Modulation of the Conductance-Voltage Relationship of the BK_Ca Channel by Mutations at the Putative Flexible Interface Between Two RCK Domains

¹ Gwangju Institute of Science and Technology (GIST)
² Tufts University School of Medicine

^* To whom correspondence should be addressed. E-mail: cspark{at}kjist.ac.kr.

Submitted on March 12, 2007
Revised on April 18, 2007
Accepted on 4 September 2007

	Abstract

Calcium-dependent gating of the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel is conferred by the large cytosolic carboxyl terminus containing two domains of the regulator of K⁺-conductance (RCK) and the high-affinity Ca²⁺-binding site (the Ca²⁺-bowl). In our previous study, we located the putative second RCK domain (RCK2) and demonstrated that it interacts directly with RCK1 via a hydrophobic 'assembly interface'. In this study, we tested the structural model of the other interface, the 'flexible interface', by strategically positioning charge pairs across the putative interface. Several charge mutations on RCK2 affected the voltage-dependent activation of the channel. In particular, the Gly-to-Asp substitution at position 803 profoundly affected channel activation by stabilizing the open conformation of the channel with minimal effects on its Ca²⁺ affinity and voltage sensitivity. Various mutations at Gly803 shifted the channel's conductance-voltage curve either left or right over a 145 mV range. Since this residue is predicted to be in the first loop of RCK2 these results strongly suggest that this loop plays a critical role in determining the intrinsic equilibrium constant for channel opening, and, they support the hypothesis that this loop is part of an interface that mediates conformational coupling between RCK1 and RCK2.

Key Words: BK channel, RCK domains, gating, modulation, site-directed mutagenesis