependence of total pump current (IP – Vm) in guinea pig myocytes. Rakowski et al. (12) showed that the voltage dependence arises because Na+ is released to the extracellular space in a long narrow channel within the pump protein. In the Na+ release state of the pump cycle, the intracellular end of this channel is closed, so when the Na+ comes off of its binding site, it can only move along the channel toward the extracellular space. However, the closed end of the channel is essentially at the same voltage as the cell. Thus the more negative the intracellular voltage, the longer the time that Na+ spends in the channel, where it can rebind and inhibit the forward cycle of the pump. Shi (13) separately measured the Vm dependence of IP1 and IP2 and found that they were similar but not identical. Although IP1 – Vm and IP2 – Vm are normally similar, autonomic input can shift IP1 – Vm (see below), so voltage dependence is a physical factor that can be altered by autonomic input. Since outward current increases as transmembrane voltage depolarizes, this is a form of negative feedback that tends to directly hyperpolarize the resting voltage while increasing the Na+ and K+ gradients that determine the resting voltage. With regard to the action potential, IP has many similarities to most K+ currents: it is outward, increases on depolarization, and tends to cause hyperpolarization, which in turn causes it to decrease. However, on the time scale of a cardiac action potential, there is no significant delay in voltage-mediated changes in IP. Of these physical factors, [Na+]i is the most pervasive effector of IP. Indeed, at steady state, Na+ efflux by the Na+-K+ pump must equal Na+ influx through all other channels and transporters. Consequently, regulatory changes in IP that do not alter influx will not alter steady-state IP; instead, [Na+]i will change until efflux equals influx. Thus autonomic input that appears to regulate IP can only cause transient changes unless it also modifies Na+ influx. One must therefore consider the entire spectrum of regulatory effects on electrical activity. If IP is uniquely affected, then [Na+]i, or perhaps [Ca2+]i, is actually the target. However, if Na+ influx is being altered, for example by a change in heart rate, at the same time that IP is changed, then the change in IP may persist at steady state, and in this circumstance it could affect some of the other targets suggested in Fig. 1.
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Autonomic regulation of the Na+-K+ pump |
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TopIntroductionIsoforms of the Na+-K+...Physical factors affecting the...Autonomic regulation of the...SummaryReferences |
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summarizes the effects of sympathetic or parasympathetic activation on the 1- and 2-isoforms of the Na+-K+ pumps in guinea pig ventricular myocytes.
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1-isoform through PKA-mediated phosphorylation; however, following PKA activation, the steps that lead to the changes in IP1 are unknown. Moreover, there are two very different responses depending on [Ca2+]i (4). At 15 nM [Ca2+]i, maximal ß-activation causes a 32% inhibition of IP1 at all physiologically relevant voltages, whereas at 1.4 µM [Ca2+]i, it causes a –37 mV voltage shift with no inhibition (7). At a [Ca2+]i of ~135 nM, half of the 1-isoforms are inhibited and half are voltage shifted. At [Ca2+]i > 1 µM, the response is essentially all voltage shift, whereas at <50 nM it is essentially all inhibition. At intermediate values of [Ca2+]i, the IP1 – Vm curves in the presence and absence of ß-activation cross, so ß-activation increases IP1 at the resting voltage but decreases it at the action potential plateau voltage. In general, these responses will, in connection with Na+/Ca2+ exchange, tend to increase steady-state [Ca2+]i when it is low and decrease it when it is high. It is interesting to note, however, that the high [Ca2+]i response of IP1 results in no change in current during the AP plateau (~0 mV); hence this response is designed to not affect plateau duration.
NE also binds to 1-adrenergic receptors (1-R), which are coupled by a Gq protein to activation of phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-diphosphate (PIP2) lipids to produce membrane-bound diacylglycerol (DAG) and cytoplasmic inositol 1,4,5-trisphosphate (IP3). IP3 causes Ca2+ release from intracellular stores, and the resulting increase in [Ca2+]i stabilizes the interaction of phosphokinase C (PKC) with the plasma membrane, where it is activated by DAG. All -effects on the Na+-K+ pump are on the 2-isoform through PKC-mediated phosphorylation (8, 15); however, as was the case with PKA, following PKC activation, the steps leading to an increase in IP2 are unknown. Because activation of PKC is [Ca2+]i dependent, so is the 1-R-mediated increase in IP2. At any given [Ca2+]i, a sufficiently high concentration of NE causes a 42% increase in IP2 at all physiologically relevant voltages. However, at a given concentration of NE, for example 10 nM, there is no effect on IP2 if [Ca2+]i is 15 nM, whereas there is a 30% increase in IP2 if [Ca2+]i is 1.4 µM (15).
Inhibition of PKC with staurosporine abolishes all 1-R effects on IP2 (15), but the nonspecific kinase inhibitor H7 has no effect on IP2 in the absence of 1-R activation, suggesting that basal activity of PKC is low (8). Conversely, when PKC is directly activated with the phorbol ester phorbol 12-myristate 13-acetate (PMA), the maximal 88% increase in IP2 is twice as large as that caused by 1-R activation (8). Moreover, in the presence of maximal 1-R activation, addition of PMA causes a further increase in IP2 that is not additive; rather, the maximal IP2 is still 88% greater than basal IP2. This suggests that the 1-receptors are coupled to a specific pool of PKC that is a subset of total PKC, which is activated by PMA. The limited coupling may be through colocalization in a membrane domain such as caveoli. It also suggests the existence of a second pool of PKC that is also coupled to IP2, but we do not know the physiological effectors that regulate this pool.
Activation of the parasympathetic nervous system causes the release of acetylcholine (ACh) in the vicinity of cardiac myocytes. ACh binds to the muscarinic ACh receptor (ACh-R) to generally reduce heart rate and cardiac output. The ACh-R is coupled through a Gi protein to inhibit adenylyl cyclase activity, reduce cAMP, and thus inhibit PKA. The effect on the Na+-K+ pumps is to reverse the ß-R-mediated effects on IP1 (6). In the absence of ß-R activation, ACh has no effect on IP1, suggesting that basal PKA activity is low. Also, direct inhibition of PKA with the synthetic peptide PKI has no effect on IP1 in the absence of ß-R activation but prevents all ß-effects (2, 3), consistent with low basal PKA activity. We do not know if there are direct effects of ACh-R activation on the PKC-mediated increase in IP2. However, since PKC activation depends on [Ca2+]i, there are almost surely indirect effects through a reduction in Ca2+ influx, a reduction in [Ca2+]i, and at least partial reversal of PKC activation.
In an organism, activation of the sympathetic or parasympathetic nervous system will have effects on cardiac myocytes that alter some of the physical factors regulating IP (see Fig. 3). Thus there can be indirect effects through, for example, a change in Na+ current causing a change in [Na+]i or a change in K+ current causing a change in [K+]o. The whole cell patch-clamp technique provides control of these physical factors; however, it is imperfect (5). We therefore varied [K+]o and [Na+]i and showed that the effects of PKA
IP1 and PKCIP2 were not indirect and that they were not through a change in the affinity of the isoform for either intracellular Na+ or extracellular K+. Thus, although the mechanisms coupling PKAIP1 and PKCIP2 are not known, they appear to be through signal transduction cascades that directly target each isoform. The kinases could either directly phosphorylate the pump proteins and alter ATPase activity of either the 1- or 2-isoform or they could phosphorylate regulatory proteins to initiate a cascade of events that are targeted to alter either IP1 or IP2.
The PKA-mediated, high-[Ca2+]i voltage shift in IP1 – Vm is particularly interesting. The voltage-independent effects of either PKA or PKC can be either a change in ATPase activity with a fixed number of pump proteins or a change in the number of pump proteins with a fixed ATPase activity, or perhaps both. Neither mechanism, however, can explain a voltage shift. As described earlier, the voltage dependence of IP is due to the release of extracellular Na+ into a voltage well (12). The more negative the voltage at the bottom of the well, the longer the released Na+ stays there; hence the greater the probability of rebinding, which slows the forward cycle of the pump. Direct PKA-mediated phosphorylation of the 1-isoform could change its free energy and thus the affinity for extracellular Na+. A reduction in affinity would require a more negative voltage to produce the same probability of rebinding; thus the result would be a negative shift in IP1 – Vm. However, intracellular Ca2+ is also involved. Binding of Ca2+ near the intracellular end of the well could cause the local voltage at the bottom of the well to become more positive, and this would also result in a negative shift in IP1 – Vm. At this stage, we can only hypothesize possible mechanisms. Elucidation of these final steps, however, may be very important to our understanding of regulation, not only in the heart, but also to our understanding the diverse effects of PKA and PKC on IP in different tissues.
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Summary |
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TopIntroductionIsoforms of the Na+-K+...Physical factors affecting the...Autonomic regulation of the...SummaryReferences |
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The ability to have specific responses is undoubtedly critical to cell function. For example, sympathetic activation increases NE, but the response of the heart to NE should not be the same as the response of the kidney. Every step in the signal transduction cascades linking NE to changes in Na+-K+ pump activity provides a means of uniquely determining the response. There are several different receptors with multiple isoforms. Each receptor is coupled to a specific G protein that can be either stimulatory or inhibitory to PKA or PKC. If the targets of PKA or PKC phosphorylation are regulatory proteins, then different cell types can express different proteins. But even if the targets are the Na+-K+ pumps, each cell type can express a different combination of isoforms and thus possess a unique response. Physiological regulation of the Na+-K+ pump is obviously complex and not well understood. One must start with the isoform of the pump and work back through the signal transduction cascade, identifying each isoform of each protein involved. When this information is ultimately available, some generalizations will likely become possible. But at this stage of our understanding, the appropriate generalization is that we should not generalize regulatory responses recorded in one type of cell to other types of cells.
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Acknowledgments |
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References |
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TopIntroductionIsoforms of the Na+-K+...Physical factors affecting the...Autonomic regulation of the...SummaryReferences |
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-and ß-adrenergic agonists in the guinea-pig ventricle. J Physiol (Lond) 516: 377–383, 1999.-Adrenergic effects on Na+-K+ pump current in guinea pig ventricular myocytes. J Physiol (Lond) 509: 117–128, 1998.This article has been cited by other articles:
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K Kjeldsen, A Norgaard, and M Gheorghiade Myocardial Na,K-ATPase: the molecular basis for the hemodynamic effect of digoxin therapy in congestive heart failure Cardiovasc Res, September 1, 2002; 55(4): 710 - 713. [Abstract] [Full Text] [PDF]
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