ANRV300-PH69-05 ARI 15 January 2007 17:10 Transporters as Channels Louis J. DeFelice1 and Tapasree Goswami2 1Department of Pharmacology and Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, Tennessee 37232; email: [email protected] 2Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115; email: tapasree [email protected] Annu. Rev. Physiol. 2007. 69:87–112 Key Words First published online as a Review in membrane, synapse, biophysics, neuroscience Advance on October 23, 2006 The Annual Review of Physiology is online at Abstract http://physiol.annualreviews.org This review investigates some key aspects of transport mechanisms This article’s doi: and recent advances in our understanding of this ubiquitous cellu- 10.1146/annurev.physiol.69.031905.164816 lar process. The prevailing model of cotransport is the alternating Copyright c 2007 by Annual Reviews. access model, which suggests that large conformational changes in All rights reserved the transporter protein accompany cotransport. This model rests 0066-4278/07/0315-0087$20.00 on decades of research and has received substantial support because many transporter characteristics are explained using its premises. New experiments, however, have revealed the existence of channels in transporters, an idea that is in conflict with traditional models. The alternating access model is the subject of previous detailed reviews. by Virginia Commonwealth University on 04/04/11. For personal use only. Here we concentrate on the relatively recent data that document pri- Annu. Rev. Physiol. 2007.69:87-112. Downloaded from www.annualreviews.org marily the channel properties of transporters. In some cases, namely, the observation of single-transporter currents, the evidence is direct. In other cases the evidence—for example, from fluctuation analysis or transporter currents too large to be described as anything other than channel-like—is indirect. Although the existence of channels in transporters is not in doubt, we are far from understanding the sig- nificance of this property. In the online Supplemental Material, we review some pertinent aspects of ion channel theory and cotransport physiology to provide background for the channels and transporters presented here. We discuss the existence of channels in transporters, and we speculate on the biological significance of this newly unveiled property of transport proteins. 87 ANRV300-PH69-05 ARI 15 January 2007 17:10 INTRODUCTION that transporter currents can be as large as channel currents. Although this does not in Cotransporters employ the electrochemical itself define channel-like activity within a gradient of certain ions to concentrate spe- transporter, traditional models for cotrans- cific substrates. Since the early 1990s, new port predict less than 1 pA of uptake current data from fluorescence microscopy and elec- for a million transporters operating at once. trophysiology have suggested that conforma- Thus, some activity extraneous to conven- tional changes predicted by the alternating tional explanations must be occurring. One access model (1) of cotransport (see also Sup- outstanding question might be to what extent plemental Figure 3) may be smaller than the uptake current is actually carried by the predicted and more similar to channel gat- substrate (in this case, 5-HT+) in addition to ing (1–5). One of the most astonishing exper- other ions. We discuss the details surround- iments of the new era of channel and trans- ing this question in the Supplemental Text, porter characterization is a study of pre- and Section B3 (follow the Supplemental Material postsynaptic signaling at an intact synapse link from the Annual Reviews home page at (6). Figure 1 shows a salient feature of this http://www.annualreviews.org). experiment. The point to note from Figure 1 is that Figure 1 shows the pre- and postsy- in real synapses large currents are associated naptic responses to the release of serotonin with neurotransmitter transporters. In this [5-hydroxytryptamine (5-HT)] in a leech review, we consider carefully what purpose synapse under voltage clamp. Before the 5- these presynaptic currents serve and whether HT-gated, postsynaptic receptor current oc- they can be explained by traditional models curs, a larger and faster presynaptic current, with fixed stoichiometry and tight coupling associated entirely with the serotonin trans- or whether they require new models with porter (SERT), occurs. This latter current channel-like properties. is associated with transporting 5-HT back into the presynaptic terminal (uptake). Thus, as early as 1993, we had a strong indication CHANNELS IN TRANSPORTERS Na+/K+ ATPase Transport pumps are bonafide enzymes, and the prime example is Na+/K+ ATPase (the Na+ pump). For supporting details, please see the Supplemental Text (Section B2) and by Virginia Commonwealth University on 04/04/11. For personal use only. also References 7 and 8. Even the archetyp- Annu. Rev. Physiol. 2007.69:87-112. Downloaded from www.annualreviews.org ical Na+ pump, however, appears to become an ion channel under certain conditions. Pa- lytoxin (PTX), a marine toxin, is found in reef corals that are armed with a stinging appara- tus (9). PTX depolarizes cells via nonselec- tive cation channels of 10 pS (10). Na+/K+ ATPase is known to be the target because pump-specific oubaine antagonizes PTX (11). Figure 1 PTX, which forms ion channels in the pump Pre- and postsynaptic responses in a serotonergic synapse. Only a + 2+ (Figure 2), presumably binds the Na pump, submillisecond delay occurs between a presynaptic Ca flash (arrow) and + + presynaptic serotonin uptake current, which is faster and larger than the forming a pore coincident with the Na /K serotonin-induced postsynaptic current (PSC). Pre- and postneurons were pathway, and activates a conductance with an + + voltage clamped at −70 mV and −60 mV, respectively. From Reference 6. affinity for Na > K . Oubaine accelerates 88 DeFelice · Goswami ANRV300-PH69-05 ARI 15 January 2007 17:10 by Virginia Commonwealth University on 04/04/11. For personal use only. Annu. Rev. Physiol. 2007.69:87-112. Downloaded from www.annualreviews.org Figure 2 + + A channel-like component of Na /K ATPase mediates the palytoxin (PTX)-induced current. (a)(To p + Left) Absence of channel activity in an outside-out, ventricular-myocyte patch held at 40 mV, with Na on both sides of the membrane and 5-mM internal MgATP, before PTX application. (Right) A single PTX-induced channel opens 1 min after 20-pM PTX is applied, characterized by long-open-time bursts with brief closures (asterisks). (Bottom) Examples of brief closures on an expanded timescale. (b) Channel currents at different voltages. Closed (c) and open (o) current levels are marked. (c)(Left and right) Histograms of baseline-corrected records 20 s long, fitted with sums of two Gaussians. (Center) Single-channel current, I (difference between peaks), plotted against V gave channel conductance of 7 pS. From Artigas & Gadsby (12, 13) and Hilgemann and colleagues (14–16). www.annualreviews.org • Transporters as Channels 89 ANRV300-PH69-05 ARI 15 January 2007 17:10 PTX washing-out effects, and preincubating tion sites. Another exchanger, NCKX2, is a the ATPase with the steroid slows subsequent Na+/Ca2+/K+ exchanger. activation of the PTX-induced conductance. As mentioned above, Ca2+ import/export Thus, PTX and ouabain likely occupy the depends partly on the Na+:Ca2+ exchange Na+ pump simultaneously, each destabiliz- ratio or transport stoichiometry. Whereas ing the other. PTX-induced channels are also Ca2+ flux equilibrium experiments indicate permeable to large organic cations, including Na+:Ca2+ ratios of 3:1, reversal potential NMDG (N-methyl-d-glucamine), suggesting data suggest 4:1 ratios (16). Using an ion- that the narrowest section of the pore must be selective electrode to quantify ion fluxes in at least 7.5 A˚ wide. giant patches, Hilgemann (19) showed that ion flux ratios during maximal transport in either direction are 3:2. Because Na+ and + 2+ Na /Ca Exchangers Ca2+ are present on both sides of the mem- The Na+/Ca2+ exchangers (NCXs) are a brane, the net current and Ca2+ flux ad- ubiquitously expressed group of transporters. ditionally are dependent upon and can be They transport Ca2+ across membranes reversed at different membrane potentials. against the ion’s electrochemical gradient by Hilgemann proposes that transport of sub- using the electrochemical gradient of Na+. strates by NCX1 is not restricted to a ratio The cotransport is therefore bidirectional and of 3:1 but that other transport stoichiome- is controlled by membrane potential and by tries are indeed possible. These include 1:1 both Na+ and Ca2+ (substrate) gradients. In at a lower rate, a Na+-conducting mode that cardiac muscle, exchangers extrude intracel- exports 1 Ca2+, and an electroneutral Ca2+ in- lular Ca2+ during the excitation-contraction flux mode that exports 3 Na+ (Figure 3). The cycle. The stoichiometry for the exchange is two minor transport modes may potentially thought to be 1 Ca2+:3 Na+; hence, the ex- contribute to and determine resting concen- changer is electrogenic, and membrane cur- trations of free Ca2+ and background inward rent is a quantitative readout of NCX ac- current in heart muscle (17, 20). This release tivity (17). Intracellular Ca2+ concentration from strict substrate coupling ratios in NCX1 also assesses NCX function (18). In cardiac therefore shifts the definition of the mech- myocytes, NCX is additionally crucial for anism involved in this molecule’s function Ca2+ homeostasis and muscle relaxation af- away from one that can be described wholly ter contraction and can play an important by the alternating access model and suggests role in excitation-contraction coupling when characteristics more akin to those of channel running in reverse. During cardiac ischemia, proteins. by Virginia Commonwealth University on 04/04/11. For personal use only. malfunction of the exchanger causes Ca2+ Noise analysis also suggests that NCX cur- Annu. Rev.