Carlos A. Villalba-Galea

Spatial Ca2+ Distribution in Contracting Skeletal and Cardiac Muscle Cells

The spatiotemporal distribution of intracellular Ca(2+) release in contracting skeletal and cardiac muscle cells was defined using a snapshot imaging technique. Calcium imaging was performed on intact skeletal and cardiac muscle cells during contractions induced by an action potential (AP). The sarcomere length of the skeletal and cardiac cells was approximately 2 micrometer. Imaging Rhod-2 fluorescence only during a very brief (7 ns) snapshot of excitation light minimized potential image-blurring artifacts due to movement and/or diffusion. In skeletal muscle cells, the AP triggered a large fast Ca(2+) transient that peaked in less than 3 ms. Distinct subsarcomeric Ca(2+) gradients were evident during the first 4 ms of the skeletal Ca(2+) transient. In cardiac muscle, the AP-triggered Ca(2+) transient was much slower and peaked in approximately 100 ms. In contrast to the skeletal case, there were no detectable subsarcomeric Ca(2+) gradients during the cardiac Ca(2+) transient. Theoretical simulations suggest that the subsarcomeric Ca(2+) gradients seen in skeletal muscle were detectable because of the high speed and synchrony of local Ca(2+) release. Slower asynchronous recruitment of local Ca(2+) release units may account for the absence of detectable subsarcomeric Ca(2+) gradients in cardiac muscle. The speed and synchrony of local Ca(2+) gradients are quite different in AP-activated contracting cardiac and skeletal muscle cells at normal resting sarcomere lengths.

Gate Closure Strictly Follows Voltage-Sensor Movements in KV Channels

Kv channels are voltage-dependent potassium pores that shape the action potential duration and are critical for cell excitability. Detection of membrane potential (V) is done by a charged (Q) voltage sensor domain (VSD) whose reorientations generate a transient gating current (IQ). Prolonged depolarization of Shaker Kv channels pushes the VSD into the relaxed state, characterized by a slowing in IQOff. Kv channels also have two gates (in series) that seal off K+ permeation: the S6 bundle crossing (BC), directly tied to the VSD, and the selectivity filter (SF). Direct comparison of K+-conduction in Shaker, reflecting the status of the BC gate, with IQ shows a strong correlation between both. As IQOff slowed down with prolonged depolarizations, BC gate closure displayed a similar 2-fold slowing when the duration of a +20mV pre-pulse was increased from 0.2 to 10 seconds. Simultaneous monitoring of the VSD movement (fluorescence recordings) and channel gate closure (ionic recordings) in the TMRM-labeled Shaker mutant M356C showed that the slowing in IQOff and gate closure occurs simultaneously. This indicates that the gate is strictly controlled by the movements of the VSD and most importantly that the BC gate remains open even when the VSD relaxes. Consequently, K+ conduction continues as long as the SF gate does not close (inactivation). Interestingly, in Kv3.1 - a channel that regulates high frequency firing in-vivo - the opposite behavior was observed: prolonged depolarization speeded up both IQOff and gate closure. Thus, the effect of VSD relaxation differs between different subtypes of Kv channels suggesting that relaxation affects the excitability of cells differently depending on their depolarization history, either reducing excitability in cells expressing Shaker or increasing it in case of Kv3.1. (Support: NIH-GM030376, FWO-G025608)