Whether “Slip-Mode Conductance” Occurs

Excitable cells rely on selective ionic conductances for electrical signaling. The work of Hodgkin and Huxley (1) propelled the modern dissection of the mechanisms of selectivity. Their formulation postulated two independent sets of ionic conductances, Na and K, whose relative permeabilities changed during the course of excitation. Mullins subsequently proposed that Na and K traverse the membrane through a single set of pores that changed from being Na-selective to K-selective (2). That view is no longer held as tenable. Several lines of experimental evidence, notably single-channel recordings, established convincingly that the membrane contains distinct sets of pores, each with its own distinctive selectivity properties (3, 4 ). Nevertheless, the prevailing view that ion channel selectivity is preserved during normal electrical activity was recently challenged in a report by L. F. Santana et al. (5). Voltage clamp experiments in rat ventricular cells led to the proposal that voltage-dependent Na channels change their selectivity in response to cyclic AMP–dependent phosphorylation. Unexpectedly, such channels conduct Ca well; similar “slip-mode conductance” responses were seen during exposure to cardiac glycosides. The extensive evidence against flexible selectivity, as well as major technical concerns over that report (5), prompted us to question whether or not this idea has a genuine biological basis. Ventricular myocytes are large cells in which voltage control is notoriously difficult to achieve (6); the large number of Ca-sensitive ionic currents (7) further complicates the interpretation of the results. To test for the existence of slip-mode conductance, we expressed human hH1 (8) or rat rH1 (9) cardiac Na channels heterologously in Chinese hamster ovary (CHO) cells. These small cells are readily voltage-clamped, have few endogenous channels, and support cyclic AMP-mediated responses (10). We performed whole-cell patch clamp recordings of membrane current (11). In 20 mM external Na, a family of typical Na currents was elicited (Fig. 1A) by depolarizing voltage pulses from 2100 mV to potentials from 280 mV to 160 mV. As shown in the I-V relation (panel B), the currents reach a maximum amplitude at 220 mV and demonstrate a reversal potential near 160 mV. Such currents increased in amplitude in response to 1 mM isoproterenol or 10 mM dibutyryl cyclic AMP (32 6 5%, n 5 9; panel C, Œ), confirming the well-established response to phosphorylation (10). However, no ionic current was measurable when the external solution was switched to 0 Na/10 mM Ca (1 6 2% of basal current, n 5 7; panel C, ƒ). That Na channels were still present and functional was verified by restoring Na to the external solution (panel C, Œ). Figure 1D shows the time course of the phosphorylation-mediated increase in peak INa and flux of Na (Œ) versus Ca (ƒ) through the modified rH1 Na channels. Similarly, there was no measureable Ca flux through phosphorylated hH1 channels in 0 Na/5 Ca solution (Fig. 1E). Results were comparable with either the human or rat channels, excluding a possible species-specific response. These experiments relied on expression of the a subunit alone. Such a test was motivated by the fact that, in rat myocardium, Na channels consist only of a subunits (12), making it unlikely that b subunits somehow contributed to the original observations of slip mode conductance in rat heart cells. Nevertheless, to exclude the possibility that b subunit coexpression is required, we performed further experiments in CHO cells that coexpress the a and b1 subunits (13, 14). In these experiments we quantified both major manifestations of ion selectivity: reversal potential (Erev), and ion flux. These two reflections of selectivity are complementary. The reversal potential in solutions of mixed ion composition yields relative ion permeabilities (4), but Erev measurements can be problematic methodologically because small changes, resulting from junction potential drift for example, are difficult to exclude. For Na currents, the very nature of the measurements necessitates that small inward and outward currents be quantified reliably, which is difficult to do in large cells (for example, cardiac myocytes) with a variety of ionic conductances. In principle, the use of TTX subtraction may help in distinguishing among various currents, but it does not prevent problems resulting from cumulative junction potential drift or endogenous TTX-sensitive Na currents. The alternative approach, that of measuring ion flux directly (as in Fig. 1), has various advantages. First, it is modelindependent: if net current is carried by a given ion, then that ion must be permeant. Secondly, net flux is the parameter of physiological importance. In the case of slip mode, Ca flux through Na channels is postulated to suffice to trigger excitationcontraction coupling (5). If that is the case, a sizable Ca current should be measurable through Na channels: there is no reason to rely solely on shifts of Erev. We took membrane current recordings from a representative CHO cell that coexpressed a 1 b1 subunits (Fig. 2A). The Na equilibrium potential was set to 0 mV by including 20 mM [Na] in both the internal and external solutions (15). Membrane current was first recorded at baseline in the absence of cyclic AMP, but with 2 mM external [Ca]. The currents reversed at 0 mV (h). The addition of 50 mM dibutyryl cAMP increased both inward and outward Na current, as expected from a simple increase in Na channel open probability. Erev did not change despite the continuing presence of Ca in the external solution (16) (F). Subsequent removal of external Ca increased the amplitude of the Na currents at negative potentials; this effect is expected from the known voltage-dependent block of Na channels by external Ca (17), but is in the opposite direction to the change that would have been expected had the channels been permeable by Ca. Once again, the current reversed at 0 mV (Œ). The single experiment shown in Fig. 2, A to C, was representative of five cells, whose mean current-voltage relations are shown (B). On removal of Ca in the continued presence of external Na, Erev did not change (16). This stability differs from the shift of 5.1 mV (18), which would have been seen had the relative Ca/Na permeabilities equaled 1.25, as stated in the report (5). Inward currents disappeared (Fig. 2B) when the cells were bathed with an external solution containing 2 mM Ca but no Na (ƒ). This observation further confirms the absence of an appreciable calcium conductance through Na channels. Results were indistinguishable between hH1 channels coassembled with human heart (hb1) or rat brain (rb1) b1 subunits. Confirmation that rb1 in fact expresses functional subunits was derived from parallel experiments in which the same b1 cDNA increased current amplitude and shifted inactivation when coexpressed with Na channel a subunits (19). Because no Erev shift was observed in mixed Na/Ca solutions, there is no basis for the notion that “slip mode” requires the modulatory presence of external Na ions. Our results contradict findings in rat ventricular myocytes (5) and in HEK cells (20). We have used CHO cells, which are known to support cyclic AMP–mediated responses and which contain few endogenous conductances. The first reports of cyclic AMP–dependent upregulation of Na currents were from expression studies in CHO cells (10). HEK cells have the virtue of being readily transfected (21), but they do not consistently support cyclic AMP–dependent responses; several lines of evidence indicate that protein kinase activity is so high in the basal condition that kinase inhibitors must be added to reveal directionally appropriate responses (22). T E C H N I C A L C O M M E N T S