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ABSTRACT: Cellular excitability originates from a concerted action of different ion channels. The genomic diversity of ion channels (over 100 different genes) underlies the functional diversity of neurons in the central nervous system (CNS) and even within a specific type of neurons large differences in channel expression have been observed. Patch-clamp is a powerful technique to study the electrophysiology of excitability at the single cell level, allowing exploration of cell-to-cell variability. Only a few attempts have been made to link electrophysiological profiling to mRNA transcript levels and most suffered from experimental noise precluding conclusive quantitative correlations. Here we describe a refinement to the technique that combines patch-clamp analysis with quantitative real-time (qRT) PCR at the single cell level. Hereto the expression of a housekeeping gene was used to normalize for cell-to-cell variability in mRNA isolation and the subsequent processing steps for performing qRT-PCR. However, the mRNA yield from a single cell was insufficient for performing a valid qRT-PCR assay; this was resolved by including a RNA amplification step. The technique was validated on a stable Ltk(-) cell line expressing the Kv2.1 channel and on embryonic dorsal root ganglion (DRG) cells probing for the expression of Kv2.1. Current density and transcript quantity displayed a clear correlation when the qRT-PCR assay was done in twofold and the data normalized to the transcript level of the housekeeping gene GAPD. Without this normalization no significant correlation was obtained. This improved technique should prove very valuable for studying the molecular background of diversity in cellular excitability.
Journal of neuroscience methods 06/2012; 209(1):227-34. · 2.30 Impact Factor
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ABSTRACT: The voltage-gated potassium channel subunit Kv2.1 forms heterotetrameric channels with the silent subunit Kv6.4. Chimeric Kv2.1 channels containing a single transmembrane segment from Kv6.4 have been shown to be functional. However, a Kv2.1 chimera containing both S1 and S5 from Kv6.4 was not functional. Back mutation of individual residues in this chimera (to the Kv2.1 counterpart) identified four positions that were critical for functionality: A200V and A203T in S1, and T343M and P347S in S5. To test for possible interactions in Kv2.1, we used substitutions with charged residues and tryptophan for the outermost pair 203/347. Combinations of substitutions with opposite charges at both T203 and S347 were tolerated but resulted in channels with altered gating kinetics, as did the combination of negatively charged aspartate substitutions. Double mutant cycle analysis with these mutants indicated that both residues are energetically coupled. In contrast, replacing both residues with a positively charged lysine together (T203K + S347K) was not tolerated and resulted in a folding or trafficking deficiency. The nonfunctionality of the T203K + S347K mutation could be restored by introducing the R300E mutation in the S4 segment of the voltage sensor. These results indicate that these specific S1, S4, and S5 residues are in close proximity and interact with each other in the functional channel, but are also important determinants for Kv2.1 channel maturation. These data support the view of an anchoring interaction between S1 and S5, but indicate that this interaction surface is more extensive than previously proposed.
Biophysics of Structure and Mechanism 04/2011; 40(6):783-93. · 2.44 Impact Factor
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ABSTRACT: Human ether-a-go-go-related gene (HERG) potassium channels contribute to the repolarization of the cardiac action potential and display unique gating properties with slow activation and fast inactivation kinetics. Deletions in the N-terminal 'proximal' domain (residues 135-366) have been shown to induce hyperpolarizing shifts in the voltage dependence of activation, suggesting that it modulates activation. However, we did not observe a hyperpolarizing shift with a subtotal deletion designed to preserve the local charge distribution, and other deletions narrowed the region to the KIKER containing sequence 362-372. Replacing the positively charged residues of this sequence by negative ones (EIEEE) resulted in a -45 mV shift of the voltage dependence of activation. The shifts were intermediate for individual charge reversals, whereas E365R resulted in a positive shift. Furthermore, the shifts in the voltage dependence were strongly correlated with the net charge of the KIKER region. The apparent speeding of the activation was attributable to the shifted voltage dependence of activation. Additionally, the introduction of negative charges accelerated the intermediate voltage-independent forward rate constant. We propose that the modulatory effects of the proximal domain on HERG gating are largely electrostatic, localized to the charged KIKER sequence.
Biophysical Journal 01/2007; 91(12):4381-91. · 3.65 Impact Factor
