Patch-Clamp Fluorometry based Determination of Relative Ion Permeability for HCN Channels

Abstract

Determining the relative permeability of different ions is a classical topic for channel biophysics. Two traditional methods have been used are: 1) single channel recording method, and 2) the reversal potential measurement based on the Goldman-Hodgkin-Katz (GHK) equation. Both methods have been used extensively, but certain limitations such as the extremely small single channel conductance prevent the broad application of either method to every type of channels. For the GHK/reversal potential method, when the differential ion concentrations across the cell membrane lead to changes in channel gating property or local biophysical parameters such as pH, the interpretation of the results could be complicated. For the determination of the relative permeability of ammonium (NH4+) compared to another ion, such as potassium (K), the GHK/reversal potential method may not produce accurate results because NH4+ in mill molar range produces a change in local pH on other side of the membrane. The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are known to be sensitive to different levels of pH. Here we have proposed a novel method for determining the relative permeability of different ions based on the technique of patch-clamp fluorometry (PCF), which enables simultaneous measurements of macroscopic current amplitude and fluorescence intensity. The slope of the current amplitude vs. fluorescence intensity is directly correlated to the relative ionic permeability. We applied this PCF-based method to the EGFP-tagged HCN channels and determined the ionic selectivity of K, Na+, and NH4+. The PK/PNa was found to be 2.92, the PK/PNa in the absence of any potassium ions was found to be 39, and the PK/PNH4 was found to be 15. Our results were consistent with previous reports. The PCF-based approach holds the potential as an alternative approach for determining the relative ion permeability.