High Resolution Scanning Patch Clamp: Life on the Nanosurface

The cardiomyocyte plasma membrane has two jobs: to conduct the action potential along its length, and transduce that action potential into the synchronous increase of cytosolic calcium that triggers contraction. Ion channels mediate these events at the surface sarcolemma and along highly ordered membrane invaginations known as T-tubules. The types of channels present in the membrane can be identified using immunocytochemistry or electron microscopy, but most are only approximately assigned to specific domains. Moreover, the resulting images likely represent mixed populations of both functional and silent channels, as well as those in reserve pools waiting to be recruited under changing physiological or pathological conditions. Because many disease conditions involve the coordinate perturbation of membrane microstructure and electrical activity, there is much to be learned by probing channel function with greater spatial resolution along the cardiomyocyte. In this issue of Circulation Research, Barghava et al.^* describe a novel method for doing just that, and report some surprising discoveries.

Scanning ion conductance microscopy (SICM)^{1, 2} has evolved in recent years to provide a wide range of cellular landscapes reminiscent of the three-dimensional images of scanning electron microscopy, but in living tissue. The probe is an electrolyte-filled pipette that reports its proximity to the cell membrane as a decrease in conductance. It gets close to the membrane, within a distance equivalent to the pipette’s internal diameter (<100 nm). The probe is lifted and translated (it “hops”) from one point to another along the X and Y coordinates, with peaks and valleys registered as different values in the Z dimension at which the conductance drops. The result is a map of the surface of a cardiomyocyte with a “super-resolution” of 20 nm, well beyond the 200–300 nm resolution provided by standard immunocytochemistry or other light microscopic methods. In one earlier report, the SICM probe was used as a patch electrode, but the small size of the pipette necessary to provide nanoscale images of the cell surface proved an obstacle to sampling more than the occasional ion channel³.

The breakthrough in the study by Bhargava and colleagues^* was to expand the size of the probe after surface mapping, thus increasing the likelihood of capturing channels resident at those locations. The same piezo-electric controller for cell surface mapping was used to move the probe away from the cell and onto the surface of the recording chamber, where the tip was progressively “clipped” to the desired size based on conductance measurements. Remarkably, something so simple as repeatedly driving the tip into the chamber bottom yielded consistently sized tips that – forget the polishing – were able to form seals with approximately 70% probability. Moreover, the pipette could be returned to a previously mapped cellular feature, namely the Z-groove or the T-tubule, or its towering crest, with an error of less than 2 nm. With T-tubules situated along Z grooves and between crests each separated by 2–3 microns, pipettes with an internal diameter of 200–300 nm were well suited to form a seal precisely within the topographically defined feature.

The study shows that increasing the patch pipette size yields significantly more functional data associated with specific membrane features. L-type calcium channels were recorded with a frequency 6 times greater in the T-tubule compared with the crest above. These values comport with those obtained from previous electrophysiology studies using detubulation to determine sarcolemmal vs. T-tubular channel distributions⁴. They also reflect the well-established role for calcium channels at the dyadic junctions between the sarcolemma and junctional sarcoplasmic reticulum to couple the electrical signal into the feed-forward calcium signal that triggers contraction⁵.

In contrast, scanning for Na channels provided unexpected results. Na channels were observed in all three topographical features but, intriguingly, on the crest they fell into large clusters separated by patches devoid of the channels. While the authors were careful not to attach a molecular label to the Na channels found in these clusters, no Na channels were found when the pipette contained 30 uM TTX. This raises the question, are these the TTX-sensitive Nav1.1 channels, which contribute only minimally to overall I_Na⁶, or the TTX-resistant (TTX-R) Nav 1.5 channels contributing the lion’s share of the whole-cell current^{7, 8}? The 30 uM concentration used could be viewed as an intermediate concentration of TTX, much greater than the IC₅₀ for TTX-sensitive (TTX-S) channels, but a little shy of what one might use to ensure an identity with the TTX-resistant Nav1.5 channels ⁷. With a larger sample size and a lower concentration of TTX, this issue could, and likely will, be readily resolved.

Why are Na channels clustered on the crests of the T-tubules? In other excitable membranes, Na channels are concentrated at sites where local currents must be amplified into action potentials with a high safety factor – the axonal initial segment^{9, 10}, the node of Ranvier¹¹, the perimeter of the neuromuscular junction endplate¹². Considering that a single action potential must trigger tightly synchronous excitation-contraction coupling throughout the cardiomyocyte¹³, a high safety factor seems like a good idea as the action potential propagates from the surface sarcolemma into the belly of the muscle along the T-tubule. It has been shown that the T-tubules present a barrier to diffusion, such that high levels of activity or in some disease states might cause an accumulation of potassium within their confined space¹⁴. A resulting local depolarization of the T-tubular membrane would be expected to increase steady-state Na channel inactivation and thereby reduce Na channel availability. The clusters of Na channels on the crest could serve to amplify the sarcolemmal signal and safeguard against action potential failure under such changing conditions. Whatever the role of these clustered channels, their discovery demonstrates new clarity of structure linked to function not realized with other techniques.

As with any technique that is more than just a gearhead’s brainchild, ideas for new applications abound. Perhaps “super-resolution scanning patch clamp” could detect smaller domains as they are called into action. Membrane caveolae comprise a host of proteins involved in signaling, exemplified by the dependence of beta-adrenergic modulation of L-type Ca channels on caveolin-3¹⁵. In these dynamic membrane compartments, the larger SICM probe might provide new insights into mechanisms underlying increased activity in response to sympathetic stimulation. Similarly the technique could capture the recruitment of ion channel reserves, such as the covert population of TTX-S channels uncovered by scorpion toxin⁶, or the channels producing I_Ks, upregulated by sympathetic activation to protect against QT prolongation and sudden cardiac death^{16, 17}. And what about ion transporters, whose function is equally amenable to probing with a patch pipette? Will this approach uncover functional coupling with neighboring ion channels, where their activity would establish a gradient with nanoscale dimensions?

Finally, let us consider the potential for super-resolution scanning patch clamp in assessing disease-induced structural and functional changes. Under conditions of heart failure and ischemic heart disease, T-tubule organization is disrupted^18-20. Ion channels normally tethered by scaffolding proteins to T-tubules are concomitantly perturbed. But T-tubule perturbation can happen early in disease processes that presage overt signs of heart failure or arrhythmia²¹. Could this new technique help define the changes in ion channel distribution or dyad coupling that follow detubulation, providing possible targets for therapeutic intervention to delay the onset of disease? Will channel clusters disperse as a substrate for dyssynchrony of Ca release, or be resorbed, shifting the balance of currents and reducing the efficacy of conduction? With an inverted microscope, a piezo-electric controller and the software developed as part of the study, this approach will enable many to climb down from our perches at 30,000 feet to peer into the nanoscale nooks and crannies of the cell surface and witness the working parts involved in a host of cellular processes or diseases.