Super-resolution scanning patch clamp reveals clustering of functional ion channels in adult ventricular myocyte.

Methods and Results We used scanning ion conductance microscopy and conventional cell-attached patch-clamp with a software modification that allows controlled increase of pipette tip diameter. The sharp nanopipette used for topography scan was modified into a larger patch pipette which can be positioned with nanoscale precision to a specific site of interest (crest, groove or T-tubules of cardiomyocytes), and sealed to the membrane for cell-attached recording of ion channels. Using this method, we significantly increased the probability of detecting activity of L-type calcium channels in the T-tubules of ventricular cardiomyocytes. We also demonstrated that active sodium channels do not distribute homogenously on the sarcolemma but rather, they segregate into clusters of various densities -most crowded in the crest region- that are surrounded by areas virtually free of functional sodium channels.

METHODS

Cardiomyocyte isolation and cell plating All animal procedures related to LTCC studies conformed to the UK Animals (Scientific Procedures) Act 1986 for rat cardiomyocytes isolation. Studies on sodium channel properties were carried out in accordance with New York University guidelines for animal use and care (IACUC Protocol 101101-02 to MD) and conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication 58-23, revised 1996). Cardiomyocytes from adult rats were isolated by the Langendorff perfusion method as described before 12 . Adult mouse ventricular myocytes were obtained by enzymatic dissociation following standard procedures. Briefly, mice were injected with 0.1 ml heparin (500 IU/ml intra-peritoneally) 20 min before heart excision and anesthetized by carbon dioxide inhalation. Deep anesthesia was confirmed by lack of response to otherwise painful stimuli. Hearts were quickly removed from the chest and placed in a Langendorff column. The isolated hearts were then perfused sequentially with low calcium, and an enzyme (collagenase, Worthington) solution. Ventricles were cut into small pieces, and gently minced with a Pasteur pipette. Calcium concentration was then increased gradually to normal values. After isolation, cardiomyocytes were plated on laminin coated coverslips or dishes and left to adhere for at least 30 minutes before the start of experiments. Cardiomyocytes were used on the same day of isolation. Cells were washed once with the external recording solution and mounted on the microscope stage for recordings. Instrumentation for super-resolution scanning patch-clam Scanning ion conductance microscopy (SICM) SICM is a non-contact scanning probe microscopy technique based on the principle that the flow of ions through the tip of a nanopipette filled with electrolytes decreases when the pipette approaches the surface of the sample 11 , 13 , 14 . The result is a three dimensional topography image of live cells with resolution of up to ≤ 20 nm 15 . All topographical images in this study were recorded using a variant of SICM called hopping probe ion conductance microscopy 16 , implemented on a software platform that controls the ICnano sample scan system 13 (Ionscope Ltd, UK). The scan head of the ICnano system consists of a three axis piezo-translation system (Physik Instrumente, UK) with a 100 × 100 μm x–y piezo-stage for sample positioning and 38 μm z-axis piezo-actuator for the vertical movement of the pipette, mounted on the stage of a conventional inverted microscope (Diaphot 200, Nikon Corporation, Tokyo, Japan). Schematic of the set-up is presented in Figure 1 . Glass nanopipettes of ~100 nm ID pulled from 1.0 mm O.D. 0.5 mm I.D. borosilicate capillary were used in all experiments. Axopatch 200A/B patch-clamp amplifiers (Molecular Devices, USA) were used to measure the pipette current as well as to record ion channel activity. Cell-attached currents were digitized using Digidata 1200B and a pClamp 10 data acquisition system (Axon Instruments; Molecular devices). Controlled modification of the pipette diameter The tip of the pipette was clipped using a software-controlled movement of the piezo-actuator. Details of the development of this method are under consideration for publication in Neuron. Briefly, after generating the topographical image of the cardiomyocyte surface, the pipette (~100 nm ID) was moved to an area clear of cells or debris. At that coordinate, the rate at which the pipette approached the sample during scanning was increased to ~500 nm/ms, and the duration of the excursion (from fall to rise) was adjusted to 500 ms. This maneuver caused the pipette tip to clip against the solid bottom of the dish ( Figure 2A,B ). The pipette resistance was continuously monitored and the clipping motion stopped once the current through the pipette reached the desired level. At that point, the pipette was repositioned to spatial coordinates that were selected based on the topography image recorded with the sharp pipette ( Figure 2C ). The lateral error of repositioning was determined by the x-y piezo actuators (PI-621.2CD; Physik Instrumente, UK). The resolution of the PI-621.2CD x-y piezo stage in the closed loop operation is 0.4 nm and the repeatability (error between repeated returns to the same point) is ± 2 nm (complete set of specifications in http://www.physikinstrumente.com/en/pdf/P620_2_Datasheet.pdf ). Of note, tip size is only a determinant of the lateral resolution of a scan 17 and it does not affect repositioning. Once the x,y coordinates of the structure are established, the precision with which the pipette is returned to a specific location is determined exclusively by the resolution and repeatability of the x-y piezo actuators. As such, though the recording pipette occupies a larger area after clipping, its center is repositioned with an accuracy of ± 2 nm.

Image recording stability

Additional experiments confirmed that the repositioning error was below the resolution limit of SICM. The results are shown in Supplemental Figure I . A specific area of an adult ventricular myocyte was scanned twice (scan 1 and scan 2) with an ~100 nm ID pipette (spatial resolution 50 nm). The two scans were pseudo-colored in red and green, respectively. The images were then superimposed, yielding a yellow pixel at those locations where red and green were in registry. Notice that the two images were virtually identical (all pixels in yellow), with no apparent offsets. These results indicate that our spatial error at repositioning was less than the overall spatial resolution of the recording system. Definition of recording sites Figure 3 illustrates the recording positions described in this manuscript. Panel A shows an image of a cardiomyocyte and the pipette, as seen optically. Panel B shows the three possible recording locations, defined by the SICM-based topology map: T-tubule, crest and Z-grooves. A depth profile along the xy plane marked by the black dotted line is shown on the right hand side. The profile reveals the periodic crests and grooves characteristic of adult cardiac myocytes. The Z-grooves correspond to the position of the Z-lines in the intracellular side 18 . The distance between crests (or Z-grooves) is ~ 2 μm and the recording probe travels into the T-tubule to a depth of ~ 100-300 nm (see vertical scale). As illustrated in panels C and D, this configuration allowed detection of ion channels (C) or the lack thereof (D) in relation to the precise location of recording. The cell-attached configuration was formed within 5-6 minutes after the SICM image was recorded. Based on estimates correlating pipette resistance to pipette tip diameter 19 we calculated that clipping the tip increased the pipette ID ~3-fold, from 100 nm to 300 nm. Assuming a hemispherical shape of the membrane patch, this increase in pipette diameter corresponds to a 10 fold increase in the area under the patch pipette, thus increasing the probability of capturing a channel (or channel cluster) under the patch.

Electrophysiological recordings

After repositioning the pipette, the non-contact mode of SICM was turned off and the pipette was lowered using the piezo actuator until it touched the membrane (indicated by an increase in the pipette resistance); as in conventional patch clamp, slight suction sealed the cell membrane onto the glass pipette. All electrophysiological recordings were obtained using the cell-attached patch-clamp configuration and were only included if seal resistance was larger than 5GΩ and leak current at 0 mV remained constant at 0 pA. For recording of LTCCs, cardiomyocytes were bathed in an external solution containing in (mmol/L): 120 K-gluconate, 25 KCl, 2 MgCl 2 , 1 CaCl 2 , 2 EGTA, 10 Glucose, 10 HEPES, pH 7.4 with NaOH, ~290 mOsm. Pipettes were filled with an internal recording solution containing in (mmol/L): 90 BaCl 2 , 10 HEPES, 10 Sucrose, pH 7.4 with TEA-OH, ~250 mOsm. The membrane under the patch was held at a voltage of −80 mV and voltage pulses were applied from −30 to +30 mV in incremental steps of 10 mV. To determine the effect of isoproterenol (ISO) on the open probability of LTCCs, repetitive depolarizing steps of 1s duration to +10 mV were applied from a holding potential of −80 mV at an inter-pulse interval of 2s. Open probability was calculated from at least 15-20 consecutive sweeps before and after 5 minutes of perfusion of 10 μmol/L ISO. Data were recorded at a sampling rate of 10 kHz, and filtered at 2 kHz. The applied voltage was corrected for a liquid junction potential of −16.7 mV. For recording of sodium currents, pipettes were filled with a solution containing (in mmol/L): NaCl 148, NaH 2 PO 4 0.4, MgCl 2 1, CdCl 2 0.2, KCl 5.4, HEPES 15, CaCl 2 1.0 and Glucose 5.5, pH 7.4 with NaOH. When indicated, tetrodotoxin (TTX; 30 μmol/L) was added to the internal pipette solution. Cells were maintained in a solution containing in (mmol/L): 0.33 NaH 2 PO 4 , 5 HEPES, 1.0 CaCl 2 and 140 KCl, pH 7.4 with KOH, thus depolarizing the membrane potential to a value estimated to be near zero. To assess the presence of fast inward currents, the membrane under the patch was held at −120 mV and voltage clamp pulses of 500 ms were applied every 3 seconds to −30 mV. To define the unitary current-voltage relation, 500 ms voltage clamp pulses were applied to −80, −70, −60 and −50 mV, from a constant holding potential of −120 mV.

Data analysis

Single channel data were analyzed using Clampex version 10.0. All graphs and statistical analysis were performed using either GraphPad prism 5 or Origin version 8.5. All data are presented as mean ± SEM for the given number of experiments. Statistical significance was calculated by a Chi-square test and significance was defined at p