A source of bias in the analysis of single channel data: assessing the apparent interaction between channel proteins

A recent study of single sodium channel currents in neuroblastoma cells suggested interaction between ion channels in close proximity to one another (T. Kiss and K. Nagy, Eur. Biophys. J. 12, 13, 1985). The opening of one channel appeared to affect the likelihood that neighboring channels might open. Some of the conclusions were based on the analysis of observed channel openings that were segregated depending on whether one channel or more than one channel was open at the same time. We hypothesized that the longer one channel remained open, the more likely another channel operating independently, would open, thereby creating the impression of an apparent coupling of channel behavior. We performed simulations and measurements of single sodium channel currents to determine whether the technique of event segregation could account for apparent channel interactions. The simulations showed that the segregation of overlapping (more than one channel open at the same time) and nonoverlapping events led to a bias in the estimated open time and the derived closing rate. To avoid the bias, we found that random pairing of opening and closing events provided an unbiased estimate of the mean closing rate. Using this random assignment approach, we showed that the mean closing rate of single sodium channels in neonatal rat myocytes decreased with depolarization over a limited range of membrane potential. This suggested that the underlying closure mechanism(s) was voltage dependent. From the analysis of open times, we found no evidence for channel interaction in the time scale of tens of milliseconds. Depolarizing steps without events occurred in runs suggesting the existence of long-lived shut state(s). Double pulse experiments with the prepulse and test pulse above threshold showed significant inactivation of channels that did not open. The rate of inactivation of shut channels was substantially slower than the closure rate of open channels. The rate of inactivation of cardiac sodium channels appeared to be strongly dependent on the initial channel state.