Nav channel mechanosensitivity: activation and inactivation accelerate reversibly with stretch

Abstract

Voltage-gated sodium channels (Nav) are modulated by many bilayer mechanical amphiphiles, but whether, like other voltage-gated channels (Kv, HCN, Cav), they respond to physical bilayer deformations is unknown. We expressed human heart Nav1.5 pore alpha-subunit in oocytes (where, unlike alphaNav1.4, alphaNav1.5 exhibits normal kinetics) and measured small macroscopic currents in cell-attached patches. Pipette pressure was used to reversibly stretch the membrane for comparison of I(Na)(t) before, during, and after stretch. At all voltages, and in a dose-dependent fashion, stretch accelerated the I(Na)(t) time course. The sign of membrane curvature was not relevant. Typical stretch stimuli reversibly accelerated both activation and inactivation by approximately 1.4-fold; normalization of peak I(Na)(t) followed by temporal scaling ( approximately 1.30- to 1.85-fold) resulted in full overlap of the stretch/no-stretch traces. Evidently the rate-limiting outward voltage sensor motion in the Nav1.5 activation path (as in Kv1) accelerated with stretch. Stretch-accelerated inactivation occurred even with activation saturated, so an independently stretch-modulated inactivation transition is also a possibility. Since Nav1.5 channel-stretch modulation was both reliable and reversible, and required stretch stimuli no more intense than what typically activates putative mechanotransducer channels (e.g., stretch-activated TRPC1-based currents), Nav channels join the ranks of putative mechanotransducers. It is noteworthy that at voltages near the activation threshold, moderate stretch increased the peak I(Na) amplitude approximately 1.5-fold. It will be important to determine whether stretch-modulated Nav current contributes to cardiac arrhythmias, to mechanosensory responses in interstitial cells of Cajal, to touch receptor responses, and to neuropathic (i.e., hypermechanosensitive) and/or normal pain reception.

FIGURE 1

Membrane stretch and Nav1.5 currents: families, I/V plots and use of B/D/A protocols. (A) Currents (running averages, n = 4) for steps from V_hold = −110 mV in 15-mV increments to successively more depolarized voltages. black, no-stretch controls; red, during stretch (using −30 mmHg). Here and throughout, imperfectly subtracted capacitive transients mark the start and end of voltage steps. For reference, the −50 mV current pair is labeled. Early current is time-expanded at right. (B) For the same patch, the average peak current (16 control, 12 stretch runs) obtained from alternating no-stretch/stretch families (n = 16 and 12 runs for 0 and −30 mmHg, respectively). The −50 mV points are labeled and +10 mV currents are shown as an inset (arrow). (C) For the same patch as in A and B, n = 40 B/D/A protocols were done next (using −30 mmHg applied continuously for the during-stretch traces) in the order −50 mV, −60 mV, −70 mV, −30 mV (the −30 mV expansion shows that stretch accelerated current onset). B/D/As are plotted black, red, and gray, respectively, here and elsewhere. (D) Stretch difference currents for these B/D/A sets; difference current above the x axis represents “stretch inhibited” (SI) and that below “stretch augmented” (SA) I_Na. (E) Finally, the beginning of a dose-response at −70 mV (n = 40) obtained in the order −30 mmHg, 0 mmHg, −40 mmHg, 0 mmHg (pink, black, red, gray, respectively). The patch ruptured during an attempt at −45 mmHg.

FIGURE 2

Nav1.5 current activation as seen in different patches. (A) A patch in which activation (no stretch) was well-resolved at the voltages tested. Three B/D/A sets (n = 40 runs) obtained in the order -60 mV, -65 mV, -70 mV (the -70 mV “after” trace is missing because the patch ruptured at run 11 destroying the running average, but visual monitoring till that point showed currents at the control amplitude). (B) A patch in which two B/D/A sets (n = 40) were obtained in the order -50 mV, -65 mV. The RC properties of the recording pipette filtered current activation, even at -65 mV, likely because the sylgard-coating, which unavoidably varies from pipette to pipette, was insufficient.

FIGURE 3

Reversible effects of stretch in a single trace B/D/A train. (A) A no-stretch B/D/A (to show the reproducibility of voltage-elicited currents) for the same patch as B. (B) An experimental B/D/A (D = −30 mmHg, n = 1) whose first two elements are expanded below. V_hold provided −110 mV of driving force for the endogenous stretch-activated cation channels, so the “clean” stretch trace shows that gadolinium had effectively inhibited these channels. Scale bars: vertical, 20 pA; horizontal, 10 ms for trains, 1.5 ms for expanded sections.

FIGURE 4

Reversible stretch effects with negative or positive pipette pressure. Three B/D/A sets (n = 80) from a patch during steps to −40 mV. Endogenous stretch-activated cation channel activity in this oocyte batch was little affected by the usual level of gadolinium, so a higher level (nominally 120 μM) was used. This right-shifted the g(V), putting −40 mV very near threshold. Other dose response data from this patch are provided as Supplementary Material. When current rundown became excessive, pipette pressure was slowly increased until, at −79 mmHg, the patch ruptured. Pressures shown here were, thus, moderate.

FIGURE 5

Stretch acceleration factors from B/D/A sets. The peak amplitude of stretch traces was normalized to the peak amplitudes of before and after traces. Then the time base of the stretch traces was expanded until the activation traces overlapped. This caused the inactivation trajectories to overlap as well. The cases illustrated (top to bottom) are from Fig 1 C, −60 mV; Fig. 2 A, −65 mV, Fig. 2 A, −60 mV; and Fig. 2 B, −65 mV (entries 2, 8, 9,and 10 of Table 1) and had stretch acceleration factors (rescaling of time axis) of 1.40, 1.40, 1.45, and 1.35, respectively. The overall data quality (noise, stationarity for before and after, minimal RC filtering of activation time course during stretch) was best in the top set and least good in the bottom set where, during stretch, activation is assumed to have been somewhat filtered.