The overall goal of this procedure is to demonstrate a technique for characterizing the frequency response of ion channels for the purpose of measuring the current flux through multiple channel types simultaneously, this is achieved by first creating the noise function that will be included in the voltage step protocol and incorporating it into a stimulus file, which is necessary in order to use custom wave forms with the clamp X software. The second step of the procedure is to apply the voltage step protocol that was just created to a model cell using standard whole cell voltage clamp techniques and record the resulting current. The third step of the procedure is to calculate the IV curve of the cell and the FFT of the portion of each sweep that contains the noise function.
The final step of the procedure is to individually correlate the amplitude of each frequency at all voltage steps with the IV curve of that cell. The results show that no frequencies highly correlate with the IV curve of the model cell. The main advantage of this technique over existing methods like standard whole cell voltage clamping, is that it may provide a means to measure more than one ion channel type simultaneously without the use of pharmacological blocking agents or voltage in activation protocols.
This method can help answer key questions in the field of ion channel physiology, such as what effect the level of expression of one ion channel type has on the activity of other ion channel types in the same tissue. The first step in the protocol is to create a noise function containing the desired frequency components. To do this, first, describe the desired frequency components in the frequency domain.
In this study, frequency components between one and 15 kilohertz are used. Then calculate the inverse fast. Fourier transform by using MATLAB's IFFT function to transform the selected frequencies to the time domain next scale, the amplitude of the noise function appropriately.
In this study, the noise function is scaled twice, once so that the zero to peak amplitude of the noise function is 50 millivolts and the second time, so that the zero to peak amplitude is 100 millivolts. The next step is to create a stimulus file using a program such as matlab. A stimulus file is just a text file with a header that tells the clamp X eight acquisition software exactly what voltage to use at every point in time.
To create the stimulus file first, create a text file with the appropriate header below the header, insert the time increments for a single sweep in the first column, the time increments should have the same temporal spacing as the sampling interval used in the measurements. This protocol uses a 200 kilohertz sampling rate. For each sweep, insert the exact voltages desired at each time step.
Each column is considered a separate sweep, so for a protocol that steps from minus 150 millivolts to plus 50 millivolts in five millivolt increments, 41 columns should be added after the time column. The specified voltages should include the noise function in clamp X.A protocol must be created for each experiment, so the next step is to create a measurement protocol that is compatible with a stimulus file generated previously. In clamp X, select the acquirer edit protocol wave zero menu option that allows you to associate a stimulus file with the current protocol.
The next step is to apply the defined stimulus to the cell. This protocol uses a model cell that has a parallel RC circuit. Attach the model cell to the measurement equipment, click the record button and clamp X to perform.
The experiment is scheduled for control purposes. Make sure to include periodic measurements that do not include any noise functions after you have collected the data, the next step is to generate the IV curves and do the fast fourier transforms. To calculate the IV curve for an individual recording, use a steady state portion of the recording temporally outside the range of the noise function to measure the current amplitude.
If the recording is not at steady state when the noise function is applied, the noise function may interfere with calculation of the IV curve, in which case the control recording should be used. Instead to calculate the IV curve for each voltage step in a recording, calculate the FFT of the portion of the recording where the noise function was inserted. Combine the Fourier transform for each voltage step into an M by N matrix where M is the number of frequencies in the FFT and N is the number of voltage steps in this configuration.
Each row of the matrix represents the FFT amplitude of a single frequency at all voltage steps in the experiment. The next step is to calculate correlation coefficients for each frequency, correlate the row with the IV curve generated previously. Record the correlation coefficient.
Next generate a plot of the correlation coefficient versus frequency to visualize frequencies which highly correlate with the IV curve. The results of the voltage clamp measurements without a noise function inserted are shown here. For the first and last 20 milliseconds of each sweep, the potential was kept at the holding potential of zero millivolts.
In between the voltage was stepped from minus 150 millivolts to plus 50 millivolts in five millivolt increments. Each voltage step was 80 milliseconds long to make the figure easier to read, only every fifth sweep is included in the figure. The red box indicates when the noise function would've been applied had there been a noise function in these sweeps.
This figure is similar to the previous figure, but with the noise function inserted into the voltage step protocol 40 milliseconds after the beginning of the voltage step, the red box shows where the noise function was inserted. The noise function had a duration of 30 milliseconds and contained frequencies between one and 15 kilohertz. This figure shows the IV curve that was calculated for the model cell.
This IV curve is linear, which is expected since the model cell is a simple parallel RC circuit. This figure shows the FFT result without the noise function for each sweep. In the model cell recordings, the FFT was calculated over the timeframe where the noise function was inserted.
The DC component, which is highlighted in red, appears to mimic the shape of the IV curve. All of the frequencies above DC appear to have amplitudes near zero for clarity only the FFT from every fifth sweep is included in the figure when the FFT is calculated for the recordings with the inserted noise function, the frequencies between one and 15 kilohertz have noticeable amplitudes. The presence of these higher frequencies, which are not normally present in voltage clamp experiments, raises the question of whether the magnitude of any of these frequencies change proportionally to the current amplitude.
These next three figures show the correlation coefficient between the amplitude of the individual frequency and the IV curve of the recording. In the first figure, no noise function is used In the second figure. The noise function had a peak amplitude of 50 millivolts, and in the third figure, the noise function had a peak amplitude of 100 millivolts.
Notice that in all cases the DC component, in other words, the response at zero hertz has a correlation coefficient greater than 0.99, and so appears to correlate nearly perfectly with the IV curve. Also, note that in all three figures, none of the other frequencies have correlation coefficients greater than 0.5. In addition, insertion of the noise function with either 50 or 100 millivolt amplitude did not change this pattern.
These results indicate that the voltage clamp circuitry and the noise function itself do not introduce high correlations to the IV curve. This validation is important in order to apply this technique to preparations containing ion channels. This figure recreates the IV curve to show the correlation or lack thereof in two sets of frequency data.
The IV curve for the model cell labeled actual IV in this figure is the dotted line that appears to superpose on the red dashed line. The red dashed line shows the DC component, which has a correlation coefficient of 0.995 to the actual IV line. The third curve displayed the jagged line shows a randomly chosen frequency of 4.399 kilohertz with low correlation.
The correlation coefficient for this line to the IV curve is 0.3212. These results show that the measurement equipment and the noise function do not by themselves cause any frequencies to correlate with the current amplitude based on this verification, the method presented here inserting a noise function to voltage steps during voltage clamp experiments can be applied first to membrane preparations containing a single ion channel type and subsequently to preparations containing multiple channel types. The results from the single ion channel experiments can be analyzed using the FFT and correlation analysis proposed here.
This analysis can be used to identify frequencies whose amplitudes correlates specifically with that particular channel type. Strong evidence has been presented suggesting multiple rate constants play a role in conduction of ions through different channels. Therefore, once frequencies have been identified, they highly correlate with one channel type and minimally correlate with other channel types.
These frequencies may then be used to estimate the current contribution of multiple channel types measures simultaneously following this procedure. Other methods like pharmacological block and pre pulses can be performed in order to answer additional questions such as how the frequency response of channels behave when an ion channels in a blocked or inactive confirmation.
