Three broad classes of ATPases are involved in active ion transport

The three classes of ion transport ATPases are the F- , V- , and P-type ATPases . Mitochondria possess F-type (F ₁ F ₀ ) ATPases that use energy stored in the proton electrochemical gradient across the inner mitochondrial membrane to synthesize ATP; the proton gradient is generated by oxidative metabolism. Vacuolar (V-type) H ⁺ -ATPases lower intraorganellar pH by concentrating protons in a variety of vesicular organelles, including lysosomes and secretory and storage vesicles. Neither the F-type nor the V-type ATPases form stable phosphorylated intermediates. In contrast, P-type ATPases do form stable phosphorylated intermediates in which the terminal phosphate of ATP has been transferred to an aspartyl residue on the ATPase (see page 118). The P-type ATPases selectively transport various, ions and other solutes into and out of cells and organelles. Examples of P-type ATPases are the plasma membrane (PM) Na ⁺ pump (Na ⁺ ,K ⁺ -ATPase), the PM and sarcoplasmic reticulum/endoplasmic reticulum (S/ER) Ca ²⁺ -ATPases (PMCA and SERCA, respectively), and the gastric mucosa proton pump (H ⁺ ,K ⁺ -ATPase). These P-type ATPases are the focus of much of this chapter.

Nearly all animal cells normally maintain a high intracellular K ⁺ concentration and a low intracellular Na ⁺ concentration

In most cells in mammals, including humans, [K ⁺ ] _i ≈ 120–130 mM, and [Na ⁺ ] _i ≈ 5–15 mM. The extracellular fluid, however, has a high [Na ⁺ ] _o (∼145 mM) and a low [K ⁺ ] _o (∼4–5 mM). Moreover, cells are not impermeable to Na ⁺ and K: Na ⁺ and K ⁺ channels and Na ⁺ gradient–dependent transport systems ( Chapters 7 and 10 ) permit Na ⁺ to enter cells and K ⁺ to exit as the ions move down their respective electrochemical gradients. Therefore all cells expend energy in the form of ATP to generate and maintain their normal Na ⁺ and K ⁺ electrochemical gradients. The transporter that accomplishes this work is the sodium pump or Na ⁺ ,K ⁺ -ATPase . In the nervous system and the kidneys, the Na ⁺ pump accounts for about 75%–85% of total ATP hydrolysis. The transport of Na ⁺ and K ⁺ by the Na ⁺ pump compensates for the leak of these ions into and out of the cell, respectively. This is known as the pump-leak mechanism of Na ⁺ and K ⁺ homeostasis. The Na ⁺ pump not only maintains constant [Na ⁺ ] _i and [K ⁺ ] _i , but also maintains cell volume. How it does this is addressed in the next section.

The Na ⁺ pump hydrolyzes ATP while transporting Na ⁺ out of the cell and K ⁺ into the cell

The Na ⁺ pump is an integral PM protein whose major (α, or catalytic) subunit has 10 membrane-spanning helices ( Fig. 11.1 ) and contains the ATP and ion-binding sites. The α-subunit is closely associated with a smaller, highly glycosylated, β-subunit that has a single membrane-spanning domain. Complexes of α- and β-subunits, in a 1:1 ratio, are required for Na ⁺ pump activity, but how the β-subunit functions is unknown. The Na ⁺ pump is frequently called the Na ⁺ ,K ⁺ -ATPase because the protein is an enzyme (specifically, an ATPase) that requires both Na ⁺ and K ⁺ for its catalytic activity (ATP hydrolysis). ^a

a The Na ⁺ ,K ⁺ -ATPase was identified in 1957 by Jens Skou. He was awarded the Nobel Prize for this work in 1997.

Schematic Model of the α-(Catalytic) Subunit of the Na ⁺ Pump.

This subunit consists of 10 membrane-spanning helical domains (cylinders in the figure). The large cytoplasmic loop between transmembrane helices 4 and 5 contains the ATP-binding domain (shown) and the aspartate phosphorylation site. The ion-binding sites are located in transmembrane helices 4, 5, 6, and 8. Residues that bind ouabain are located on the external surfaces of helices 1, 2, 5, 6, and 7; thus, bound ouabain may block access to the cation-binding sites.

(Modified from Lingrel, J.B., Croyle, M.L., Woo, A.L., & Argüello, J.M. (1998). Ligand binding sites of Na, K-ATPase. Acta Physiologica Scandinavica: Supplementum, 643 , 69–77.)

The Na ⁺ pump hydrolyzes one ATP molecule to ADP and inorganic phosphate (P _i ) while transporting three Na ⁺ ions out of the cell and two K ⁺ ions into the cell. The transport cycle begins with the binding of ATP (as a Mg ²⁺ -ATP complex) at the hydrolytic site on the α-subunit ( Fig. 11.1 ). When three Na ⁺ ions bind to the pump on the cytoplasmic side, the ATP is cleaved and its terminal phosphate is transferred to the α-subunit, forming a phosphorylated intermediate. This phosphorylation enables the protein to undergo a conformational change so that the bound Na ⁺ becomes transiently inaccessible (“occluded”) to both the cytoplasm and the extracellular fluid (ECF). The Na ⁺ -binding site then opens to the ECF. This conformational change also markedly reduces the Na ⁺ affinity, while greatly increasing K ⁺ affinity. Thus the three Na ⁺ ions are able to dissociate even though [Na ⁺ ] _o ≈ 145 mM. Then, when two K ⁺ ions bind, the protein undergoes another conformational change. As the α-subunit–phosphate bond is cleaved, P _i is released into the cytoplasm, and the K ⁺ -binding sites close to the ECF and become transiently occluded, and then open to the cytoplasm. The two K ⁺ ions are released into the cytoplasm because the affinity for K ⁺ decreases markedly during this conformational change. This sequence of steps in the Na ⁺ pump cycle is illustrated in Fig. 11.2 A.

(A) Sequence of steps in the Na ⁺ pump cycle illustrates the mechanism of the pump. The cycle begins with the binding of ATP to the large cytoplasmic loop ( a ) , followed by the binding of three Na ⁺ ions ( gray circles ) from the cytosol ( a ) . This enables the terminal phosphate of ATP to be transferred to the α-subunit, and the three Na ⁺ ions to be transiently occluded ( b ). The three Na ⁺ ions are then released to the extracellular fluid (ECF; c ). Two K ⁺ ions ( blue circles ) from the ECF bind ( d ) and, following cleavage of the P _i , are transiently occluded ( e ) . The cycle ends with the release of the two K ⁺ ions into the cytosol ( f ) . (B) Net reaction mediated by the Na ⁺ pump. Note that the ouabain-binding site faces the ECF.

The net reaction for the Na ⁺ pump can be written as:

and can be diagrammed as shown in Fig. 11.2 B. Note that this chemical reaction is reversible and can generate ATP if the product concentrations are greatly increased and the substrate concentrations are greatly reduced.

As a result of the 3 Na ⁺ :2 K ⁺ coupling ratio, inhibition of the Na ⁺ pump will lead to a net gain of solute (as Na ⁺ salts, to maintain electroneutrality) and a rise in osmotic pressure. The cells will therefore gain water and swell (see discussion of the Donnan effect in Chapter 4 ). Thus the Na ⁺ pump also is essential for maintaining cell volume (this is the “pump-leak mechanism”).

The Na ⁺ pump is “electrogenic”

The reaction sequence ( Equation 11.1 ) reveals that during each Na ⁺ pump cycle, one more positive charge leaves the cell than enters. This net flow of charge (i.e., outward “pump current”) across the membrane generates a small voltage (cytoplasm negative). The Na ⁺ pump is therefore said to be electrogenic. Indeed, this voltage adds to the V _m , so that the actual resting V _m is slightly more negative than the V _m calculated from the GHK equation ( Chapter 4 ). The maximum voltage that can be generated by the Na ⁺ pump with a coupling ratio of 3 Na ⁺ :2 K ⁺ , under steady-state conditions, is approximately 10 mV. In practice, however, the contribution of the electrogenic Na ⁺ pump to the resting V _m (i.e., in the steady state) in most cells is only a few millivolts (1–4 mV) and is usually ignored. When [Na ⁺ ] _i rises significantly, as in neurons after a long burst of action potentials, the rate of Na ⁺ transport by the Na ⁺ pump can increase considerably. Under these non–steady-state conditions, the Na ⁺ pump may transiently hyperpolarize the cells by 20 mV or more, thereby temporarily reducing the ability of stimuli to excite the cells.

The Na ⁺ pump is the receptor for cardiotonic steroids such as ouabain and digoxin

The Na ⁺ pump α-subunit is uniquely sensitive to a class of drugs known as cardiotonic steroids . Two examples, digoxin and ouabain , were originally discovered in plants, but ouabain also is synthesized in humans and other mammals and is increasingly recognized as a hormone ( Box 11.1 ). Cardiotonic steroids inhibit the Na ⁺ pump and, as described later, thereby induce a cardiotonic effect (increased force of contraction of the heart, or positive inotropic effect ). This is the key to cardiotonic steroid therapeutic efficacy in heart failure. In addition, ouabain binding to the Na ⁺ pump can initiate cellular signaling by activating various protein kinase cascades that modulate protein expression and phosphorylation.

There are four molecular isoforms of the Na ⁺ pump α-subunit , α ₁ to α ₄ , which differ in their affinities for Na ⁺ and K ⁺ . These isoforms have been conserved during vertebrate evolution. All cells express α ₁ and one other isoform; α ₁ is responsible for maintaining the low [Na ⁺ ] _i in “bulk” cytoplasm.

Expression of specific α-subunit isoforms is upregulated or downregulated under various physiological and pathophysiological conditions. For example, in the heart, thyroid hormone increases, and heart failure decreases α ₂ expression. In kidney distal tubules, aldosterone upregulates α ₁ , which then promotes Na ⁺ reabsorption. Also, several hormones such as dopamine, vasopressin, and serotonin (5-hydroxytryptamine [5-HT]), modulate the activity of the Na ⁺ pump in a tissue- and isoform-specific manner. These hormones activate or inactivate the pump by promoting phosphorylation of the pump at sites other than the site that is phosphorylated during ion transport. New understanding about the significance of the isoforms is beginning to emerge ( Box 11.2 and Fig. 11.3 ).

Ca ²⁺ storage in the sarcoplasmic/endoplasmic reticulum is mediated by a Ca ²⁺ -ATPase

In Chapter 10 we noted that the cytosolic free (ionized) Ca ²⁺ concentration ([Ca ²⁺ ] _i ) in most cells at rest is approximately 100 nM (10 ⁻⁷ M or 0.0001 mM). The total intracellular Ca ²⁺ concentration is generally about 1000 to 10,000 times higher than this, however, or about 0.1 to 1 mM. Thus more than 98% of the intracellular Ca ²⁺ is sequestered in intracellular organelles, although a small amount is buffered (i.e., bound to cytoplasmic proteins, such as calmodulin, and to other molecules). The primary Ca ²⁺ storage site is the ER or SR, but a small amount is also normally concentrated in mitochondria. The S/ER is a system of interconnected tubules and sacs within the cytoplasm that plays a central role in Ca ²⁺ signaling. Some elements of the S/ER lie just beneath the PM and are specialized for Ca ²⁺ signal initiation or amplification. When cells are activated (e.g., by hormones, neurotransmitters, or depolarization), Ca ²⁺ is often released from the S/ER stores. This released Ca ²⁺ can trigger such processes as contraction and secretion ( Chapters 12 and 15 ). Subsequently, the Ca ²⁺ is resequestered in the S/ER.

This Ca ²⁺ sequestration is accomplished via an S/ER Ca ²⁺ pump, SERCA. SERCA uses one ATP to transport two Ca ²⁺ ions from the cytosol to the S/ER lumen and two protons (H ⁺ ions) from the lumen to the cytosol by a transport mechanism analogous to that of the Na ⁺ pump.

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Electrical consequences of ionic gradients Ion channels Synaptic physiology I Synaptic physiology II Summary of elementary circuit theory Answers to study problems

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