of the L-type Ca channel by measuring conductance at various permeant ion con- ...... architecture into a single term, the capture radius of the channel, R c.
Permeation in the Dihydropyridine-sensitive Calcium Channel Multi-ion Occupancy but No Anomalous Mole-Fraction Effect between Ba 2+ and Ca 2 + DAVID T. YUE a n d EDUARDO MARBAN From the Departments of Medicine and Biomedical Engineering, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 ABSTRACT We investigated the mechanism whereby ions cross dihydropyridinesensitive (L-type) Ca channels in guinea pig ventricular myocytes. At the singlechannel level, we found no evidence o f an anomalous mole-fraction effect like that reported previously for whole-cell currents in mixtures o f Ba and Ca. With the total concentration o f Ba + Ca kept constant at 10 (or 110) mM, neither conductance nor absolute unitary current exhibits a paradoxical decrease when Ba and Ca are mixed, thereby weakening the evidence for a multi-ion permeation scheme. We therefore sought independent evidence to support or reject the multi-ion nature o f the L-type Ca channel by measuring conductance at various permeant ion concentrations. Contrary to the predictions o f models with only one binding site in the permeation pathway, single-channel conductance does not follow MichaelisMenten kinetics as Ba activity is increased over three orders of magnitude. Twofold variation in the Debye length o f permeant ion solutions has litde effect on conductance, making it unlikely that local surface charge effects could account for these results. Instead, the marked deviation from Michaelis-Menten behavior was best explained by supposing that the permeation pathway contains three or more binding sites that can be occupied simultaneously. The presence o f three sites helps explain both a continued rise in conductance as [Ba 2+] is increased above 1 10 mM, and the high single-channel conductance ( - 7 pS) with 1 mM [Ba~§ ] as the charge carrier; the latter feature enables the L-type channel to carry surprisingly large currents at physiological divalent cation concentrations. Thus, despite the absence o f an anomalous mole-fraction effect between Ba and Ca, we suggest that the L-type Ca channel in heart cells supports ion flux by a single-file, multi-ion permeation mechanism.
Address reprint requests to Dr. Eduardo Marban, Hunterian 116, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205. J. G~:N.PHYSIOL~) The Rockefeller University Press 90022-1295/90/05/0911/99$2.00 Volume 95 May 1990 911-939
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THE JOURNAL OF GENERAL PHYSIOLOGY. VOLUME 9 5 .
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INTRODUCTION
Dihydropyridine-sensitive, or L-type, Ca channels demonstrate remarkable conduction properties that point to a mechanism whereby ions bind to specific sites in the permeation pathway as they traverse the pore. Under physiologic conditions L-type channels are quite selective for Ca 2+ and yet support a large flux of Ca 2+ into the cell. In spite of their high selectivity for divalent over monovalent cations when both are present (Goldman permeability ratios >1,000; Lee and Tsien, 1984), L-type channels can also carry large monovalent currents in the absence of divalent cations (Kostyuk et al., 1983; Almers et al., 1984; Coronado and Affolter, 1986; Hess et al., 1986; Hadley and H u m e , 1987). Two general classes of models are capable of explaining these and other distinctive features of ion permeation through L-type Ca channels. The first proposes that divalent cations, when present, bind to a highaffinity site that makes the pore Ca-selective by allosteric modulation. In the absence of Ca 2+ (or another divalent cation, e.g., Ba 2+) the pore loses its selectivity and the channel can support a large flux of monovalent ions (Kostyuk et al., 1983; Kostyuk and Mironov, 1986). The second mechanism envisions a pore studded with two or more sites that bind ions as they traverse the channel. The affinity with which a given ion binds to each site determines selectivity, while high fluxes are maintained as multiple ions occupy the pore simultaneously and propel each other through by electrostatic repulsion. Such single-file, multi-ion permeation models, elaborated earlier for K (Hille and Schwarz, 1978) and Na channels (Begenisich and Cahalan, 1980), have found particular appeal in their application to Ca channels (Hess and Tsien, 1984; Almers and McCleskey, 1984). A major distinguishing feature that has favored multi-ion occupancy is the "anomalous mole-fraction effect" (AMFE) between Ba 2+ and Ca ~+. This phrase describes the observation that, at a constant concentration of divalent cations, Ca channel current is paradoxically smaller when mixtures o f Ba ~+ and C a 2+ a r e presented to the channel, c o m p a r e d with the currents in pure solutions of either. Such a decrease in the current carried by L-type Ca channels has been observed in mixtures of Ba 2+ and Ca 2+ in both cardiac (Hess and Tsien, 1984; McDonald et al., 1986; Campbell et al., 1988) and skeletal (Almers and McCleskey, 1984) muscle, as well as in neurons (Byerly et al., 1985). Although gating and premeation are difficult to distinguish in macroscopic currents, the changes in flux seemed clear enough to be interpreted as evidence for multi-ion occupancy. The interaction a m o n g permeant divalents has particular significance since, when Ba or Ca ions are present, the simplest allosteric model is reduced to a single-site mechanism inconsistent with an AMFE (Hille and Schwarz, 1978). The paradoxical increase in monovalent current in the absence o f divalents represents another manifestation of anomalous molefraction behavior (Coronado and Affolter, 1986), but interpretation of these phen o m e n a are complicated because maintenance of normal channel architecture could depend critically on the presence of divalents (Kostyuk et al., 1983; Pietrobon et al., 1988). Thus, the AMFE between divalents constitutes a crucial feature favoring a multi-ion permeation hypothesis. Unfortunately, the conditions under which anomalous interactions between Ba 2+ and C a 2+ have been observed give reason to wonder whether they reflect genuine permeation properties of the pore. The majority of data have been obtained at the
YuE ANDMAY.BAN Pe.aneation in L-Type Calcium Channels
913
whole-cell level, where gating and permeation cannot always be dissected unambiguously. Hess and Tsien (1984) used fluctuation analysis to estimate single-channel current (i) and found that i near 0 mV decreased from ~0.07 pA in 10 mM [Ca 2+ ] to - 0 . 0 3 pA in 7 mM [Ba 2+ ] + 3 m M [Ca ~+ ] (total [divalent] = 10 m M ) . Given the fivefold smaller value of their noise-estimated i in 10 mM [Ca 2§ ] as compared with direct measurements of unitary currents (Hess et al., 1986), technical limitations may have biased the estimates o f i. Few single-channel recordings have confirmed directly whether such an AMFE is a genuine feature o f native L-type Ca channel permeation (Friel and Tsien, 1989), and these apparently conflict with results from skeletal t-tubule Ca channels reconstituted in bilayers that indicate no AMFE between Ba 2+ and Ca ~§ (Ma and Coronado, 1987, 1988). Interpretation of anomalous interactions is further complicated by considerations of voltage dependence. At both single- and whole-cell levels, the AMFE between Ba 2+ and Ca ~+ has only been reported at depolarized potentials (>__-10 mV). Interpretation o f observations in this potential range is extremely difficult for two reasons, as illustrated by the hypothetical single-ion channel in Fig. 1 (details in Appendix). The values for the free energy profile in the inset of Fig. 1 A were chosen to mimic known open-channel current-voltage relations from cell-attached patch recordings with 110 mM [Ca ~+] or [Ba 2+] in the pipette. Note first that, because the reversal potential with Ca ~+ as the external charge carrier is larger than with Ba e§ while the inward conductance with Ca ~+ is smaller than with Ba 2+ (Hess et al., 1986), current-voltage relations corresponding to various mixtures of extracellular Ba ~+ and Ca 2+ converge upon each other at positive potentials. Fig. 1 A plots three current-voltage relations corresponding to Ba 2+ mole fractions of 0 (a, 110 mM [Ca~+]), 0.7 (b, 77 mM [Ba~+] + 33 mM [Ca2+]), and 1 (c, 110 mM [Ba~+]). The differences at positive potentials are minuscule. Hence, even if the channel were a simple single-site pore, slight shifts in voltage control, or in the cell resting potential in the case of cell-attached patches, could deceptively give the appearance o f an AMFE. More importantly, Fig. 1 B reveals that at voltages >__-10 mV, an increasing amount of outward K § flux (+) competes with inward Ba 2+ flux (0) in determining net current (solid line). At these potentials, the relative currents at different mole fractions reflect not only divalent ion permeation, but also K + occupancy o f the channel. Conductance determinations in solutions of symmetrical divalents (Fig, 1 C, 0), if experimentally accessible, would provide the clearest test of its mole-fraction dependence, but most measurements have been performed under asymmetric conditions with three permeant ions present. Analytical proofs that single-ion channels cannot produce an AMFE have not been derived for the case of three types of ions vying for occupancy of the pore, and it is not clear whether this rule holds under all such situations. Because of these concerns, we tested for an AMFE between Ba ~+ and Ca 2+ at the single-channel level, u n d e r conditions where differences between multi- and singleion behavior would be unambiguous. We recorded unitary currents through L-type Ca channels in guinea pig ventricular myocytes, the same preparation in which an AMFE was found at the whole-cell level by Hess and Tsien (1984) and by McDonald and co-workers (1986). Because of the constraint o f asymmetrical ionic conditions entailed by the cell-attached configuration (required to avoid loss o f activity with
914
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transpatch leakage c u r r e n t (see Methods). This leads to corrections o f + 15 mV f r o m the nominally zero potential, which have b e e n i n c o r p o r a t e d into Fig. 4 and the comparison to follow. Fig. 6 focuses on the mole-fraction d e p e n d e n c e o f single-channel c u r r e n t at 0 A
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YUEAND MARBAN Permeation in L-Type Calcium Channels
921
important to sample segments o f data for the histogram analysis according to the following criteria. Well-resolved openings and short segments o f bracketing current baselines on both sides were included in the histogram analysis, so that two peaks a p p e a r in the histograms, one corresponding to the baseline and the other to current during channel openings. The open-channel current amplitude was then taken as the difference between the medians o f two overlapping Gaussian functions fitted to the data by iterative, nonlinear least-squares analysis (Draper and Smith, 1981). In Fig. 6 A, representative openings used in the analysis a p p e a r to the right o f the histograms. That the double Gaussian functions provide excellent fits to the histograms, with few events in the valley between the peaks, attests to the good resolution of o p e n and closed amplitudes free f r o m contamination by filtered gating transitions (Yellen, 1984). The results o f such histogram analysis reveal that there is no detectable AMFE even with current at 0 mV as the endpoint: the best fits to the data in Fig. 6 A yield current amplitudes o f - 0 . 3 3 9 , - 0 . 3 4 5 , and - 0 . 8 5 6 pA for mole fractions o f 0, 0.7, and 1, respectively. Results f r o m a total o f nine patches were consistent, as demonstrated by the pooled data plotted in Fig. 6 B. Thus, we conclude that the "anomalous mole-fraction effect" as observed at the 20th
W O Z _ 20 M in Ma and Coronado, 1987; cf. Rosenberg et al., 1988). Deviations from Michaelis-Menten kinetics are even more readily apparent in the Eadie-Hofstee transformation of our data, shown in Fig. 8 B. If the pore bound only one ion at a time, the Eadie-Hofstee plot would be linear. Instead, the relationship in Fig. 8 B deviates flagrantly from linearity: an attempt to regress one region (high, mid, or low conductance) would fail to fit the two others. Before interpreting these results in the context o f intrapore properties, we considered the possibility that local negative charges n e a r or in the o u t e r m o u t h o f the channel could have enhanced the ion activity facing the entrance o f the conduction pathway, particularly at low Ba concentrations. Although ionic strength, and t h e r e f o r e Debye length, was held constant over the concentration range f r o m 1 to 55 Ba, changes in divalent cation concentrations are themselves sufficient to alter
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THE
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surface potential (see, e.g., McLaughlin, 1977, f o r a t r e a t m e n t o f G o u y - C h a p m a n Stern theory). Such local c h a r g e effects c o u l d influence the shape o f the relationship b e t w e e n p e r m e a n t ion activity a n d channel c o n d u c t a n c e (e.g., G r e e n et al., 1987), as has b e e n shown for m o n o v a l e n t cation p e r m e a t i o n t h r o u g h L-type Ca A
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FIGURE 8. Relation of Ba conductance to Ba activity in the L channel channel. (A) Pooled [3data (unfilled symbols) for conductance vs. Ba activity (a~; Ld means _+SD are plotted for 0 n >-_three patches, except for Z < the two points without error bars. The filled symbol repre0 sents the average of two 0 patches (range less than symZ 9 bol size) using a pipette solu0 tion with one-half the Debye I' I I I 0 length of the solutions corre0 120 sponding to the unfilled syma B a ( rnH ) bols at the same a~. Ionic 50 strength was held constant for points between 1 and 55 Ba, so El. that the same activity coefficient would apply during this LLI sharply rising, critical phase of (D io the plot, thereby making inferZ .< ences from this portion of the I-curve independent of exact (,.3 + knowledge of activity coeffiE3 cients. For points between 70 4* Z and 400 Ba concentrations, acO tivity coefficients were adjust(_] ed for varying ionic strength as I I I I 0 I I described in the Materials and o 3O Methods. Here, exact knowlCONDUCTANCE (pS)/eE9 a (mH) edge of activity coefficient is less critical because of the shalC low rise of the curve; the con+ 2 0 mV ductances attained at the higher divalent activities could not be produced by a single-ion model +140 at any (even infinite) activity as shown below in B. For the unfilled symbols, the actual activms ~ ity coefficient equaled 0.299 for [Ba] _
