Identification of minuscule inward currents as precursors to membrane electroporation-induced currents: Real-time prediction of pore appearance

Background/Aims: The objective of this study is to examine the current signals in response to large hyperpolarizations with the aid of principal component analysis (PCA) to search for or even predict current fluctuations related to membrane electroporation-induced current (I _MEP ). Methods: The characteristics of principal eigenvalues generated for I _MEP and the current signals at 10 sec prior to the start of initial I _MEP (I _Pre ) were examined. As membrane hyperpolarizations were applied at 0.1 Hz, the appearance of I _MEP coincided with the higher principal eigenvalues extracted in PCA. Results: Subsequent addition of LaCl ₃ (100 μM) greatly reduced I _MEP and associated principal eigenvalues. In real-time analysis for a single frame (i.e, 300 msec), in response to large hyperpolarization, multiple runs of heralded minuscule inward currents (I _min ) occurring before large rise in current amplitudes were detected. With PCA, such heralded I _min was noted to coincide with the extreme principal eigenvalues. The duration of I _min together with large principal eigenvalues was influenced by different levels of membrane hyperpolarization. In GH3 cells, palmitoyl-L-carnitine (PALCAR), a long-chain acylcarnitine, effectively increased the I _MEP amplitude with an EC ₅₀ value of 2.4 μM. However, in PALCAR-treated cells, the I _min together with higher principal eigenvalues disappeared, while in isoflurane-treated cells, I _min occurring before large rise of current amplitude remained intact. Similarly, the PCA analysis from I _Pre in RAW 264.6 macrophages showed the presence of herald I _min accompanied by the extreme principal eigenvalues. Conclusion: It is clear from this study that these large principal eigenvalues are representative of MEP-associated formation of electropores. Therefore, different compositions around the surface membrane of cells may alter the appearance of I _min followed by I _MEP emergence.