The chapter hypothesis is (i) that the maxi-K and Katp channels will be expressed and active in human osteoblast-like cell lines and primary osteoblasts, and (ii) that Katp ligands will have a functional role in MG63 cell proliferation.
This chapter aims to establish the patch-clamp technique with MG63 and SaOS-2 cells, to demonstrate the abundance and activity o f K+ channels in MG63 and SaOS-2 osteoblast-like cell membranes, and show some o f the first important data demonstrating that K+ channels are also active in primary human osteoblasts. RT-PCR will be used to confirm the expression o f the maxi-K channel a and p i -4 subunits and Katpchannel Kir6 and SUR subunits.
The putative roles of Katp channels in osteoblast proliferation will also be explored, with the objective o f testing whether KAtpchannels are important in osteoblast cell growth by means o f haemocytometry and MTS dye-conversion assays.
C H A P T E R 3: F U N C T I O N A L K* C H A N N E L S IN O S T E O B L A S T C E L L M E M B R A N E S
3.2 Methods
3.2.1 Electrophysiology
MG63, SaOS-2 and HOB-c cells were seeded at densities o f between 3,000 and 5,000 cells per coverslip and incubated for at least 24 hours before patch-clamp recording. Stock solutions o f drugs were prepared in distilled water unless otherwise stated, and were obtained from Sigma. HOB-c primary human osteoblasts with passage numbers between 4 and 10 were used.
The patch-clamp methods described in chapter 2 (2.4) were used, with freshly- pulled electrodes with a resistance o f between 4 and 8 MQ (measured with High K+ in the pipette and Locke solution in the bath). Criteria for successful seals included a seal resistance o f > 1 GQ, low variance o f noise when channels were closed (RMS noise < 1), and stability over a range o f holding potentials.
3.2.1.1 Single channel analysis
Using Strathclyde electrophysiology Software WinEDR v2.8.2, recordings were initially scanned at low resolution by eye for potentially interesting events, anomalies, etc. followed by software construction o f distribution o f current amplitude histograms for all digitised points to determine single
channel current amplitudes and the number o f channels active in the patch.
Peaks were fitted with Gaussian probability density functions using the least
curve, estimates o f the single channel open probability could be made from area under the curve, corresponding to the percentage o f time spent in that current state, and provided that only one channel appeared in the patch.
Occasionally, in recordings from less-stable patches, or where current states were close together, the all points histogram was not successful at resolving current peaks, and the Patlak average method (Patlak, 1988) was used to construct current amplitude histograms. This method allows effective resolution o f peaks by averaging a sequence o f digitised data points (user defined from 2 - 256) and excluding averages with a standard deviation that exceeds the variance of a portion o f closed state record. This effectively excludes transitional states, allowing construction o f resolved peaks.
Transition detection was used to measure the transitions from channel closed to open states, and the dwell times in these states. This was achieved by placing a threshold at 50% o f the open single channel current level. This provided valuable open probability data (as NP0) and the dwell times for kinetic analysis. Logarithmic dwell time histograms (as described by Sigworth & Sine, 1987) were constructed to determine how the length o f open and closed states o f the maxi K channel varied with changes in potential. Histograms were fitted with exponential probability density functions (least squares method) to reveal open (t0) or closed (xQ) time components.
C H A P T E R 3: F U N C T I O N A L K + C H A N N E L S IN O S T E O B L A S T C E L L M E M B R A N E S
3.2.1.2 Filtering
The recording apparatus made use o f in-built filters to decrease the amount o f random electrical noise in the recordings. However, though filtering can be a very useful tool, heavy filtering may erase short-duration channel openings and in doing so, decrease the quality o f the data record. Also, in some cases rather than complete erasure o f short-duration openings, filtering results in failure to record the full amplitude o f a channel current, which has clear consequences for I/V plots and conductance calculations. Therefore, filtering has to be a payoff between improved signal/noise and minimising loss o f data quality.
The effective cut-off frequency o f filtering used in the experimental apparatus for the single-channel recordings detailed in this chapter can be calculated by the following equation (Colquhoun, 1994):
1 / /c2 = 1 / / 12 + 1 / / 2 2 . . . + 1 / / » 2
where f c is the effective cut-off frequency and / i , f i and / n are the cut-off frequencies o f each filter in the system. Thus, in the system used here with a 20 kHz filter and a 5 kHz filter, the calculated effective cut-off is 4.85 kHz.
Another way to describe the level o f filtering is by the rise time, which is the time taken for a signal to rise from 10% o f its low value to 90% o f its maximum value. The rise time, tT, is given by (Colquhoun, 1994):
tr = 0.3321 / / c
Therefore, a 4.85 kHz filter has a rise time o f 68 ps. The f c and /r values are important in determining the minimum duration o f an event which can be detected at a specified fraction o f its full amplitude, which is often called the minimum resolvable event. Colquhoun & Sigworth (1983) showed that for the errors in duration o f events caused by filtering to become negligible and for the signal to reach at least 90% o f its full amplitude, the minimum event duration needs to be above 1.3tT and for a reliable amplitude measurement, an opening must be above 2tT.
The minimum resolvable event for an opening at 90% o f its full amplitude for the 4.85 kHz effective filter is given by 1.3/r which is 88 ps. Event durations shorter than double this value (for greater confidence), i.e. shorter that 176 ps, were not included in the kinetic analysis.